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Philips HeartStart Intrepid监护除颤器TBI建议说明书

Application notePhilips HeartStart Intrepidmonitor/defibrillator TBI advisory OverviewThe TBI advisory feature on the HeartStart Intrepidassists in the monitoring of patients who are determinedat high risk of having suffered a traumatic brain injury.The TBI advisory provides visual guidance to helpprevent the following conditions:• Hypoxia – low blood oxygen saturation(as measured by SpO 2)• Hypotension – low systolic blood pressure• Hyperventilation-induced hypocapnia – decreasedcarbon dioxide in the blood (as measured by EtCO 2).The TBI advisory is available for both adult and infant/child patients on devices in Monitor Mode configured with SpO 2, NBP, and EtCO 2. The TBI advisory display reflects the appropriate target limits for each TBI Care parameter.IntroductionEvery year, 2.2 million people in the UnitedStates suffer a traumatic brain injury (TBI), with approximately 52,000 deaths and 280,000 hospitalizations.1 The economic burden of TBI is more than USD 60 billion per year.1 TBI occurs when an external mechanical force inflicts sudden trauma to the head disrupting the normal function of the brain.2However, subsequent preventable (secondary) injury to the brain can add dramatically to the primary injury, and the extent of damage during this time is significantly influenced by the early care the patient receives.3 The emphasis on early recognition and early treatment of TBI should be similar to that of other pathologies where survivability and recovery are time-dependent, i.e., sudden cardiac arrest.4TBI PathophysiologyThe brain accounts for the consumption of 20% of bodily oxygen and 25% of the blood glucose. Because of this, cerebral blood flow (CBF) is critical for ensuring adequate brain metabolism and preventing cerebral ischemia. CBF is a function of cerebral perfusion pressure (CPP) and cerebral vascular resistance (CVR), CPP is calculated by subtracting intracranial pressure (ICP) from mean arterial blood pressure (MAP) (CPP= MAP-ICP). Normal ICP ranges from 10-15 mmHg, and normal MAP ranges from 70-95 mmHg; therefore, the average CPP is approximately 60-80 mmHg. CBF is regulated through several mechanisms in the healthy patient; however, disruption of those mechanisms can have catastrophic consequences on the brain and its control of body systems.ICP and TBIThe cerebrum, cerebellum, and brainstem use approximately 80% of the intracranial space (1,200 cc), with blood vessels and cerebral spinal fluid utilizing the remaining volume at 12% (150 cc) and 8% (90 cc), respectively. Cerebral swelling and hemorrhage secondary to TBI increase the cerebral volume and require the body to compensate for the increased volume by reducing the volume of other brain structures and cerebrospinal fluid within the cranium. If the amount of room available to compensate forthe increasing volume is less than the expanding volume, then an increase in ICP will occur. Initially,the brain compensates for an increase in cerebral mass by compressing the cerebral venous blood vessels and, as cerebral volume continues to increase, cerebrospinal fluid is pushed from the skull into the spinal space. The initial compensatory mechanisms can maintain ICP close to normal; however, once those mechanisms are exhausted, ICP quickly rises. The continued increase in ICP impedes arterial flow into the cranial space and constricts cerebral blood vessels. These mechanisms decrease CBF and, subsequently, the cardiocerebrovascular system attempts to compensate for the decreased CBF by increasing SBP. This further increases ICP.The decreased CBF also causes cerebral ischemiaand neuronal hypoxia. If spontaneous ventilationis compromised, pCO2increases, and this results in cerebral vasodilation and additional increases in ICP. Cerebral edema secondary to neuronal injury and cerebral ischemia leads to a further exacerbation of ICP. Ultimately, the continued increase of ICP will lead to profoundly compromised CBF, resulting in death. Figure 1.552Three critical factors of TBI (the 3 H-Bombs)The National EMS TBI Guidelines published by theBrain Trauma Foundation emphasize the importanceof maintaining adequate cerebral oxygenation andperfusion by avoiding three critical factors.7• Hypoxia• Hypotension• Hyperventilation (that leads to hypocapnea andcerebral vasoconstriction)A single incidence of any one of these factorsdramatically increases patient mortality.3 However,maintaining End-Tidal Carbon Dioxide (EtCO2), arterialblood oxygenation (SpO2), and a systolic bloodpressure (SBP) within guidelines established by theTraumatic Brain Injury Foundation have demonstratedimproved outcomes in patients with severe TBI.3HypocapniaPreventing hyperventilation induced hypocapnia(EtCO2<32 mmHg) is critical because of its directcorrelation to CBF.3,7 For example, a patient who hasa pCO2 of 40 mmHg has a CBF of 50 ml per 100g ofbrain tissue per minute.8 However, if pCO2drops to30 mmHg, then CBF will drop to 40ml per 100g ofbrain tissue per minute, a 20% decrease in CBF.8 Therelationship between CBF and pCO2is so delicate thata 1 mmHg change in pCO2can alter CBF up to 5% anddecreasing pCO2by relatively little (i.e., <32 mmHg) isassociated with significant mortality.3,8-10Unfortunately, unrecognized inadvertenthyperventilation in TBI patients is still a majorproblem, at least in the prehospital environment. Inthe San Diego RSI Trial, 59% of patients intubatedbefore arriving at the hospital experienced at leastone incident of hyperventilation resulting in anEtCO2value of less than 25 mmHg.9 The importanceof monitoring EtCO2cannot be overstated. Onestudy observed that prehospital intubation in asetting where capnography was not used reporteda mortality rate of 33%; conversely, the ability tomonitor patients’ EtCO2level and use a ventilator wasassociated with a statistically significant, 18% lowerrelative rate of death.11Figure 1: Relationship between increased intracranial pressure and decreased cerebral perfusion.34Hypotension and hypoxiaMultiple studies have demonstrated a strong correlationbetween a single episode of hypotension (SBP <90mmHg) or hypoxia (SpO 2<90%) and decreased patientsurvival.7,12-14 Independently, hypotension or hypoxiahave a dramatic deleterious effect on patient mortality.However, when a patient experiences both hypotensionand hypoxia, the adjusted probability of death doublesagain (beyond that of either alone).15The Excellence in Prehospital Injury Care (EPIC)TBI study examined the outcome of 13,151 patientswho had suffered major (moderate/severe/critical)TBI over a seven-year period. The researcherscompared the mortality rate of patients whoexperienced neither hypoxia nor hypotension(n=11,545), hypotension only (n=604), hypoxia only(n=790), or hypotension and hypoxia (n=212) in theprehospital environment.15 The study concludedthat having at least one episode of hypotension orhypoxia (alone) resulted in a 20.7% (n=125) and 28.1%(n=222) mortality rate, respectively. The combinationof both hypoxia and hypotension was associated witha 43.9% (n=93) mortality rate.15 Comparatively, patients who did not experience hypoxia or hypotension had a 5.6% (n=644) rate of death.15 The adjusted analysis found that a patient that experiences both hypotension and hypoxia in the prehospital environment has a 600% (aOR = 6.1) higher likelihood of dying than someone who experiences neither.15Another important finding of the EPIC TBI study was the absence of any identifiable SBP threshold in relation to patient mortality. The study identified that over the range of 40 mmHg to 120 mmHg SBP there was a consistent decrease in survival for every 10 mmHg drop and a progressive association between decreasing SBP and both unadjusted and adjusted risk of death.1 In other words, a patient with a SPB of 60 mmHg is 4 times as likely to die than a patient with a SBP of 135 mmHg.1 The implications are obvious: the injured brain is highly sensitive to a lack of perfusion, and the levels of systemic blood pressure associated with increased mortality may be far above the “classic” threshold for “hypotension” (SBP<90 mmHg).1Importance of treating and preventing hypotension, hypoxia, and hypocapnia Researchers from the EPIC TBI study worked with ArizonaEMS agencies to implement prehospital TBI guidelinesfocusing on three areas of prevention and treatment (thethree “H-Bombs”):• Preventing and treating hypoxia by use of high-flowO 2 and emphasis on basic airway maneuvers• Preventing and treating hypotension by rapidlyinfusing isotonic fluids• Preventing hyperventilation by using appropriateventilation rates, adjuncts, and EtCO 2In the EPIC Study, adjusted survival to hospital dischargedoubled (aOR 2.03) for patients with severe TBI (headregion severity score = 3-4) and tripled (aOR 3.28) forpatients with severe TBI that also received positivepressure ventilation (PPV) via bag-valve mask orendotracheal intubation (ETI). The increased survival ofpatients with severe TBI receiving PPV was attributedto the emphasis on maintaining EtCO 2 between 35 and 45 mmHg, rate-timers that gave visual cues for proper manual ventilation and using flow-controlled ventilation bags to help prevent hyperventilation and overventilation.One of the most important findings of the EPIC Study was that the initial improvement of patient survival faded over time, and this was most likely because of the diminished emphasis on following the prehospital TBI guidelines. The researchers could not require retraining of the study participants. Therefore, a method of ensuring that hypoxia, hypotension, and hypocapnea are recognized early should be available to all prehospital providers. The authors concluded that their findings make it likely that the use of monitoring technology that can give cues and real-time feedback to providers during the resuscitation of brain injured patients will improve outcomes by helping to prevent secondary brain injury from hypoxia, hypotension, and hyperventilation.10HeartStart Intrepid Traumatic Brain Injury advisory OverviewThe TBI advisory feature on the HeartStart Intrepidassists in the monitoring of patients who are determinedat high risk of having suffered a traumatic brain injury.The TBI advisory provides visual guidance to helpprevent the following conditions:• Hypoxia – low blood oxygen saturation (asmeasured by SpO2)• Hypotension – low systolic blood pressure• Hyperventilation-induced hypocapnea – decreasedcarbon dioxide in the blood (as measured by EtCO2).The TBI Advisory is available for both adult and infant/child patients on devices in Monitor Mode configuredwith SpO2, NBP, and EtCO2. The TBI Advisory displayreflects the appropriate target limits for each TBI Care parameter.Notes:• These limits must be pre-established andpre-configured prior to using this feature (see “Configuration – TBI Advisory” in the HeartStart Intrepid Instructions for Use).• If a physiological alarm condition occurs, it will have a higher priority than the TBI advisory and will obscure the TBI Indicators.56Enabling the TBI advisory1. TBI: Ensure an SpO 2 sensor, NBP cuff, and EtCO 2 tubing are connected to the patient2. Press the [Enable TBI] soft key.If no patient age has been entered, the Patient Age number selector appears.3. If prompted, enter the patient age.The patient age is displayed in the status area next to patient name/ID.When age is entered, mode text changes from Monitor to Monitor TBI.Notes:• The TBI Advisory is only available in Monitor Mode and is not available during the acquisition of a 12-lead ECG.• Devices must be configured with SpO 2, NBP, andEtCO 2 measurement parameters, and all threeparameters must be in use.• TBI Advisory can only be enabled when TBI limits are pre-configured on the device.• The neonatal patient category is not supported.Displaying the TBI advisoryThe TBI advisory display appears in the numeric parameter area. A TBI limit bar is displayed at the top of the corresponding measure box for NBP , EtCO 2, and SpO 2.TBI for systolic blood pressure (SBP)Systolic blood pressure that falls outside theestablished TBI limits is indicated by a yellow bar, and TBI SBP > High Limit or TBI SBP < Low Limit is displayed, as shown in Figure 2.Systolic blood pressure within limits the setting for the TBI configuration is indicated by TBI SBP on a green bar, as shown in Figure 3.TBI for EtCO 2 If the EtCO 2 value falls outside the established TBI limits, TBI EtCO 2 > High Limit or TBI EtCO 2 < Low Limit is displayed in a yellow TBI bar as shown in Figure 4.If the EtCO 2 value is within the limits setting for the TBI configuration, TBI EtCO 2 is displayed in a green bar as shown in as shown in Figure 5.WARNING: To ensure correct functioning of the CO 2 measurement, use only approved CO 2 accessories as listed in the HeartStart Intrepid Instructions for Use, Chapter 18:Supplies and Accessories.Figure 5: TBI within limits.Figure 2: TBI outside limits.Figure 3: TBI within limits.Figure 4: TBI outside limits.7TBI for SpO 2For SpO 2, there is no upper limit, only a lower limitthreshold for SpO 2 is set. If the SpO 2 value falls belowthe established TBI limit, TBI SpO 2 < Low Limit isdisplayed on a yellow TBI bar as shown in Figure 6.If the SpO 2 value is above the limit set for the TBIconfiguration, the TBI bar is green, and the text TBI SpO 2is displayed on a green bar as shown in Figure 7.Figure 6: SPO 2below TBI limit.Figure 7: SPO 2 above TBI limit.References1. Spaite, D. W., Hu, C., Bobrow, B. J., Chikani, V., Sherrill,D., Barnhart, B., . . . Adelson, P. D. (2017). Mortality andprehospital blood pressure in patients with majortraumatic brain injury: Implications for the hypotension threshold. JAMA Surgery, 152(4), 360-368.2. Rosenfeld, J. V., Maas, A. I., Bragge, P., Morganti-Kossmann, M. C., Manley, G. T., & Gruen, R. L. (2012).Early management of severe traumatic brain injury.The Lancet, 380(9847), 1088-1098.3. Spaite, D. W., Bobrow, B. J., Stolz, U., Sherrill, D., Chikani,V., Barnhart, B., . . . Denninghoff, K. R. (2014). Evaluation of the impact of implementing the emergency medical services traumatic brain injury guidelines in arizona:The excellence in prehospital injury care (epic) study methodology. Academic Emergency Medicine, 21(7), 818-830. doi:doi:10.1111/acem.12411.4. Gaither, J. B., Spaite, D. W., Bobrow, B. J., Denninghoff,K. R., Stolz, U., Beskind, D. L., & Meislin, H. W. (2012).Balancing the potential risks and benefits of out-of-hospital intubation in traumatic brain injury:The intubation/hyperventilation effect. Annals ofEmergency Medicine, 60(6), 732-736.5. Bledsoe, B. E., Porter, R. S., & Cherry, R. A. (2001). Head,facial, and neck trauma. In Paramedic care: Principles & practices trauma emergencies (1 ed., Vol. 4, pp. 285-286). Upper Saddle River, NJ: Prentice Hall.6. Sanders, M. J. (2012). General principles ofpathophysiology. In K. McKenna, L. M. Lewis, & G.Quick (Eds.), Mosby’s paramedic textbook (4th ed., pp.211-256). Burlington, MA: Jones & Bartlett Learning.7. Carney, N., Totten, A. M., O’Reilly, C., Ullman, J. S.,Hawryluk, G. W., Bell, M. J., . . . Ghajar, J. (2017).Guidelines for the management of severe traumaticbrain injury, fourth edition. Neurosurgery, 80(1), 6-15.doi:10.1227/NEU.0000000000001432.8. Giardino, N. D., Friedman, S. D., & Dager, S. R.(2007). Anxiety, respiration and cerebral bloodflow: Implications for functional brain imaging.Comprehensive Psychiatry, 48(2), 103-112.9. Davis, D. P., Heister, R., Poste, J. C., Hoyt, D. B., Ochs, M.,& Dunford, J. V. (2005). Ventilation patterns in patients with severe traumatic brain injury following paramedic rapid sequence intubation. Neurocritical Care, 2(2),165-171. 10. Spaite, D. W., Bobrow, B. J., Keim, S. M., Barnhart, B.,Chikani, V., Gaither, J. B., . . . Hu, C. (2019). Association of statewide implementation of the prehospital traumatic brain injury treatment guidelines with patient survival following traumatic brain injury: The excellence inprehospital injury care (epic) study. JAMA Surgery,e191152-e191152. doi:10.1001/jamasurg.2019.1152.11. Poste, J. C., Davis, D. P., Ochs, M., Vilke, G. M.,Castillo, E. M., Stern, J., & Hoyt, D. B. (2004). Airmedical transport of severely head-injured patientsundergoing paramedic rapid sequence intubation.Air Medical Journal, 23(4), 36-40. doi:https://doi.org/10.1016/j.amj.2004.04.006.12. Shutter, L. A., & Narayan, R. K. (2008). Blood pressuremanagement in traumatic brain injury. Annals ofEmergency Medicine, 51(3, Supplement), S37-S38.doi:https:///10.1016/j.annemergmed.2007.11.013.13. Cormio, M., Robertson, C. S., & Narayan, R. K. (1997).Secondary insults to the injured brain. Journal ofClinical Neuroscience, 4(2), 132-148. doi:https://doi.org/10.1016/S0967-5868(97)90062-X.14. Manley, G. (2001). Hypotension, hypoxia, and headinjury: Frequency, duration, and consequences.Archives of Surgery, 136(10), 1118-1123.15. Spaite, D. W., Hu, C., Bobrow, B. J., Chikani, V., Barnhart,B., Gaither, J. B., . . . Sherrill, D. (2017). The effect ofcombined out-of-hospital hypotension and hypoxia on mortality in major traumatic brain injury. Annalsof Emergency Medicine, 69(1), 62-72. doi:10.1016/j.annemergmed.2016.08.007.8。

戊酸雌二醇联合米索前列醇对绝经期妇女取环的作用分析

·临床研究·医学食疗与健康 2023年8月第21卷第22期作者简介：曾敏（1981.12-），女，本科，副主任医师，研究方向为妇科戊酸雌二醇联合米索前列醇对绝经期妇女取环的作用分析曾敏（湖南省岳阳市人民医院，湖南岳阳 414000）【摘要】目的：评定药物戊酸雌二醇合米索前列醇方案用于绝经期妇女取环中的效果。

方法：纳入岳阳市人民医院2020年1月至2022年10月的100例绝经期取环妇女，采用简单随机化法分组法，50例米索前列醇方案者为对照组，50例戊酸雌二醇联合米索前列醇方案者为试验组，评定宫颈软化程度、疼痛程度、取环成功情况、手术情况及不良反应发生情况等指标，2组存在的差异。

结果：试验组绝经期妇女宫颈软化1级的几率高于对照组，2级、3级的几率低于对照组，差异有统计学意义（P ＜0.05）；试验组0级疼痛几率高于对照组，2级、3级疼痛几率低于对照组，差异有统计学意义（P ＜0.05）；试验组取环成功率（100.00%）比较于对照组取环成功率（92.00%）更高，差异有统计学意义（P ＜0.05）；试验组手术出血量、手术用时均低于对照组，差异有统计学意义（P ＜0.05）；试验组不良反应发生率（22.00%）比较于对照组不良反应发生率（20.00%），差异无统计学意义（P ＞0.05）。

结论：绝经期妇女取环中应用药物戊酸雌二醇合米索前列醇方案，能促使宫颈更好的软化，降低术中疼痛程度与手术出血量、手术用时，提高取环成功率，且不会增加不良反应的发生率，安全有效。

【关键词】戊酸雌二醇；米索前列醇；绝经期；取环【中图分类号】R711 【文献标识码】A 【文章编号】2096-5249（2023）22-041-04对于宫内节育环是一种较为常用的避孕工具，具有经济、可逆、简便、有效等特点，但是妇女过了孕龄期后，宫内节育环即失去了价值，此时则需要将节育环取出[1-2]。

节育环是机体中的异物，若长时间不取，则会对身心健康造成影响，出现节育环嵌顿、变形等的情况，甚至会对子宫造成损伤，引发腰痛、小腹下坠腹痛、阴道不规则出血等症状，降低生活质量，因此，将绝经期妇女的节育环取出很有必要[3-4]。

Hypogonadotropic Hypogonadism in Type 2

subnormal testosterone concentrations in these men are associated with a two to three times elevated risk of cardiovascular events and death in two early studies.Short-term studies of tes-tosterone therapy in hypogonadal men with type2diabetes have demonstrated an increase in insulin sensitivity and a decrease in waist circumference.However,the data on the effect of tes-tosterone replacement on glycemic control and cardiovascular risk factors such as cholesterol and C-reactive protein concentrations are inconsistent.As far as sexual function is concerned,testos-terone treatment increases libido but does not improve erectile dysfunction and thus,phospho-diesterase inhibitors may be required.Trials of a longer duration are clearly required to definitively establish the benefits and risks of testosterone replacement in patients with type2diabetes and low testosterone.(J Clin Endocrinol Metab96:2643–2651,2011)S ubnormal free testosterone concentrations in asso-ciation with inappropriately low LH and FSH con-centrations and a normal response to GnRH of LH and FSH in type2diabetes were first described in2004(1). These abnormalities were independent of the duration and severity of hyperglycemia[glycosylated hemoglo-bin(HbA1c)].Magnetic resonance imaging in these hy-pogonadal patients showed no abnormality in brain or the pituitary(1).This association of hypogonadotropic hypogonadism(HH)with type2diabetes has now been confirmed in several studies and is present in25–40% of these men(2–5).In this context,it is important that The Endocrine Society now recommends the measure-ment of testosterone in patients with type2diabetes on a routine basis(6).These observations were recently extended to younger patients with type2diabetes be-tween the ages of18and35yr who had HH at a rate of 33%when the usual normal range for middle age was employed,whereas the rate was58%when age-specific normal range for free testosterone for the young was em-ployed(7).With the advent of more specific liquid chro-matography tandem mass spectrometry assay for measur-ing total testosterone,the reference ranges for total and free testosterone have recently been revised downward. Using this methodology,in our most recent study,we have found that29%of men with type2diabetes have sub-normal free testosterone concentrations,as measured by equilibrium dialysis(8);25%had HH,whereas4%had hypergonadotropic hypogonadism.Type2diabetic men with low testosterone levels have also been found to have a high prevalence of symptoms suggestive of hypogonadism such as fatigability and erectile dysfunction(2).In all of the above studies,total testosterone and free testosterone concentrations wereISSN Print0021-972X ISSN Online1945-7197Printed in U.S.A.Copyright©2011by The Endocrine Societydoi:10.1210/jc.2010-2724Received November19,2010.Accepted July8,2011.Abbreviations:BMD,Bone mineral density;BMI,body mass index;CRP,C-reactive protein; HbA1c,glycosylated hemoglobin;HH,hypogonadotropic hypogonadism;HOMA-IR,ho-meostasis model assessment for insulin resistance;PSA,prostate-specific antigen.J Clin Endocrinol Metab,September2011,96(9):2643–2643inversely related to body mass index(BMI)and age. However,the presence of low testosterone concentra-tion was not entirely dependent upon obesity because 25%of nonobese patients(31%of lean and21%of overweight)also had HH(1).HH is relatively rare in type1diabetes and,therefore,is not a function of di-abetes or hyperglycemia per se(9).Thus,in view of the inverse relationship between BMI and testosterone con-centrations in both type1and type2diabetes,HH is probably related to insulin resistance(1,4,9).Previous studies have shown that hypogonadism is associated with upper abdominal adiposity,insulin resistance,and the metabolic syndrome(10,11).Treatment of systemic insulin resistance by rosiglitazone leads to a modest in-crease in testosterone concentrations in men with type 2diabetes(12),without the restoration of testosterone concentrations to normal.A recent study investigated the prevalence of low tes-tosterone concentrations in a large number of obese and diabetic men(mean age,60yr;range,45–96yr)(13);44% of diabetic and33%of age-matched nondiabetic men had subnormal free testosterone concentrations,respectively. Forty percent of obese men and50%of obese diabetic men had subnormal free testosterone concentrations.Thus, obesity is associated with a high prevalence of hypogo-nadism,and the presence of diabetes adds to that risk. Possible Pathophysiological Mechanisms Underlying HH in Type2DiabetesRole of estradiolBecause testosterone and androstenedione in the male can be converted to estradiol and estrone,respectively, through the action of aromatase in the mesenchymal cells and preadipocytes of adipose tissue,it has been suggested that excessive estrogen secretion due to aromatase activity in the obese may potentially suppress the hypothalamic secretion of GnRH(14).This hypothesis was examined in a recent study that compared the estradiol concentrations in240type2diabetic men with and without HH(8).Total estradiol concentrations were measured by immunoassay, and free estradiol concentrations were calculated using SHBG.Total and free estradiol concentrations in men with HH were significantly lower than in those without HH(8).To confirm these findings,total estradiol concen-trations were measured in a subset of102men by the liquid chromatography tandem mass spectrometry assay,and free estradiol concentrations were measured by equilib-rium dialysis.Estradiol concentrations were25%lower in men with HH.Free estradiol concentrations were directly related to free testosterone concentrations,irrespective of age or BMI.The diminished availability of the substrate, testosterone,may therefore be the major determinant fac-tor of estradiol concentrations in these men.A study in elderly men(European Male Ageing Study)has also found lower estradiol concentrations in hypogonadal men(15). Thus,it appears that the low testosterone concentrations in HH of diabetes,as in aging,are not the consequence of estradiol-dependent suppression of the hypothalamo-hy-pophyseal-gonadal axis.Furthermore,HH in type2dia-betic men with a normal weight is not likely to be associ-ated with increased estradiol concentrations(1).Role of insulin resistanceThe selective deletion of the insulin receptor from neu-rons in mice leads to a reduction in LH concentrations by 60–90%and low testosterone concentrations(16).These animals respond to GnRH challenge by normal or supra-normal release of LH.In addition,these animals had atro-phic seminiferous tubules with markedly impaired or ab-sent spermatogenesis.In addition,it is known that the incubation of hypothalamic neurons with insulin results in the facilitation of secretion of GnRH(17,18).Thus,in-sulin action and insulin responsiveness in the brain are necessary for the maintenance of the functional integrity of the hypothalamo-hypophyseal-gonadal axis.Role of inflammatory mediatorsTNF-␣and IL-1␤have been shown to suppress hypo-thalamic GnRH and LH secretion in experimental animals and in vitro(19,20).It is therefore relevant that C-reactive protein(CRP)concentrations are markedly increased in hypogonadal type2diabetic men compared with men with type2diabetes and normal testosterone(6.5vs.3.2 mg/liter)(21).These data were confirmed by another study from Australia in which the median CRP concen-tration in type2diabetic patients with low total testos-terone was7.7mg/liter compared with4.5mg/liter in men with normal testosterone(4).Free testosterone concen-trations were inversely related to CRP concentrations(rϭϪ0.27;Pϭ0.02).It is thus possible that inflammatory mediators may contribute to the suppression of the hypo-thalamo-hypophyseal axis and the syndrome of HH in type2diabetes.The presence of inflammation may also contribute to insulin resistance because several inflamma-tion-related mediators,such as suppressor of cytokine sig-naling-3,I␬B kinase␤,and c-Jun N-terminal kinase-1in-terfere with insulin signal transduction(22,23)and contribute to insulin resistance.These mediators are also known to be increased in obesity(24).In summary,it is likely that there are several interlinked causative mechanisms underlying HH in men with type2 diabetes.It should also be noted that human chorionic2644Dandona and Dhindsa Hypogonadism in Type2Diabetes J Clin Endocrinol Metab,September2011,96(9):2643–2651gonadotropin-induced testosterone secretion by Leydig cells is inversely related to insulin sensitivity(as measured by hyperinsulinemic euglycemic clamp)among men with varying degrees of glucose tolerance(25).Thus,the lesion resulting in hypogonadism in obesity and type2diabetes may occur at several levels of the hypothalamic-pitu-itary-gonadal axis.However,the absence of an increase in gonadotropin concentrations indicates that the pri-mary defect in type2diabetes and obesity is at the hy-pothalamo-hypophyseal level.What Comes First:Hypogonadism or Type 2Diabetes?Because even young men with type2diabetes and patients with newly discovered type2diabetes have a high prev-alence of HH and obesity is associated with HH,it is pos-sible that HH precedes diabetes.Several epidemiological studies have shown that low testosterone at baseline ap-proximately doubles the odds of development of type2 diabetes(26–28).The data,however,are more consistent with total testosterone than with free testosterone(29). It is possible that low SHBG concentrations may medi-ate a portion of this association.SHBG polymorphisms that lead to lower SHBG concentrations are strongly predictive of the development of type2diabetes, whereas those that lead to higher SHBG concentrations are protective(30,31).Does Hypogonadism Matter?Possible Consequences of Hypogonadism in Type2 DiabetesIt is well accepted that low testosterone concentrations are associated with symptoms such as fatigue,lack of libido, and erectile dysfunction.Recent studies have described pathophysiological effects of subnormal testosterone con-centrations beyond those related to sexual health,as dis-cussed below.Symptoms of sexual dysfunctionCross-sectional studies have found a high prevalence of low libido(64%),erectile dysfunction(74%),and fatigue (63%)in hypogonadal men with type2diabetes(2).How-ever,the presence of these symptoms was similarly high in eugonadal men with type2diabetes as well(48,65,and 57%,respectively).The treatment of erectile dysfunction with phosphodiesterase-5inhibitors such as sildenafil in men with type2diabetes is known to be not as effective as that in nondiabetic subjects(32).Cardiovascular diseaseRecent evidence from longitudinal observational stud-ies shows that low testosterone concentration is prospec-tively associated with an increase in the incidence of car-diovascular ughlin et al.(33)prospectively followed794elderly men(mean age,71yr)for20yr in a community setting.The hazard ratio for men in the lowest quartile of bioavailable testosterone was1.44for all-cause mortality and1.36for cardiovascular mortality.Another prospective study[Osteoporotic Fracture in Men(MrOS) Swedish cohort(34)]that included3014men(mean age, 75yr;mean follow-up,4.5yr)showed a65%increased risk of mortality in men with low free testosterone(Ͻ6.1 ng/dl).Subnormal free testosterone concentrations are as-sociated with a69%increased risk of stroke or transient ischemic attack(35).Many cross-sectional,retrospective, case-control and smaller studies have also demonstrated an association of low testosterone with increased mortal-ity(36–38).However,the relationship between cardio-vascular mortality and low testosterone was not seen in two longitudinal studies(39,40).These studies were done in relatively younger populations(mean ages,52and55 yr)and had much lower mortality rates,which can pos-sibly explain the lack of an association(39,40).A recent study in930men with coronary artery disease reported that a low testosterone at baseline was associated with increased mortality after7yr of follow-up(21vs. 12%)(41).Only one study has looked at the association between subnormal testosterone concentrations and car-diovascular mortality specifically in men with type2di-abetes(42):in153men with type2diabetes and known coronary artery disease,subnormal free testosterone con-centration at baseline increased cardiovascular mortality by three times over2yr.Insulin sensitivityHH in men with type2diabetes is associated with a higher BMI(3–4kg/m2),12%more sc fat mass(measured by dual-energy x-ray absorptiometry),and higher waist-to-hip ratio compared with eugonadal men with type2 diabetes(1,2,43).In one study involving type2diabetic men from the United Kingdom,74%of hypogonadal men were obese compared with54%of eugonadal men(2).As of yet,no study has measured visceral,im,or hepatic fat content in type2diabetic men with and without HH. Many studies have documented that hypogonadism is as-sociated with insulin resistance(reviewed in Refs.44and 45).No study has compared the insulin resistance in type 2diabetic men with subnormal or normal testosterone concentrations.J Clin Endocrinol Metab,September2011,96(9):2643–2645HematocritHypogonadal type2diabetic men have a lower hemat-ocrit than those with normal testosterone concentrations (21).The prevalence of normocytic normochromic ane-mia in such patients is38%compared with3%in those with normal testosterone concentrations.A large study (464men)also found a direct correlation between free testosterone concentrations and hemoglobin in men with type2diabetes and renal insufficiency(46).Testoster-one regulates erythropoiesis(47).However,it has not yet been determined whether the association of anemia with hypogonadism in men with type2diabetes is causal or is secondary to other confounding factors such as inflammation.In these men,hemoglobin is positively related to testosterone but negatively related to CRP concentrations(21).Bone densityHypogonadism is associated with a decrease in bone mineral density(BMD)and an increase in fracture rate (48,49).Furthermore,trabecular bone architecture(mea-sured by high-resolution magnetic resonance imaging)de-teriorates much more in hypogonadal men compared with eugonadal men(50).Hypogonadal men usually have lower estradiol concentrations compared with eugonadal men because testosterone is the substrate for estradiol for-mation by aromatization(15).In epidemiological studies, estradiol concentrations correlate more robustly with BMD than testosterone concentrations in men(51).This is especially true of trabecular bone.However,testoster-one appears to be an independent predictor of cortical bone density(52,53).One study in men with type2dia-betes has shown that free testosterone concentrations are positively associated with BMD in arms and ribs,but not with hip,spine,or total body BMD values(43).Another study has shown a positive relation of lumbar spine BMD with free testosterone concentrations in men with type2 diabetes(54).No study has evaluated the relation between BMD and free estradiol concentrations in these men.It is possible that BMD in men with type2diabetes might relate more strongly to estradiol than to testosterone con-centrations,as has been shown in elderly nondiabetic men. No data are available on the fracture rates of hypogonadal men with type2diabetes.Prostate-specific antigen(PSA)Type2diabetic men have20%lower PSA concentra-tions than nondiabetic men(55).PSA concentrations are lower in hypogonadal than in eugonadal type2diabetic men(0.89vs.1.1ng/ml)(56).It is interesting that the incidence of prostatic carcinoma is lower in men with di-abetes.This is in contrast to the increased incidence of cancer in diabetics in various organs including the co-lon,the kidney,the breast,the endometrium,and the pancreas(57).The diminished incidence of prostate cancer in diabetics may receive a contribution from the high prevalence of HH and low testosterone concentra-tions.However,epidemiological studies do not support a causative role of testosterone in prostate cancer in nondiabetic populations(58).Should Testosterone Be Measured inEvery Patient with Type2Diabetes? Because the frequency of subnormal free testosterone con-centrations in type2diabetes is at least25%,we believe that free testosterone concentration should be measured in every patient with type2diabetes.This is consistent with The Endocrine Society guidelines.The prevalence of hy-pothyroidism is between5and8%in this population,and yet we screen every one for this condition.An Androgen Deficiency in Aging Male(ADAM)questionnaire should be administered in every patient with a low testosterone so that the presence of clinical hypogonadism can be estab-lished.One can argue that if the case for the replacement of testosterone in patients with HH is not proven,as dis-cussed below,is there a case for measuring its concentra-tions in every patient with type2diabetes?We believe that there is because,like hypothyroidism,patients may slide gradually into this clinical state without any overt symp-toms that may be revealed through direct questioning.“Asymptomatic”men may realize that they had been symptomatic only after a trial with testosterone.Such pa-tients may potentially benefit from testosterone replace-ment therapy,as discussed below.Should Men with Type2Diabetes andLow Testosterone Be Replaced with Testosterone?Issues to Be Considered in View of the Above DataThe Endocrine Society recommends that men with low testosterone and symptoms of androgen deficiency be con-sidered for therapy with testosterone(6).The guidelines do not recommend treatment of asymptomatic men with low testosterone.The Institute of Medicine recommends that more short-term studies in selected populations should investigate the benefits and risks of testosterone therapy.Trials in men with type2diabetes and obesity are important in this regard because both are commonly as-sociated with hypogonadism.A few studies on testoster-one replacement in type2diabetic men with low testos-terone have emerged and are described below.2646Dandona and Dhindsa Hypogonadism in Type2Diabetes J Clin Endocrinol Metab,September2011,96(9):2643–2651Insulin resistanceThree studies have shown a decrease in insulin resis-tance after testosterone therapy in hypogonadal men with type2diabetes.Kapoor et al.(59)studied the effects of treatment with im testosterone for3months in24hy-pogonadal type2diabetic men in a placebo-controlled, double-blind,crossover trial.Homeostasis model assess-ment for insulin resistance(HOMA)-IR decreased by1.73 after testosterone therapy compared with placebo.In an-other trial,32men with the metabolic syndrome and newly diagnosed type2diabetes with total testosterone concentration of less than350ng/dl(12nmol/liter)were prescribed diet and exercise(60).Half of them were also given transdermal testosterone for1yr.Testosterone ther-apy resulted in greater improvements in insulin sensitivity (measured by HOMA-IR;Ϫ0.9)compared with diet and exercise alone.A prospective,randomized,double-blind multicenter trial of transdermal testosterone(3g metered-dose2%gel for1yr)therapy in220hypogonadal men with type2diabetes or metabolic syndrome has recently been published[Testosterone Replacement in Hypogo-nadal Men with Either Metabolic Syndrome or Type2 diabetes study(TIMES2)(61)].The primary endpoint of the study was a change in insulin sensitivity,as measured by HOMA-IR.Patients were evaluated every3months.A total of136men in the study had type2diabetes,176men had metabolic syndrome,and92men had both.Testos-terone therapy resulted in a15%(Pϭ0.01)decrease in HOMA-IR at6months and at1yr time-points in men with type2diabetes as well as in those with metabolic syn-drome.One study in lean hypogonadal type2diabetic men with a mean BMI of24kg/m2did not show any change in insulin sensitivity after treatment with low-dose im testosterone(100mg every3wk)for3month(62).This dose is inadequate and may account for the lack of effect. It is,however,possible that the change in insulin sen-sitivity due to testosterone therapy occurs only in obese, and presumably insulin-resistant,men.Thus,it appears that insulin resistance improves with testosterone ther-apy in obese men with type2diabetes.These studies have calculated HOMA-IR to measure insulin resis-tance.This needs to be confirmed by trials that use hy-perinsulinemic-euglycemic clamp methodology.It is also not clear whether the effect is due to a change in body composition or independently of it.Glycemic controlIn three of the above-mentioned studies,glycemic con-trol was also evaluated by measuring HbA1c and fasting glucose.The small study by Kapoor et al.(59)showed a decrease in fasting glucose(28mg/dl)and HbA1c(0.37%) compared with placebo with3months of testosterone re-placement.The trial in men with new onset type2diabetes with transdermal testosterone did show a decrease in HbA1c from7.5to6.3%over a period of1yr(60).This was in conjunction with diet and exercise,but no hypo-glycemic medications.The comparison group in this study was a diet and exercise group.There was a decrease in HbA1c from7.5to7.1%in this group.The mean fasting glucose decreased by34and29mg/dl in the testosterone and diet/exercise groups,respectively(Pϭ0.06for com-parison among groups).However,the larger trial (TIMES2)did not show a clear effect of testosterone re-placement on HbA1c(61).Medication changes were not allowed for the first6months of the study.Patients with type2diabetes showed a trend toward improvement in HbA1c at1yr(Ϫ0.4%;Pϭ0.057)but not at6months (Pϭ0.6).Although no changes were made in patient’s medications for the first6months,the study protocol al-lowed medication changes between6and12months; therefore,no clear conclusions can be made regarding the effect of testosterone therapy on glycemic control from this trial.There were no changes in fasting glucose or in-sulin.Thus,there appears to be a mild decrease in HbA1c with testosterone therapy in men with type2diabetes,but the data are inconsistent and currently testosterone re-placement cannot be recommended for glycemic control. Symptoms and sexual dysfunctionIn the TIMES2trial,there was an improvement in the International Index of Erectile Function score in the tes-tosterone replacement group,mainly due to an increase in sexual desire,but other symptoms did not change.The smaller trial of im testosterone by Kapoor et al.(59)in hypogonadal men with type2diabetes showed an im-provement in symptoms as measured by the ADAM ques-tionnaire.Although there are no specific studies assessing the effect of testosterone replacement on the effectiveness of phosphodiesterase IV inhibitors like sidenafil,studies in hypogonadal nondiabetics do show this benefit(63). Body composition and abdominal adiposity Heufelder et al.(60)showed a decrease in waist cir-cumference of14cm in men with new onset type2diabetes treated for1yr with transdermal testosterone,diet,and exercise.The control group that was prescribed only diet and exercise lost5cm.Kapoor et al.(59)showed a de-crease by1.63cm in waist circumference after im testos-terone treatment.In the TIMES2trial,there was a small but statistically significant decrease in waist circumference (0.8cm)in type2diabetic men treated with testosterone. Significantly,BMI did not change in any of the studies despite the decrease in abdominal girth.J Clin Endocrinol Metab,September2011,96(9):2643–2647Cardiovascular outcomesA recent meta-analysis of testosterone therapy trials ranging from3months to3yr did not show any change in the rates of death,myocardial infarctions,revasculariza-tion procedures,or cardiac arrhythmias compared with placebo/nonintervention groups(64).However,none of these trials was powered to show a difference.Surpris-ingly,a recent trial of testosterone replacement therapy designed to study the effects of testosterone replacement for6months on muscle mass and strength in elderly men (Ͼ65yr old)with limited mobility had to be discontinued prematurely due a higher incidence(22vs.5%)of cardio-vascular-related adverse events in the testosterone treat-ment arm compared with the placebo arm(65).This trial was not included in the previously mentioned meta-anal-ysis.The study population had a high prevalence of chronic conditions,and it is possible that the results could have been due to chance alone.However,other studies in elderly populations have not shown an increase in cardiac events after testosterone replacement(66–68).The TIMES2trial(61)reported that cardiovascular events oc-curred less commonly with testosterone than with placebo (4.6vs.10.7%;Pϭ0.095);however,this effect was short of significance.A recent study presented at the British En-docrine Societies meeting is of interest(69).This study investigated the effect of baseline testosterone concentra-tions and testosterone replacement therapy in hypogo-nadal men with type2diabetes on all-cause mortality.A total of578men with type2diabetes with a mean age of 61yr were followed for5.8Ϯ1.3yr;338men had normal testosterone concentrations at baseline;240were hypogo-nadal,of which58men received testosterone replacement therapy;and72men(12%)died during follow-up.The mortality rate in eugonadal men and untreated hypogo-nadal men was9and20%,respectively.Hypogonadal men treated with testosterone had a mortality rate of 8.6%,significantly lower than that in the untreated hy-pogonadal group.Testosterone replacement in the setting of heart failure has also recently been reported to have beneficial effects on exercise capacity,muscle strength, and HOMA-IR(70).One study showed a decrease of15mg/dl in total cholesterol but no change in low-density lipoprotein cholesterol,high-density lipoprotein cholesterol,or triglycerides after testosterone therapy for3months (59).In the TIMES2trial,men with metabolic syndrome had a15%decline in lipoprotein(a)and a7%decline in total and low-density lipoprotein concentrations.Men with type2diabetes had similar trends,but the results were not significant.There was,however,a6%decline in high-density lipoprotein concentrations in both the metabolic syndrome and type2diabetes groups(61).No changes have been seen in blood pressure after tes-tosterone treatment(59,61).Heufelder et al.showed a decrease in CRP concentra-tions(Ϫ0.5mg/dl)and an increase in adiponectin(0.9␮g/ml)after testosterone therapy(60).However,CRP, IL-6,resistin,and TNF-␣concentrations did not change after im testosterone replacement for3months in a trial by Kapoor et al.(71).There was also a decrease in adiponec-tin after testosterone therapy.The reasons for the discrep-ancies between studies are not clear but could be related to the differences in study design,route of testosterone ad-ministration,and duration of therapy.Safety issuesThe TIMES2trial(61)did not show an increase in age-adjusted PSA values.PSA concentrations exceeded normal limits in four subjects at12months(three in the testos-terone treatment arm and one in placebo).Mean PSA con-centrations did not change after1yr of therapy in the study by Heufelder et al.(60)either.In this context,it is impor-tant that the replacement of testosterone in hypogonadal patients in general does not lead to an increased risk of prostatic carcinoma,although the trials have been too lim-ited in duration and number of patients(64). ConclusionsHH is found in25%of men with type2diabetes.An additional4%have hypergonadotropic hypogonadism. Low testosterone concentrations in men with type2dia-betes are associated with an increased prevalence of symp-toms of hypogonadism,obesity,very high CRP concen-trations,mild anemia,and decreased BMD.In addition, these men have an elevated risk(two to three times)of cardiovascular events and death in two small studies. Short-term studies of testosterone therapy have demon-strated an increase in libido.In addition,there is an in-crease in insulin sensitivity.Some,but not all studies,have shown an improvement in glycemia,body composition, and cardiovascular risk factors such as cholesterol and CRP concentrations.Trials of a longer duration are clearly required to definitively establish the benefits and risks of testosterone replacement in patients with type2diabetes and HH.AcknowledgmentsAddress all correspondence and requests for reprints to: Paresh Dandona,B.Sc.,M.D.,D.Phil.(Oxon),F.R.C.P.,Di-rector,Diabetes-Endocrinology Center of Western New York,Chief of Endocrinology,State University of New York2648Dandona and Dhindsa Hypogonadism in Type2Diabetes J Clin Endocrinol Metab,September2011,96(9):2643–2651。

镓盐对骨质疏松症大鼠骨组织中羟基磷灰石及胶原含量的影响

镓盐对骨质疏松症大鼠骨组织中羟基磷灰石及胶原含量的影响庞炜;康乐;付友兰;王倩云;贾红兵【摘要】BACKGROUND: In recent years, it is discovered that gallium nitrate can decrease bone conversion.OBJECTIVE: To observe the effects of gallium nitrate on the content of hydroxyapatite and collagen in rats with osteoporosis.METHODS: The rats were divided into control group, osteoporosis group and gallium nitrate group. Osteoporosis rat modelswere made by cutting off the bilateral ovaries in the latter two groups. The rats in the gallium nitrate therapy group were treatedwith 1 mg/kg gallium nitrate, three times a week through abdominal cavity; those in the control and osteoporosis groups wereadministrated with normal saline. The rats in the three groups had free access to water and standard food. All the contents ofhydroxypatite and collagen in osseous tissue were determined and analyzed.RESULTS AND CONCLUSION: The content of hydroxyaptite between the osteoporosis group and normal control group wasdifferent significantly (P ＜ 0.05); there were no significant differences among gallium nitrate therapy group, control group andosteoporosis group (P ＞ 0.05), but the content of hydroxyaptite in the gallium nitrate group had increasing trend compared withthat in the normal group. The level of collagen in osteoporosis group was obviously lower than that in normal control group andgallium nitrate therapy group (P ＜ 0.05). When osteoporosis happened, the level of collagen in osteoporosis rats decreased andthe content of hydroxyapatite was in the trend of increasing,and gallium nitrate could inhibit the process.%背景:硝酸镓是近10多年来发现的一种能降低骨转换的药物.目的:观察硝酸镓对骨质疏松大鼠骨羟基磷灰石及胶原含量的影响.方法:实验分为3组.正常组大鼠开腹切除1小块脂肪,其余大鼠切除双侧卵巢制作大鼠骨质疏松症动物模型.正常组及骨质疏松组腹腔注射生理盐水,硝酸镓组腹腔注射硝酸镓,3次/周,常规饲料喂养12周后取大鼠双侧股骨.结果与结论:骨质疏松组大鼠骨组织中羟基磷灰石含量明显高于正常组(P < 0.05);硝酸镓组与正常组相比有升高趋势(P > 0.05),与骨质疏松组间差异无显著性意义(P > 0.05),骨质疏松组骨中胶原较正常组及硝酸镓组明显降低(P < 0.05).结果表明,发生骨质疏松时骨中胶原含量明显降低,羟基磷灰石含量有升高趋势,硝酸镓可以抑制这一过程.【期刊名称】《中国组织工程研究》【年(卷),期】2011(015)034【总页数】3页(P6289-6291)【关键词】硝酸镓;骨质疏松症;羟基磷灰石;胶原;大鼠【作者】庞炜;康乐;付友兰;王倩云;贾红兵【作者单位】解放军第三二三医院骨科,陕西省西安市,710054;解放军第三二三医院骨科,陕西省西安市,710054;解放军第三二三医院骨科,陕西省西安市,710054;解放军第三二三医院骨科,陕西省西安市,710054;解放军第三二三医院骨科,陕西省西安市,710054【正文语种】中文【中图分类】R3180 引言骨代谢的过程是成骨细胞形成新骨和破骨细胞吸收旧骨的过程，骨量的多少取决于同一骨重建单位中骨形成与骨吸收的平衡。

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Albert, A. (1958) Chemical aspects of selective toxicity.
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Cl Estermustine Sodium Phosphate Emcyt® - Pharmacia & Upjohn
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与安慰剂对照治疗子宫内膜异位症骨盆痛

Dienogest in the treatment of endometriosis-associated pelvic pain:a 12-week,randomized,double-blind,placebo-controlled studyThomas Strowitzki a ,*,Thomas Faustmann b ,Christoph Gerlinger c ,Christian Seitz daDepartment of Gynecological Endocrinology and Reproductive Medicine,University of Heidelberg,Vossstrasse 9,69115Heidelberg,Germany bBayer Schering Pharma AG,Global Medical Affairs Women’s Healthcare,Berlin,Germany cBayer Schering Pharma AG,Global Biostatistics,Berlin,Germany dBayer Schering Pharma AG,Global Clinical Development Women’s Healthcare,Berlin,Germany1.IntroductionEndometriosis is a common condition in women of reproduc-tive age,associated with recurrent episodes of pain,dysmenorrhea,and dyspareunia [1–3].Approved treatments for the symptoms of endometriosis include hormonal therapies such as gonadotropin-releasing hormone (GnRH)agonists,androgens (i.e.,danazol),and progestins [4,5].These therapies are,however,frequently associ-ated with sub-optimal efﬁcacy and tolerability that may impact on quality of life and treatment adherence [3,6].GnRH agonists in the absence of ‘‘add-back’’therapy are associated with symptoms of estrogen deprivation and with bone demineralization that limits therapy to 6months,while danazol may cause undesirable androgenic effects and adverse lipid changes [1,4,5].Progestins are regaining popularity because of their long-term efﬁcacy in pain control,although certain progestins are associated with andro-genic effects and weight gain [1,7].Dienogest is a selective progestin that combines the pharma-cologic properties of 19-norprogestins and progesterone deriva-tives,offering potent progestogenic effects without androgenic,mineralocorticoid,or glucocorticoid activity [8,9].Previous trials demonstrated that dienogest provides effective reductions in endometriosis-associated pelvic pain (EAPP)and laparoscopic measures of pathology [10–16].A dose-range study indicated that 2mg/day is the optimal dose for dienogest in the treatment of endometriosis [11].Recently,dienogest 2mg daily demonstrated equivalent efﬁcacy to the GnRH agonists,buserelin acetate and leuprolide acetate,for relieving the pain of endometriosis in two 24-week,randomized studies [12,13].Based on the consistent efﬁcacy and safety proﬁle demonstrated in clinical trials,dienogest offers potential in the long-term treatment of endome-triosis.A placebo effect is well documented in studies of pain control [17].For example,Koninckx et al.[18]reported a 30%reduction in pain severity associated with placebo even in women with deep endometriosis.Novel treatments for the symptoms of endometri-osis should therefore demonstrate superiority relative to placebo to verify their efﬁcacy.The current study is the ﬁrst placebo-European Journal of Obstetrics &Gynecology and Reproductive Biology xxx (2010)xxx–xxxA R T I C L E I N F O Article history:Received 2September 2009Received in revised form 2February 2010Accepted 2April 2010Keywords:Dienogest Progestins Endometriosis Pelvic pain PlaceboA B S T R A C TObjective:To investigate the efﬁcacy and safety of oral dienogest 2mg compared with placebo in the treatment of endometriosis-associated pelvic pain (EAPP).Study design:This was a 12-week,randomized,double-blind,placebo-controlled,multicenter (n =33)study in Germany,Italy,and Ukraine of 198women aged 18–45years with laparoscopically conﬁrmed endometriosis and EAPP score !30mm on a visual analog scale (VAS).Dienogest 2mg or placebo was administered orally once daily.The primary efﬁcacy variable was absolute change in EAPP from baseline to Week 12,as determined by the target variables of change in VAS score and change in intake of supportive analgesic medication (ibuprofen)for pelvic pain.Results:Mean reductions in VAS score between baseline and Week 12in the full analysis set were 27.4mm and 15.1mm in the dienogest and placebo groups,respectively—a signiﬁcant score difference of 12.3mm in favor of dienogest (P <0.0001).Changes in intake of supportive analgesic medication were modest in both groups.The primary efﬁcacy measure of absolute change in EAPP demonstrated the superiority of dienogest over placebo.Dienogest was generally well tolerated and few adverse events were associated with therapy.Conclusions:Dienogest at a dose of 2mg daily for 12weeks was signiﬁcantly more effective than placebo for reducing EAPP.ß2010Elsevier Ireland Ltd.All rights reserved.*Corresponding author.Tel.:+496221567910;fax:+496221564099.E-mail address:Thomas_strowitzki@med.uni-heidelberg.de (T.Strowitzki).Contents lists available at ScienceDirectEuropean Journal of Obstetrics &Gynecology andReproductive Biologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /e j o g r b0301-2115/$–see front matter ß2010Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.ejogrb.2010.04.002controlled investigation of dienogest2mg daily in the treatment of endometriosis.2.Materials and methods2.1.Study designThis international,randomized,double-blind,placebo-con-trolled study investigated the efﬁcacy and safety of dienogest 2mg once daily orally in the treatment of women with endometriosis and EAPP.The study was conducted at33centers in Germany(n=19),Italy(n=8),and Ukraine(n=6).The study protocol was approved by local independent ethics committees and all participants provided written informed consent.The study was conducted in accordance with the amended version of the Declaration of Helsinki and complied with Good Clinical Practice.2.2.PatientsWomen aged18–45years,between menarche and menopause and in good general health,with or without infertility,were eligible for study inclusion.Inclusion criteria included histologically proven endometriosis(stages I–IV,using revised American Society of Reproductive Medicine[r-ASRM]scoring)[19],determined at diagnostic laparoscopy within12months of study baseline.Patients were required at screening and baseline to have an EAPP score !30mm on a visual analog scale(VAS;0mm represents absence of pain and100mm indicates unbearable pain).Exclusion criteria included amenorrhea of three or more months,a primary need for surgical treatment of endometriosis, previous use of hormonal agents within1–6months of screening (dependent on the class of agent)or abnormalﬁndings at gynecological examination other than endometriosis.2.3.Study medicationAfter screening,patients were observed for a4-week treat-ment-free period until the baseline visit,when they were assigned in1:1ratio to receive a single daily tablet of dienogest2mg or placebo.Assignment of patients to a treatment group was by means of a blocked randomization list generated by a Central Randomization Service.Treatment started on Day2of theﬁrst menstruation after the baseline visit.Dienogest or placebo tablets were subsequently taken at the same time every day for12weeks.To preserve blinding,the two treatments were indistinguishable in appear-ance.Each center had both dienogest and placebo tablets pre-coded.Treatment compliance was monitored by tablet counts and patient diaries.Patients were offered supportive analgesic medication in the form of self-administered ibuprofen tablets up to1200mg/day.Patient diaries were used to record intake.2.4.Efﬁcacy variablesThe primary efﬁcacy variable was absolute change in EAPP assessed from baseline to study end.EAPP was evaluated at Weeks 0,4,8,and12by assessment of patient-reported pain score on VAS and intake of supportive analgesic medication(ibuprofen tablets/ 28days).Biberoglu and Behrman(B&B)scale scores[20]were recorded as a secondary efﬁcacy variable between baseline and study end to assess changes in severity of symptoms(pelvic pain,dysmenor-rhea,and dyspareunia)and signs(pelvic tenderness and indura-tion).B&B scale scores were additionally summed to derive scores for‘‘pelvic pain’’,‘‘physical signs’’,and‘‘total symptom and sign severity’’.Further secondary efﬁcacy variables included change in quality of life using the SF-36Health Survey Questionnaire,global assessments of efﬁcacy by patients and investigators using the Clinical Global Impressions(CGI)scale,and withdrawals due to lack of efﬁcacy.2.5.Safety variablesThe incidence and severity of adverse events(overall and drug-related)and withdrawals due to adverse events were recorded throughout the study and reported using Medical Dictionary for Regulatory Activities terminology.Laboratory assessments,including hematology,clinical chem-istry,serum estradiol(E2),and urine analysis parameters,vital sign assessments,and physical and gynecologic examinations were performed at screening and study end.Bleeding patterns (frequency and intensity)were recorded daily in patient diaries and evaluated using the World Health Organization(WHO)90-day reference period method[21].2.6.Statistical analysisThe primary efﬁcacy variable was individual absolute change in EAPP,determined by the target variables of change in VAS score and change in intake of supportive analgesic medication(over the preceding28days)between baseline and study end.Statistical methods were based on the assumption that intake of ibuprofen as supportive medication could potentially confound the measure-ment of pelvic pain using the VAS.Therefore,both VAS score and intake of supportive analgesic medication were analyzed simulta-neously.Statistical analyses used the testing procedure of Roehmel et al.[22]to ensure that any improvement in VAS score for dienogest relative to placebo was not due to an increase in supportive analgesic medication.If passed successfully,the procedure of Roehmel proves the superiority of dienogest over placebo in reducing pelvic pain(measured by both VAS and supportive analgesic medication intake)with strict control of the type I error rate alpha.It also provides point estimates and conﬁdence intervals(CIs)to assess the magnitude of effect on the two endpoints,in accordance with European Medicines Agency guidance[23].The procedure of Roehmel is more powerful and requires fewer patients than the classical Bonferroni correction in the case of two endpoints.The testing procedure of Roehmel was carried out one-sided with a signiﬁcance level alpha of0.025.At an expected drop-out rate of 25%,the sample size was calculated to include at least88patients per treatment arm,yielding a power of90%.Missing data for VAS and supportive analgesic medication were replaced using the last-observation-carried-forward approach.Descriptive statistics reported secondary efﬁcacy and safety outcomes.Bleeding patterns were analyzed according to Gerlinger et al.[24].Results are presented as means and standard deviations unless stated otherwise.Efﬁcacy analyses were conducted on the full analysis set(FAS) including all patients receiving at least one dose of study medication and with at least one observation post-dosing.A per-protocol set(PPS)was analyzed for selected efﬁcacy variables in support of FAS analyses;the PPS included all FAS patients without major protocol violations.Safety analyses were performed on the FAS.3.Results3.1.Patient characteristicsOf215patients screened,198were randomized to treatment and included in the FAS(n=102,dienogest;n=96,placebo).TheT.Strowitzki et al./European Journal of Obstetrics&Gynecology and Reproductive Biology xxx(2010)xxx–xxx 2study was completed by 188patients.Main reasons for study discontinuation are shown in Fig.1.The PPS included 144patients (n =74,dienogest;n =70,placebo).Demographic characteristics and disease severities,including mean VAS score,were broadly similar at baseline in the two groups (Table 1).The majority of patients in both groups had stage III or IV endometriosis based on r-ASRM criteria.Compliance with study medication measured by tablet counts was high (dienogest:99.7%;placebo:99.1%).Use of concomitant medication was reported by similar proportions of patients in the dienogest (46.1%)and placebo (46.9%)groups.3.2.Primary efﬁcacy variableDienogest was signiﬁcantly superior to placebo in reducing EAPP in both the FAS (P =0.00165)and the PPS (P =0.00007).The beneﬁcial effect of dienogest relative to placebo in reducing EAPPwas mainly based on the decrease in VAS score and not on the decrease in supportive analgesic medication.3.2.1.Change in VAS scoreDienogest and placebo were both associated with clinically relevant reductions in VAS score.However,VAS score reductions were signiﬁcantly greater in the dienogest than placebo group (27.4mm and 15.1mm,respectively;group difference:12.3mm;95%CI 6.4–18.1;P <0.0001)(Fig.2).The signiﬁcantly greater effect of dienogest than placebo for VAS score reduction was also demonstrated in the PPS,where the group difference in score at study end was 13.2mm (95%CI 7.3–19.2);P <0.0001.3.2.2.Change in intake of supportive analgesic medicationIntake of supportive analgesic medication at baseline was modest in both groups (Table 1).Intake during the study decreased by 4.4Æ6.4tablets/28days in the dienogest group and by 3.7Æ8.2Fig.2.Change in VAS score (mean ÆSEM)over 12weeks in dienogest and placebo groups(FAS).Fig.1.Patient disposition.Table 1Demographic characteristics and disease severities at baseline (FAS).ParameterDienogest 2mg (n =102)Placebo (n =96)Age (years,mean ÆSD)31.5Æ6.731.4Æ6.0Height (cm,mean ÆSD)165.5Æ5.2166.7Æ5.3Weight (kg,mean ÆSD)62.4Æ10.362.6Æ10.8Body mass index (kg/m 2,mean ÆSD)22.7Æ3.522.5Æ3.5Blood pressure (mmHg,mean ÆSD)Systolic 115.1Æ9.4115.9Æ9.5Diastolic72.8Æ6.772.1Æ7.3r-ASRM stage of endometriosis (%women)Stage I 12.78.3Stage II 16.719.8Stage III 45.144.8Stage IV25.526.0VAS score (mm,mean ÆSD)56.8Æ18.057.0Æ17.8Supportive analgesicmedication/28days (number ofibuprofen 400mg tablets,mean ÆSD)9.9Æ7.49.4Æ8.6FAS,full analysis set;SD,standard deviation;r-ASRM,revised American Society of Reproductive Medicine;VAS,visual analog scale.T.Strowitzki et al./European Journal of Obstetrics &Gynecology and Reproductive Biology xxx (2010)xxx–xxx3tablets/28days in the placebo group (group difference:0.74tablets/28days;95%CI À1.412–2.895;P =NS).3.3.Secondary efﬁcacy variablesProﬁles of symptom and sign severity on the B&B scale were similar in the dienogest and placebo groups at baseline.During treatment,there was a redistribution in the proportions of patients in ‘‘moderate’’or ‘‘severe’’categories toward ‘‘mild’’or ‘‘none’’categories for B&B symptoms of pelvic pain,dysmenorrhea,and dyspareunia in both groups.However,there was a tendency to more frequent shifts toward lower severity categories in the dienogest than placebo group (Fig.3).B&B signs also showed reductions in overall severity by Week 12in both groups,with a tendency toward greater reduction in severity of pelvic tenderness in the dienogest group (Fig.4).Sum scores on the B&B scale demonstrated decreased intensity over time in both groups,but more pronounced reductions in the dienogest than placebo group.At study end,proportions of patients in the ‘‘none’’category in the dienogest and placebo groups,respectively,were 11.8%versus 2.1%for ‘‘pelvic pain’’,17.6%versus 11.5%for ‘‘physical signs’’,and 7.8%versus 2.1%for ‘‘total symptom and sign severity’’.Quality-of-life analyses indicated greater improvements in the dienogest group for two of eight SF-36categories:bodily pain (21.8Æ22.8%,dienogest;10.3Æ20.5%,placebo)and role emotional (18.4Æ33.9%,dienogest;9.6Æ46.4%,placebo).Mental sum scale and physical sum scale scores showed similar improvements in bothgroups.Fig.3.B&B severity proﬁle [20]at baseline and Week 12in dienogest and placebo groups for single symptoms (A)pelvic pain,(B)dysmenorrhea,and (C)dyspareunia (%patients reporting each severity)(FAS).Fig.4.B&B severity proﬁle [20]at baseline and Week 12in dienogest and placebo groups for single signs (A)pelvic tenderness and (B)induration (%patients,as assessed by physician)(FAS).T.Strowitzki et al./European Journal of Obstetrics &Gynecology and Reproductive Biology xxx (2010)xxx–xxx4The proportion of patients ‘‘very much improved/much improved’’at Week 12,assessed by physicians on the CGI,was 52.9%in the dienogest and 22.9%in the placebo group.Dienogest also showed beneﬁt versus placebo in assessments of overall satisfaction,where proportions of patients ‘‘very much satisﬁed/much satisﬁed’’were 43.1%and 20.8%,respectively.One patient withdrew due to lack of efﬁcacy,which occurred in the placebo group at 46days.3.4.Safety variablesDienogest was generally well tolerated,with no reported serious or unexpected adverse events.Incidences of adverse event-related withdrawals were low in both groups (n =2,dienogest:breast pain and uterine bleeding;n =1,placebo:blood human chorionic gonadotropin increased).Adverse events were predomi-nantly mild-to-moderate in intensity in both groups.Incidences of the most common adverse events are shown in Table 2.Adverse events considered potentially drug-related occurred in 15patients (14.7%)in the dienogest group and 7patients (7.3%)in the placebo group,and included headache (2.9%,dienogest;3.1%,placebo),breast discomfort (2.0%; 1.0%),nausea (2.0%; 1.0%),asthenia (2.0%;0%),and depression (2.0%;0%).One incidence of hot ﬂush was reported in the dienogest group.Mean values for laboratory parameters (including lipid con-centrations;Table 3)were generally normal at screening and remained within normal limits in both groups.Mean E2levels were comparable in the two groups at screening (0.273Æ0.244nmol/L,dienogest;0.247Æ0.325nmol/L,placebo).By Week 12,mean changes in E2levels were À0.070Æ0.282nmol/L and 0.070Æ0.554nmol/L in the dienogest and placebo groups,respectively.The mean number of days,number of episodes,and duration of episodes with bleeding/spotting were comparable between the groups (Table 4).The mean number of days,number of episodes,and duration of episodes with spotting only were greater in the dienogest than placebo group.Analysis of bleeding patterns according to the WHO 90-day reference period method identiﬁed the proportions of women with ‘‘infrequent bleeding’’(18.4%,dienogest;0%,placebo),‘‘frequent bleeding’’(19.4%;9.8%),‘‘irregular bleeding’’(37.8%;28.3%),‘‘prolonged bleeding’’(24.5%; 5.4%),‘‘amenorrhea’’(1%;0%),and ‘‘normal bleeding’’(23.5%;60.9%).There were no withdrawals associated with altered bleeding patterns.4.DiscussionIn this 12-week,placebo-controlled study,dienogest 2mg daily orally was associated with signiﬁcant reductions in EAPP in patients with endometriosis.Consistent with the beneﬁcial effects on EAPP,dienogest was associated with improvements in pelvic pain,dysmenorrhea,dyspareunia,and pelvic tenderness on the B&B scale.Improvements in global assessments of efﬁcacy and quality of life reported by investigators and patients offer supportive evidence for the beneﬁt of dienogest in endometriosis.Notably,an improved quality of life is increasingly recognized as an important objective,alongside improvement in symptoms,in the treatment of endometriosis [1–3].The improvement in EAPP associated with dienogest was accompanied by an acceptable safety and tolerability proﬁle,consistent with observations in previous reports [10–16].Treat-ment-related adverse events were generally mild-to-moderate in intensity.Dienogest demonstrated a modest suppression of estradiol,in agreement with other studies where estradiol levels remained within the lower end of the normal range [13,16].In contrast,a relative increase in estradiol was observed in the placebo group,which is compatible with normal ﬂuctuations of the menstrual cycle.The moderate suppression of estradiol with dienogest may represent a potential advantage over other therapies,such as GnRH agonists,which require estrogen add-back if used longer than 6months.Also in contrast to GnRH agonists,dienogest was not associated with an increased incidence of hot ﬂushes,in agreement with other studies [10,13].Table 3Mean (ÆSD)lipid concentrations at baseline and after 12weeks of dienogest or placebo treatment.Parameter (normal reference range)Dienogest 2mg Placebo BaselineStudy end Baseline Study end Triglycerides;mmol/L (0.8–1.94)0.96Æ0.51 1.11Æ0.680.99Æ0.73 1.08Æ0.86Total cholesterol;mmol/L (4.14–6.73) 4.79Æ0.95 4.87Æ1.03 4.94Æ1.12 4.95Æ1.11HDL-C;mmol/L (1.09–2.28) 1.48Æ0.31 1.48Æ0.34 1.55Æ0.38 1.55Æ0.33LDL-C;mmol/L (1.97–5.65)2.89Æ0.732.93Æ0.772.95Æ0.752.95Æ0.76SD,standard deviation;HDL-C,high-density lipoprotein cholesterol;LDL-C,low-density lipoprotein cholesterol.Table 4Bleeding patterns associated with dienogest or placebo treatment.ParameterDienogest 2mg Placebo Number of days withbleeding/spotting,mean ÆSD 22.5Æ14.722.4Æ9.6Number of bleeding/spotting episodes,mean ÆSD2.6Æ1.6 2.8Æ1.1Duration of bleeding/spotting episodes,days,mean ÆSD 6.1Æ3.4 6.0Æ2.1Number of spotting-only days,mean ÆSD12.0Æ9.79.7Æ8.7Number of spotting-only episodes,mean ÆSD 1.2Æ1.30.6Æ1.2Duration of spotting-only episodes,days,mean ÆSD3.9Æ3.62.9Æ1.8Table 2Incidences and rates of most frequent adverse events (i.e.,at least 2%of patients in the dienogest or placebo group,FAS).Adverse eventDienogest 2mg (n =102)Placebo (n =96)Eventsn (%)patients Events n (%)patients Headache 1811(10.8)65(5.2)Cystitis 33(2.9)00Nausea33(2.9)11(1.0)Nasopharyngitis 22(2.0)65(5.2)Bronchitis 22(2.0)33(3.1)Inﬂuenza 22(2.0)33(3.1)Depression22(2.0)22(2.1)Breast discomfort 22(2.0)11(1.0)Asthenia 22(2.0)00Vomiting 0033(3.1)Gastritis 0022(2.1)Proteinuria0022(2.1)Vaginal candidiasis22(2.1)FAS,full analysis set.Order of presentation is based on the frequency of patients in the dienogest group.T.Strowitzki et al./European Journal of Obstetrics &Gynecology and Reproductive Biology xxx (2010)xxx–xxx5In contrast to reports for danazol,dienogest had little effect on plasma lipid levels in the current study,consistent with other studies [11,15].It is beneﬁcial for any medication to display neutral effects on plasma lipids,particularly when used in the long term.As expected,the bleeding data showed a higher incidence of desynchronized bleeding patterns with dienogest relative to placebo,but also a tendency to milder intensity of bleeding over time.No patients withdrew due to bleeding abnormalities in the dienogest group.A potential limitation of this study is the short treatment duration,as it would have been difﬁcult to justify a prolonged placebo treatment period in patients experiencing chronic pain.A second potential limitation relates to the variable number of patients recruited (range 1–28patients)across study centers.While different levels of patient recruitment at individual centers had the potential to introduce systematic bias,this variation also reﬂected the different sizes of the participating centers.The investigators in this study adhered strictly to the protocol in order to maintain objectivity in data collection,while avoiding bias in recruitment and randomization.Baseline characteristics regarding endometriosis disease stage did not differ from those in an active-controlled study of dienogest that we published recently [13].Notably,a large proportion of the patients screened were subsequently randomized to treatment,while overall incidences of protocol deviations were low and were balanced between treatment groups.Strengths of the study include the comparison of active treatment versus placebo in a double-blind design and the use of multiple efﬁcacy and safety measures based on previous trial experience.Despite knowledge of a substantial placebo effect,there remains a scarcity of placebo-controlled studies of medications in the treatment of pain in endometriosis [4,5].This study therefore represents an important contribution to evidence-based prescribing.In conclusion,in this 12-week,randomized,double-blind,placebo-controlled study,dienogest 2mg/day orally was associat-ed with signiﬁcant improvements in EAPP and other efﬁcacy measures.Dienogest may represent an effective and well-tolerated treatment for the painful symptoms of endometriosis.Role of the funding sourceFunding for this study (A32473)was provided by Bayer Schering Pharma AG.The authors and the sponsor designed the study.The authors had access to all data and participated in the analysis and interpretation of data Conﬂict of interest statementTS has no commercial interest,ﬁnancial interest,and/or other relationship with manufacturers of pharmaceuticals,laboratory supplies,and/or medical devices or with commercial providers of medically related services.TF,CG,and CS are full-time employees of Bayer Schering Pharma AG.AcknowledgementEditorial support was provided by PAREXEL MMS.References[1]Kennedy S,Bergqvist A,Chapron C,et al.ESHRE guideline for the diagnosis andtreatment of endometriosis.Hum Reprod 2005;20:2698–704.[2]Mounsey AL,Wilgus A,Slawson DC.Diagnosis and management of endome-triosis.Am Fam Physician 2006;74:594–600.[3]Sinaii N,Cleary SD,Younes N,Ballweg ML,Stratton P.Treatment utilization forendometriosis symptoms:a cross-sectional survey study of lifetime experi-ence.Fertil Steril 2007;87:1277–86.[4]Prentice A,Deary AJ,Bland E.Progestagens and anti-progestagens for painassociated with endometriosis.Cochrane Database Syst Rev 2000;2:CD002122.[5]Prentice A,Deary AJ,Goldbeck-Wood S,Farquhar C,Smith SK.Gonadotrophin-releasing hormone analogues for pain associated with endometriosis.Cochrane Database Syst Rev 2000;2:CD000346.[6]Hummelshoj L,Prentice A,Groothuis P.Update on endometriosis.Women’sHealth 2006;2:53–6.[7]Vercellini P,Fedele L,Pietropaolo G,Frontino G,Somigliana E,Crosignani PG.Progestogens for endometriosis:forward to the past.Hum Reprod Update 2003;9:387–96.[8]Oettel M,Carol W,Elger W.A 19-norprogestin without 17a-ethinyl group II:dienogest from a pharmacodynamic point of view.Drugs Today 1995;31:517–36.[9]Sasagawa S,Shimizu Y,Kami H,et al.Dienogest is a selective progesteronereceptor agonist in transactivation analysis with potent oral endometrial activity due to its efﬁcient pharmacokinetic proﬁle.Steroids 2008;73:222–31.[10]Cosson M,Querleu D,Donnez J,et al.Dienogest is as effective as triptorelin inthe treatment of endometriosis after laparoscopic surgery:results of a pro-spective,multicenter,randomized study.Fertil Steril 2002;77:684–92.[11]Ko¨hler G,Faustmann TA,Gerlinger C,Seitz C,Mueck AO.A dose-ranging study to determine the efﬁcacy and safety of dienogest 1,2and 4mg daily in endometriosis.Hum Reprod 2010;108:21–5.[12]Harada T,Momoeda M,Taketani Y,et al.Dienogest is as effective as intranasalbuserelin acetate for the relief of pain symptoms associated with endometri-osis-a randomized,double-blind,multicenter,controlled trial.Fertil Steril 2009;91:675–81.[13]Strowitzki T,Marr J,Gerlinger C,Faustmann T,Seitz C.Dienogest is as effectiveas leuprolide acetate in treating the painful symptoms of endometriosis:a 24-week,randomized,multicentre,open-label trial.Hum Reprod 2010;25:633–41.[14]Irahara M,Harada T,Momoeda M,Tamaki Y.Hormonal and histological studyon irregular genital bleeding in patients with endometriosis during treatment with dienogest,a novel progestational therapeutic agent.Reprod Med Biol 2007;6:223–8.[15]Ko¨hler G,Go ¨retzlehner G,Brachmann K.Lipid metabolism during treatment of endometriosis with the progestin dienogest.Acta Obstet Gynecol Scand 1989;68:633–5.[16]Schindler AE,Christensen B,Henkel A,Oettel M,Moore C.High-dose pilotstudy with the novel progestogen dienogest in patients with endometriosis.Gynecol Endocrinol 2006;22:9–17.[17]Turner JA,Deyo RA,Loeser JD,Von Korff M,Fordyce WE.The importance ofplacebo effects in pain treatment and research.JAMA 1994;271:1609–14.[18]Koninckx PR,Craessaerts M,Timmerman D,Cornillie F,Kennedy S.Anti-TNF-alpha treatment for deep endometriosis-associated pain:a randomized pla-cebo-controlled trial.Hum Reprod 2008;23:2017–23.[19]Revised American Society for Reproductive Medicine classiﬁcation of endo-metriosis:1996.Fertil Steril 1997;67:817–21.[20]Biberoglu KO,Behrman SJ.Dosage aspects of danazol therapy in endometri-osis:short-term and long-term effectiveness.Am J Obstet Gynecol 1981;139:645–54.[21]Rodriguez G,Faundes-Latham A,Atkinson LE.An approach to the analysis ofmenstrual patterns in the critical evaluation of contraceptives.Stud Fam Plann 1976;7:42–51.[22]Roehmel J,Gerlinger C,Benda N,Laeuter J.On testing simultaneously non-inferiority in two multiple primary endpoints and superiority in at least one of them.Biom J 2006;48:916–33.[23]EMEA,CPMP.Points to consider on adjustment for baseline covariates.EMEACPMP/EWP/2863/99,7Westferry Circus,Canary Wharf,London E144HB,UK;2003.[24]Gerlinger C,Endrikat J,Kallischnigg G,Wessel J.Evaluation of menstrualbleeding patterns:a new proposal for a universal guideline based on the analysis of more than 4500bleeding diaries.Eur J Contracept Reprod Health Care 2007;12:203–11.T.Strowitzki et al./European Journal of Obstetrics &Gynecology and Reproductive Biology xxx (2010)xxx–xxx6。

碱基切除修复抑制剂甲氧胺联合β-榄香烯治疗恶性脑胶质瘤的实验研究

序言β-榄香烯属国家二类非细胞毒性抗肿瘤新药，临床研究证实其对包括脑胶质瘤在内的多种肿瘤疗效确切，且无其他传统化疗药常有的骨髓抑制、肝肾功能损害等毒副作用。

但目前临床应用的榄香烯乳注射液因其存在静脉炎发生率很高、剂型性质不稳定等缺点，其进一步的应用受到了较大的限制。

碱基切除修复抑制剂甲氧胺（Methoxyamine），可通过裂解核酸内切酶破坏DNA碱基切除修复过程，从而抑制肿瘤细胞对损伤作用的修复反应。

据此，可认为抑制DNA 碱基切除修复可能是增强肿瘤细胞化疗敏感性的潜在靶点，目前多项实验报道也已证实了甲氧胺可增强烷化剂和放疗的抗肿瘤效果。

近年来，通过纳米技术构建的纳米脂质体在提高药物溶解度、增加药物稳定性、降低药物副作用、缓控释给药等方面较普通的脂质体有了显著的提高。

研究表明，纳米脂质体对正常细胞和组织无损伤作用，并可长时间吸附于靶细胞周围，因此使药物能充分向靶组织渗透，也可以通过静电吸附效应与细胞膜接触而融合而进入细胞内。

因此将药物包封于纳米脂质体被认为可以改变被包封药物的体内分布，提高药物治疗指数，降低药物毒性。

基于增强β-榄香烯的疗效，减少毒副作用的目的，本课题研究内容分两部分：（一）联合碱基切除修复抑制剂甲氧胺，探讨是否在体内外抗瘤活性上具有协同作用，以期减少榄香烯用量，降低毒副反应，为其在临床的应用提供实验和理论依据。

（二）、利用纳米脂质体技术构建新型的β-榄香烯-纳米脂质体药物传递系统，初步探讨其体外抗瘤活性。

II碱基切除修复抑制剂甲氧胺联合β-榄香烯治疗恶性脑胶质瘤的实验研究中文摘要胶质瘤是成人神经系统最常见的原发性肿瘤，手术全切除率很低，复发率高，当前多种治疗效果仍不理想。

榄香烯属国家二类非细胞毒性抗肿瘤新药，临床研究发现其对多种肿瘤疗效确切，而且还具有提高和改善机体免疫功能，与放化疗协同作用等独特效果。

但是肿瘤细胞具有强大的DNA损伤修复机制，会对化疗药物产生抗性。

因此抑制这种内在的DNA修复过程，如碱基切除修复抑制剂甲氧胺的联合应用有利于提高化疗药物的抗瘤效果。

基于网络药理学研究_丹参_姜黄_药对降尿酸作用机制_韩晶雪

［19］BARNHAM K J，MASTERS C L，BUSH A I．Neurodegenerative dis-eases and oxidative stress．Nat．Rev．Drug Discov［J］．2004，3：205－214．［20］王袖和，童红霞，徐浩，等．雌激素保护神经作用研究现状［J］．神经解剖学杂志，2018，34（2）：263－266．［21］ZHAO T Z，SHI F，HU J，et al．GPER1mediates estrogen－induced neuroprotection against oxygen－glucose deprivation in the primaryhippocampal neurons［J］．Neuroscience，2016，328：117－126．［22］BROTFAIN E，GRUENBAUM S E，BOYKO M，et al．Neuroprotec-tion by estrogen and progesterone in traumatic brain injury and spi-nal cord injury［J］．Curr Neuropharmacol，2016，14：641－653．［23］MEHTAB KHAN，RAHAT ULLAH，SHAFIFIQ UR REHMAN，et al．17β－Estradiol Modulates SIRT1and Halts Oxidative Stress－Mediated Cognitive Impairment in a Male Aging Mouse Model［J］．Licensee MDPI，Basel，Switzerland，2019．DOI：10．3390/cells8080928［24］YAN WEN－HAO，WU JUN，SONG BO，et al．Treatment with a brain－selective prodrug of17β－estradiol improves cognitive func-tion in Alzheimer＇s disease mice by regulating klf5－NF－kB path-way［J］．Naunyn－Schmiede berg＇s Arch Pharmacol，2019．DOI：10．1007/s00210－019－01639－w［25］ZHENG J Y，LIANG K S，et al．Chronic Estradiol AdministrationDuring the Early Stage of Alzheimer＇s Disease Pathology Rescues A-dult Hippocampal Neurogenesis and Ameliorates Cognitive Deficitsin Aβ1－42Mice［J］．Mol Neurobiol，2017，54（10）：7656－7669．［26］吴勉华，王新月．中医内科学［M］．北京：中国中医药出版社，2012：156－162．［27］王丹丹．补肾益精方含药血清对血脑屏障的影响及该方治疗轻度认知功能障碍的疗效评价［D］．南京：南京中医药大学，2016．［28］迟淑梅，沈涌．补肾填精益髓方联合西药治疗阿尔兹海默病临床研究［J］．新中医，2018，50（7）：71－74．［29］高虹．补肾益智汤干预老年轻度认知障碍向痴呆转化的临床研究［J］．中西医结合心血管病杂志，2015，3（24）：70－71．［30］李贞．醒脑益寿方治疗肾精亏虚型轻度认知障碍的临床观察［D］．武汉：湖北中医药大学，2019．［31］盛望，王瑾茜，李旭华，等．肾脑复元汤对阿尔兹海默病大鼠海马区神经保护作用及对碱性成纤维生长因子、脑源性神经营养因子表达的影响［J］．世界中医药，2018，13（11）：2835－2838．［32］阳松威，郭建生，王晓倩，等．补肾疗更浸膏对去势更年期模型大鼠神经内分泌功能失调的作用［J］．中成药，2016，38（3）：651－654．［33］邢亚明，赵地，孙跃．补肾中药对去势大鼠雌性激素、骨代谢及破骨细胞活性的影响［J］．中国煤炭工业医学杂志，2019，22（4）：413－148．DOI：10．13193/j．issn．1673-7717．2021．03．042基于网络药理学研究“丹参－姜黄”药对降尿酸作用机制韩晶雪1，2，刘鹏3，刘毓1，2，胡琳2，王新伟1，赵婷婷2（1．黑龙江省中医药科学院，黑龙江哈尔滨150036；2．中日友好医院，北京100029；3．北京中医医院顺义医院，北京101300）摘要：目的运用网络药理学方法研究“丹参－姜黄”药对降尿酸的作用机制。

辅助生殖技术相关并发症的研究进展

DOI:10.12280/gjszjk.20200730谢奇君，李欣，赵纯，凌秀凤△【摘要】辅助生殖技术（ART）已被广泛应用于不孕症的治疗，包括人工授精（AI）和体外受精-胚胎移植（IVF-ET），及其相关衍生技术如胞浆内单精子注射（ICSI）、胚胎植入前遗传学筛查（PGS）、卵母细胞体外成熟（IVM）、胚胎辅助孵化（AH）等技术。

随着ART的普及和妊娠率的逐步提高，其带来的并发症及其安全性也越来越受到重视。

能够及时发现和处理并发症，可以最大限度地使不孕症患者获得安全优质的妊娠结局。

目前，ART相关并发症发生的原因仍存在争议，有部分研究认为是由于ART导致的多胎妊娠或不孕症病因，也有研究认为是由于ART本身。

故对近年来相关文献进行分析，总结ART相关并发症的最新研究进展。

【关键词】生殖技术，辅助；并发症；不育，女（雌）性；体外受精；胚胎移植Research Progress of Complications Related to Assisted Reproductive Technology XIE Qi-jun,LI Xin,ZHAO Chun,LING Xiu-feng.Reproductive Center,Women′s Hospital of Nanjing Medical University(NanjingMaternity and Child Health Care Hospital),Nanjing210004,ChinaCorresponding author:LING Xiu-feng,E-mail:************************【Abstract】Assisted reproductive technology(ART)has been widely used in the treatment of infertility,including artificial insemination(AI)and in vitro fertilization-embryo transfer(IVF-ET),and many derivativetechniques such as intracytoplasmic sperm injection(ICSI),preimplantation genetic screening(PGS),in vitromaturation(IVM),and assisted hatching(AH).With the popularity of ART and the gradual improvement ofpregnancy rate,more and more attention has been paid to the complications of ART and the safety of ART.Detection and treatment of complications in time can maximize the safety and high quality of pregnancy,and thegood outcomes for patients.At present,the causes of ART-related complications are still controversial.Somestudies believe that the increased rate of multiple pregnancy caused by ART or the factors related to infertility arethe main causes.However,other studies believe that the factors of ART itself cannot be overlooked.In this review,we analyzed the complications related to ART and the causes.【Keywords】Reproductive techniques，assisted；Complications；Infertility，female；Fertilization in vitro；Embryo transfer（ＪＩｎｔＲｅｐｒｏｄＨｅａｌｔｈ蛐ＦａｍＰｌａｎ，2021，40：204-208）·综述·基金项目：国家自然科学基金（81871210）作者单位：210004南京医科大学附属妇产医院（南京市妇幼保健院）生殖中心通信作者：凌秀凤，E-mail：************************△审校者辅助生殖技术（assisted reproductive technology，ART）是一种应用各种技术处理精子或卵子，以帮助不孕症夫妇实现生育的方法，包括人工授精（artificial insemination，AI）、体外受精-胚胎移植（invitro fertilization-embryo transfer，IVF-ET）及相关技术，如胞浆内单精子注射（intracytoplasmic sperminjection，ICSI）、胚胎植入前遗传学筛查（preimplantation genetic screening，PGS）、卵母细胞体外成熟（in vitro maturation，IVM）、胚胎辅助孵化技术（assisted hatching，AH）和卵母细胞玻璃化冷冻技术等。

123例多胎妊娠行选择性减胎患者妊娠结局临床因素分析1

华中科技大学硕士学位论文123例多胎妊娠行选择性减胎患者妊娠结局临床因素分析姓名：赵霞申请学位级别：硕士专业：妇产科学指导教师：艾继辉2011-05-06华中科技大学硕士学位论文123例多胎妊娠行选择性减胎患者妊娠结局的临床因素分析硕士研究生：赵霞导师：艾继辉副教授华中科技大学同济医学院附属同济医院生殖中心中文摘要目的：通过对我院因不孕症接受ART治疗后多胎妊娠并行选择性减胎的患者的临床资料进行统计分析，评估不同的临床因素对多胎妊娠选择性减胎后患者妊娠结局的影响。

方法：回顾分析于2002年1月至2009年12月因不孕症于华中科技大学同济医学院附属同济医院生殖中心行IVF/ICSI+ET助孕治疗后由于多胎妊娠行选择性减胎的患者，共123例，统计分析年龄、授精方式、新鲜/复苏周期、减胎时机、初始妊娠个数、减胎后保留胎儿个数等临床因素与妊娠结局之间的关系。

结果：在选择的临床因素指标中，减胎时机和减胎后保留胎儿个数与接受减胎患者的妊娠结局明显相关。

孕8周后进行选择性多胎减胎后的患者，其流产率为64.29%，较孕8周前行选择性减胎患者的流产率19.27%，明显升高；选择性多胎妊娠减胎后，孕早期发生自发性减胎的概率低，仅为3.25%；三胎妊娠减为单胎妊娠的流产率低，仅为0.9%；选择性多胎妊娠减胎后保留单胎妊娠和双胎妊娠的分娩孕周分别为37.1±2.6w:35.7±2.48w,初生儿体重分别为 3.20±0.41kg:2.48±0.36kg。

MFPR后单胎妊娠未出现重大围产期并发症及新生儿畸形病例。

结论：选择性多胎妊娠减胎过程中，减胎时机以及减胎后保留胎儿的个数显著影响减胎患者的妊娠结局，孕8周后进行选择性多胎减胎后的患者，其流产率明显升高；多胎妊娠减胎后，孕早期发生自发性减胎的概率低；选择性多胎妊娠华中科技大学硕士学位论文减胎后保留单胎妊娠的妊娠结局明显优于双胎妊娠。

益生菌对阿尔茨海默病作用的研究进展

益生菌对阿尔茨海默病作用的研究进展发布时间：2021-12-14T06:08:15.523Z 来源：《中国结合医学杂志》2021年12期作者：宋鑫萍1,2，李盛钰2，金清1[导读] 阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

近年来研究显示，益生菌在适量使用时作为有益于宿主健康的微生物，在防治阿尔茨海默病方面具有积极影响，其作用机制可能通过调节肠道菌群，影响神经免疫系统，调控神经活性物质以及代谢产物，通过肠-脑轴影响该病发生和发展。

宋鑫萍1,2，李盛钰2，金清11.延边大学农学院，吉林延吉 1330022.吉林省农业科学院农产品加工研究所，吉林长春 130033摘要：阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

本文综述了近几年来国内外益生菌对阿尔茨海默病的作用进展，以及其预防和治疗阿尔茨海默病的潜在作用机制。

关键词：益生菌；阿尔茨海默病；肠道菌群；机制Recent Progress in Research on Probiotics Effect on Alzheimer’s DiseaseSONG Xinping1,2，LI Shengyu2，JI Qing1*(1.College of Agricultural, Yanbian University, Yanji 133002，China)(2.Institute of Agro-food Technology, Jilin Academy of Agricultural Sciences, Chanchun 130033, China)Abstract：Alzheimer’s disease has become one of the major diseases threatening the life and health of the global elderly. The number of patients is increasing year by year, and the economic cost of nursing is high, which poses a major challenge to the global economy. In recent years, studies have shown that probiotics, as microorganisms beneficial to the health of the host, have a positive impact on the prevention and treatment of Alzheimer’s disease. Its mechanism may be through regulating intestinal flora, affecting the nervous immune system, regulating the neuroactive substances and metabolites, and affecting the occurrence and development of the disease through thegut- brain axis. This paper reviews the progress of probiotics on Alzheimer’s disease at home and abroad in recent years, as well as its potential mechanism of prevention and treatment.Key words：probiotics; Alzheimer’s disease; gut microbiota; mechanism阿尔茨海默病（Alzheimer’s disease, AD），系中枢神经系统退行性疾病，属于老年期痴呆常见类型，临床特征主要包括：记忆力减退、认知功能障碍、行为改变、焦虑和抑郁等。

分子印迹技术的研究进展及其在分离中的应用

分子印迹技术的研究进展及其在分离中的应用杨苏宁;丁玉【摘要】Molecular imprinting technology（MIT） is a new and great development potential separation technology.Because of its reservation,recognition and practicability,molecularly imprinted polymers with a good prospect had been widely used in separation technology and had aroused extensive attention.In this paper the application of molecular imprinting technique in the separation of various aspects was reviewed,including its origin and basic principle.Besides,the developing trend of MIT was also predicted.%分子印迹技术（MIT）是一种新的、很有发展潜力的分离技术。

分子印迹聚合物因其具有预定性、识别性和实用性的优点,已广泛应用于分离技术中,显示出良好的应用前景,引起了人们的广泛关注。

介绍了分子印迹技术的产生、原理及其在分离技术方面的应用,并对其进行了展望。

【期刊名称】《山西化工》【年(卷),期】2011(031)004【总页数】4页(P30-32,38)【关键词】分子印迹技术;原理;分离;应用【作者】杨苏宁;丁玉【作者单位】中国矿业大学化工学院,江苏徐州221008;中国矿业大学化工学院,江苏徐州221008【正文语种】中文【中图分类】Q658引言人们研究分子印迹技术(即分子烙印技术，molecular imprinting technology，MIT)的历史由来已久，可以追溯到上个世纪。

β-Estradiol_17-valerate_SDS_MedChemExpress

Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:May-24-2017Print Date:May-24-20171. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :β-Estradiol 17-valerateCatalog No. :HY-B0672CAS No. :979-32-81.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureGHS Classification in accordance with 29 CFR 1910 (OSHA HCS)Acute toxicity, Oral (Category 4),H302Acute aquatic toxicity (Category 1),H400Chronic aquatic toxicity (Category 1),H4102.2 GHS Label elements, including precautionary statementsPictogramSignal word WarningHazard statement(s)H302 Harmful if swallowed.H410 Very toxic to aquatic life with long lasting effects.Precautionary statement(s)P264 Wash skin thoroughly after handling.P270 Do not eat, drink or smoke when using this product.P273 Avoid release to the environment.P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor/ physician if you feel unwell.P330 Rinse mouth.P391 Collect spillage.P501 Dispose of contents/ container to an approved waste disposal plant.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:Estradiol valerianateFormula:C23H32O3Molecular Weight:356.50CAS No. :979-32-84. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature:Powder-20°C 3 years4°C 2 yearsIn solvent-80°C 6 months-20°C 1 monthShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance White to off-white (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. 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英文：硫辛酸改善PC12细胞中GSH的水平：帕金森治疗的新启示

Pre-treatment with R -Lipoic Acid Alleviates the Effects of GSH Depletion in PC12Cells:Implications for Parkinson’sDisease TherapyS.Bharath 1,*,B.C.Cochran 1,M.Hsu 1,J.Liu 2,3,B.N.Ames 2,3,J.K.Andersen 11Buck Institute for Age Research,8001Redwood Blvd.,Novato,CA 94945,USA2Division of Biochemistry and Molecular Biology,University of California,Berkeley,CA 94720,USA 3Children’s Hospital Oakland Research Institute,Oakland,CA 94609,USAReceived 22August 2001;accepted 12April 2002AbstractOxidative stress is believed to play a key role in the degeneration of dopaminergic neurons in the substantia nigra (SN)of Parkinson’s disease (PD)patients.An important biochemical feature of PD is a signiﬁcant early depletion in levels of the thiol antioxidant compound glutathione (GSH)which may lead to the generation of reactive oxygen species (ROS),mitochondrial dysfunction,and ultimately to subsequent neuronal cell death.In earlier work from our laboratory,we demonstrated that depletion of GSH in dopaminergic PC12cells affects mitochondrial integrity and speciﬁcally impairs the activity of mitochondrial complex I.Here we report that pre-treatment of PC12cells with R-lipoic acid acts to prevent depletion of GSH content and preserves the mitochondrial complex I activity which normally is impaired as a consequence of GSH loss.#2002Elsevier Science Inc.All rights reserved.Keywords:Parkinson’s disease;Glutathione (GSH);R -lipoic acid;Mitochondrial complex IINTRODUCTIONParkinson’s disease (PD)is a progressive,neurode-generative disorder which involves the loss of dopa-minergic neurons of the substantia nigra (SN)pars compacta (Beal,1992)The disease condition occurs mostly in late midlife with clinical features including motor impairment,gradual loss of cognition,and depression.Oxidative stress appears to play an impor-tant role in neuronal degeneration associated with PD (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).Dopaminergic neurons are particularly prone to oxidative stress due to oxidation of dopamine leading to the generation of reactive oxygen species(ROS).ROS act to oxidize various biological macro-molecules thereby disturbing homeostasis within the cell ultimately leading to cell death (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).It has been observed that the SN of early PD patients has signiﬁcantly decreased levels of glutathione (GSH)(Perry and Yong,1988).GSH is a tripeptide localized both within the cytoplasm and mitochondria of neurons and other cell types.It acts as the major non-proteinac-eous antioxidant and redox modulator within the brain.The depletion of GSH in the SN is the earliest known indicator of oxidative stress in presymptomatic PD,preceding both decreases in mitochondrial complex I activity and dopamine levels (Jenner,1993).GSH is synthesized by a two-step reaction involving the enzymes g -glutamyl cysteine ligase (g -GCL)and glutathione synthetase.g -GCL is the rate-limiting enzyme in this process and brain GSH appearstoNeuroToxicology 23(2002)479–486*Corresponding author.Tel.:þ1-415-209-2000;fax:þ1-415-209-2231.E-mail address :bsrinivas@ (S.Bharath).0161-813X/02/$–see front matter #2002Elsevier Science Inc.All rights reserved.PII:S 0161-813X (02)00035-9primarily arise through synthesis from its constituent amino acids via this enzyme(Meister,1988).g-GCL is a dimer composed of a heavy catalytic subunit and a light regulatory subunit(Huang et al.,1993).GSH is synthesized in the cytosol and transported into the mitochondria via an energy-dependent transporter (Meister,1988).In earlier studies from our laboratory, we reported on the effects of partial depletion of GSH levels via g-GCL inhibition on mitochondrial physio-logy in PC12cells(Jha et al.,2000).When GSH levels in PC12were down regulated by simultaneous dox-ycycline(Dox)-induced expression of antisense g-GCL light and heavy subunit mRNAs and protein levels, it resulted in decreased mitochondrial GSH levels, increased oxidative stress,and decreased mitochon-drial function.Decreased mitochondrial activity in these cells appeared to be due to a selective inhibition of complex I activity as a result of thiol oxidation of the enzyme complex.These results suggested that the early observed GSH losses in the SN of PD patients could be linked to decreases in complex I activity(via increased ROS)and subsequent mitochondrial dysfunction which ultimately leads to dopaminergic cell death associated with the disease(Jha et al.,2000;Beal,1992).R-lipoic acid plays a fundamental role in mitochon-drial metabolism as a coenzyme for pyruvate dehy-drogenase and alpha-ketoglutarate dehydrogenase and as a substrate for the NADPH-dependent enzyme glutathione reductase.In recent years,R-lipoic acid has received considerable attention as an antioxidant (Fuchs et al.,1997;Packer et al.,1995;Sen et al., 1999).It has been demonstrated to have neuroprotec-tive effects on neuronal cells(Wolz and Krieglstein, 1996,1997;Tirosh et al.,1999;Sen et al.,1998). Studies using both in vitro and in vivo models have suggested that pretreatment with R-lipoic acid increases cellular levels of GSH,probably by prevent-ing its depletion thereby protecting mitochondrial integrity(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).To address the question of the possible mechanism of R-lipoic acid-mediated protection,we have analyzed the effect of pretreatment with R-lipoic acid on GSH-depleted PC12cells.We have used buthionine sulfox-imine(BSO),an irreversible inhibitor of the enzyme g-GCL,to decrease GSH levels within PC12.BSO is a widely used pharmacological agent for decreasing GSH content in peripheral tissues as well as in cell culture models(Minchinton et al.,1984;Lee et al., 1988;Thanislass et al.,1995;Andersen et al.,1996). We demonstrate that pre-treatment of GSH-depleted cells with R-lipoic acid prevents depletion of the GSH pool within the cytoplasm and mitochondria.We also show that this pretreatment leads to preservation of mitochondrial complex I activity lost due to GSH depletion.MATERIALS AND METHODSPC12cells were obtained from American Type Culture Collection(ATCC),Rockville,MD.All tissue culture materials were procured from Life Technolo-gies or Cellgro.Chemicals were obtained from Sigma Chemical Company.The GSH assay kit was obtained from OXIS Research Inc.R-lipoic acid was a gift from ASTA Medica(Frankfurt/Main,Germany).Cell CulturePC12cells were cultured in DMEM medium con-taining10%horse serum,5%fetal bovine serum(FBS) and antibiotics(2units penicillin/ml and0.02mg streptomycin/ml)at378C and5%CO2.Cells were subcultured once a week by gentle cell scraping.Stock solutions of buthionine-S,R-sulfoximine(BSO)and R-lipoic acid were prepared in Hanks balanced salt solution(HBSS).Preparation of MitochondriaMitochondria were prepared by the method of Trounce et al.(1996).Brieﬂy,PC12cells were har-vested,washed in Buffer H(5mM HEPES,210mM mannitol,70mM sucrose,1mM EGTA and0.5% BSA)then resuspended in the same buffer.The cell suspension was then homogenized and centrifuged at 800Âg for5min at48C.The supernatant which was enriched in mitochondria was then centrifuged at 10,000Âg for20min at48C.The resultant mitochon-drial pellet was resuspended in buffer H and stored as aliquots atÀ208C.Estimation of Total Glutathione(GSHþGSSG) in Whole Cells and MitochondriaTotal cellular and mitochondrial glutathione estima-tion was carried out using a kit from OXIS Research Inc.following the instructions provided by the manu-facturer.Cells were plated at1Â106cells/ml in either 6-well dishes or90mm diameter plates.All estima-tions were conducted in triplicate.Cells were incubated with BSO and/or R-lipoic acid for the indicated time intervals at378C.Cells were then harvested,disrupted480S.Bharath et al./NeuroToxicology23(2002)479–486by sonication and cellular proteins precipitated by tricholoroacetic acid.The soluble fraction,which is enriched for total glutathione,was estimated using the OXIS kit.Brieﬂy,samples wereﬁrst buffered and then the reducing agent,tris(2-carboxyethyl)phosphine (TCEP)was added to reduce any oxidized glutathione (GSSG).The chromogen4-chloro-1-methyl-7-triﬂuor-omethylquinolinium methylsulfate was then added which forms thioethers with all thiols present in the sample.Upon addition of base to raise the pH to greater than13,a b-elimination speciﬁc to the GSH-thioether results in a chromophoric thione.The absorbance of this thione measured at420nm is directly proportional to total glutathione concentration.All values obtained were normalized per protein and tabulated. Mitochondrial Complex I Enzyme Assay Complex I enzyme assays were carried out as described by Trounce et al.(1996).Brieﬂy,the assay was initiated by addition of aliquots of sonicated cell samples to50mM potassium phosphate pH7.4, 500m M EDTA,1%BSA,200m M NADH and200m M decylubiquinoneþ2m M rotenone in the presence of KCN with0.002%dichloroindophenol(DCIP)as a secondary electron acceptor.The decrease in the absorbance at600nm was recorded as a measure of enzyme reaction rate at308C for10min and speciﬁc activity was calculated.The results were plotted as relative rotenone-sensitive speciﬁc activity.c-Glutamyl Cysteine Ligase Enzyme Assayg-GCL assays were carried out as described by Seelig and Meister(1985).Brieﬂy,cells obtained after various treatments were washed in1ÂPBS and resuspended in0.1M Tris–HCl,pH8.0.The cell suspension was sonicated and used as a source for g-GCL enzyme activity.The enzyme activity was determined at378C by adding cell lysates to a reaction mixture containing0.1M Tris–HCl buffer,pH8.0, 150mM KCl,5mM sodium ATP,2mM phosphoenol-pyruvate,10mM L-glutamate,10mM a-amino buty-rate,20mM MgCl2,2mM EDTA,0.2mM NADH, 17U pyruvate kinase and17U lactate dehydrogenase. The absorbance at340nm was monitored as a measure of enzyme activity.Assays run in the absence of a-aminobutyrate served as blank.All the g-GCL activ-ity values were normalized per protein.RESULTSPC12cells represent a good model system for the study of oxidative stress in dopamine-containing cells as they exhibit several physiological properties of dopaminergic neurons(Greene and Tischler,1976). PC12cells were treated with100m M of BSO for different time points and total glutathione content measured.Following BSO treatment,there was a gradual decrease in the levels of total cellular GSH content with time(Fig.1);the maximum decrease was observed at24h(data not shown).For subsequent experiments,addition of100m M of BSO for a time period of6h was employed to maintain approximately 50%depletion in total cellular GSH content,similar to the decreases observed in the PD SN.The unbound form of R-lipoic acid has two–SH groups and has been found to be an effective thiol anti-oxidant(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).Therefore,R-lipoicacid Fig.1.The effect of100mM BSO on total cellular glutathione levels in PC12cells at various time points.Significance of difference fromthe control was determined by ANOV A single factor analysis.Data is expressed as meanÆS:E:M:(n¼3).ÃP<0:005vs.untreated cells;ÃÃP<0:001vs.untreated cells.S.Bharath et al./NeuroToxicology23(2002)479–486481pretreatmentwas testedtoseeifthiscould helpovercome the dramatic decreases in BSO-induced glutathione levels.PC12cells were incubated with 100m M of freshly prepared R -lipoic acid for 24h and then sub-jected to BSO treatment for 6h as previously described.Pretreatment with R -lipoic acid prevents depletion of glutathione levels within BSO-treated PC12cells (Fig.2a ).Mitochondrial glutathione content estimated from BSO-treated cells pre-treated with R -lipoic acid versus untreated cells demonstrate that mitochondrial levels of glutathione were decreased as signi ﬁcantly as cellular glutathione levels by BSO treatment and that they too are completely preserved upon pre-treatment with R -lipoic acid (Fig.2b ).These results emphasize the role of R -lipoic acid as a potential anti-oxidant with the ability to help cells maintain their total GSH pool.It has been well documented in PD that mitochon-drial dysfunction is a hallmark of disease pathogenesis (Beal,1992).This abnormality is manifested by a selective decrease in the enzymatic activity of mitochondrial complex I (Beal,1992;Jenner,1993;Hillered and Chan,1988;Haas et al.,1995;Jha et al.,2000).Since pretreatment with R -lipoic acid signi ﬁ-cantly prevented BSO-mediated depletion of cellular and mitochondrial total glutathione levels,it was logi-cal to analyze whether this type of pretreatment could lead to preservation of mitochondrial complex I activ-ity following BSO treatment.Hence,mitochondrial complex I enzyme assays were carried out on cells which had been pretreated with R -lipoic acid versus un-pretreated cells prior to BSO application.Fig.3shows data demonstrating that pre-incubation ofPC12Fig.2.The effect of pre-treatment with 100mM R -lipoic acid on the total glutathione (GSH þGSSG)levels in BSO treated and untreated (a)whole cell extracts and (b)mitochondria.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean þS :E :M :(n ¼3).(a)ÃP <0:0001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:001vs.BSO alone treated cells.(b)ÃP <0:001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:005vs.BSO alone treatedcells.Fig.3.The effect of pre-treatment with 100m M R -lipoic acid on mitochondrial complex I activity in BSO-treated and untreated PC12cells.Relative rotenone-sensitive specific activities are plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:005vs.control.482S.Bharath et al./NeuroToxicology 23(2002)479–486cells with R -lipoic acid preserves impaired complex I activity caused by BSO insult.The effect of R -lipoic acid demonstrated here might be due to either an increase in the activity of g -GCL,the rate-limiting enzyme in the glutathione production,or due to the direct anti-oxidant activity of R -lipoic acid which may prevent glutathione depletion.To distinguish between these two,we carried out enzyme assays for g -GCL from extracts obtained after various treatments.The result of these experiments (Fig.4)demonstrate that upon BSO treatment alone there is a decrease ($50%)in the g -GCL activity whereas cells treated with either R -lipoic acid alone or R -lipoic acid þBSO do not show any difference in the activity compared to control cells.DISCUSSIONOne of the earliest detectable events during the course of PD is a signi ﬁcant decrease in cellular levels of GSH in the SN (Pearce et al.,1997;Jenner,1993).This may contribute to oxidative stress and ensuing neuronal cell death of dopaminergic neurons in this brain region.Based on our earlier published ﬁndings,we proposed that early GSH depletion in the SN may be directly responsible for selective inhibition of mito-chondrial complex I activity and the concomitant loss of mitochondrial function which leads to neuronal cell death in this disorder (Jha et al.,2000).Selective reductions in GSH levels which precede losses in mitochondrial complex I activity have been reported to occur not only in PD but also in associated toxin models of the disease such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)administration (Perry and Yong,1988;Hallman et al.,1985;Sriram et al.,1998).Whether the inhibition of complex I activity and subsequent decreases in ATP levels fol-lowing MPTP administration can be totally accounted for by decreases in GSH levels is unclear as complex I also appears to be directly inhibited by interaction with 1-methyl-4-phenyl pyridium (MPP þ)formed during monoamine oxidase-B-mediated oxidation of MPTP (Jha et al.,1999).However,decreasing GSH levels by treatment with the pharmacological agent BSO has been shown to potentiate the neurodegenerative effects of MPTP in the SN and to alone cause sublethal damage to the nigrostriatal system (Wullner et al.,1996;Andersen et al.,1996).GSH is an anti-oxidant molecule well known to protect proteins during oxidative stress.It acts by conjugating oxidized thiol groups of target proteins forming mixed disul ﬁdes which are then processed by GSH reductase,thioredoxin or protein disulphide isomerase to reduced protein thiol residues and GSH (Ravindranath and Reed,1990;Jung and Thomas,1996).Thus,GSH serves as a major non-protein cellular defense against oxidative stress by maintaining the reduced state of cellular thiol proteins.Since GSH depletion in the SN is well documented in PD,it is of paramount importance to search for and test com-pounds which could prevent depletion of cellular levels of GSH in PD patients.To screen for such compounds,we have utilized the PC12cell culture model system in which activity of g -GCL has been partially impaired by BSO treatment.Although g -GCL activity levels do not appear to be speci ﬁcally impaired in sporadic cases of PD,the overall effect of BSO treatment in the dopaminergic PC12cells mimics that which is seen in the Parkinsonian brain,i.e.a decrease in GSHlevelsFig.4.The effect of pre-treatment with 100m M R -lipoic acid on g -glutamyl cysteine ligase activity in BSO-treated and untreated PC12cells.Relative %specific activity is plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:0001vs.control.S.Bharath et al./NeuroToxicology 23(2002)479–486483leading to increased oxidative stress(Wullner et al., 1996;Andersen et al.,1996).Furthermore,though acute depletion of GSH appeared to have no effect on overall cell viability or growth even after48h(Jha et al.,2000),prolonged mitochondrial dysfunction would likely eventually result in decreased cell viability like that observed in dopaminergic SN neurons in PD. As shown in Figs.1and2,increased exposure to BSO results in a notable decrease in the GSH content both in whole cell preparations and in the mitochondria. Signiﬁcant changes occur in the mitochondrial phy-siology during oxidative stress and in diseases like PD (Beal,1992).In this current study,we found that BSO treatment eliciting an increase in oxidative stress via a 50%decrease in total cellular GSH content resulted a 40%decrease in the speciﬁc activity of mitochondrial complex I(Fig.3).This correlates well with our earlier ﬁndings in which we observed a similar decrease in mitochondrial complex I activity following50%GSH depletion,with no apparent effects on the activities of the other mitochondrial complexes(Jha et al.,2000). Mitochondrial complex I is considered to be the mitochondrial complex the most severely affected by oxidative stress(Lenaz et al.,1997).In synaptic mitochondria,complex I exerts a major control over oxidative phosphorylation such that at only25%inhi-bition,energy metabolism is disturbed resulting in decreased ATP synthesis(Davey et al.,1998).R-lipoic acid,a thiol containing compound,is a widely used antioxidant(Fuchs et al.,1997;Packer et al.,1995;Sen et al.,1999).R-lipoic acid functions as a redox regulator of proteins such as myoglobin, prolactin,thioredoxin,and NF-kappa B transcription factor(Bushtamante et al.,1998;Packer et al.,1995, 1997).It has been utilized to treat or prevent peripheral neuropathy and cardiac autonomic neuropathy(Ziegler and Gries,1997),insulin resistance in type II diabetes (Jacob et al.,1996),retinopathy and cataracts(Maitra et al.,1996),glaucoma(Head,2001),HIV/AIDS (Packer et al.,1995),cancer(Ames,1999),liver disease (Bushtamante et al.,1998),Wilson’s disease(Yamamoto et al.,2001),cardiovascular disease(Matalon et al., 1984)and lactic acidosis caused by inborn errors of metabolism(Yoshida et al.,1990).It has also been used for treatment of Alzheimer type dementia(Hager et al., 2001).R-lipoic acid administration has been reported to result in increased ambulatory activity and improved memory in aged animals and to partially restore age-associated mitochondrial decay in both the liver and heart(Hagen et al.,1999,2000;Suh et al.,2001). It therefore seems likely that thiol antioxidants such as R-lipoic acid which can enhance mitochondrial function,scavenge free radicals,and increase the levels of the antioxidants GSH and ascorbate might be useful neuroprotective agents via their reducing actions onvital protein thiol residues(Han et al.,1997;Xu and Wells, 1996).Our results together with previous studies suggest that R-lipoic acid may be an effective neuroprotective agent in age-associated neurodegeneration.Utilizing the PC12cell model system,we propose that R-lipoic acid administration could be an effective way of circumventing or delaying mitochondrial dys-function associated with PD.Treatment with R-lipoic acid alone seems to signiﬁcantly increase GSH levels only in whole cell preparations but not in mitochon-drial extracts(see Fig.2a and b).However,pretreat-ment of cells with R-lipoic acid appears to prevent BSO-mediated GSH depletion in both whole cells and mitochondria.These data suggest that only during BSO treatment is there a R-lipoic acid-mediated increase in mitochondrial GSH to counter BSO generated ROS production.We were curious to know whether the effect of R-lipoic acid on GSH levels was due to an induction of g-GCL activity.Han et al.(1997)have shown that R-lipoic acid increases de novo synthesis of glutathione by improving cystine utilization.It is also possible that reduction of R-lipoic acid to dihydrolipo-ate in vivo might cause oxidation of GSH to GSSG in turn inducing GCL activity.However,since total glu-tathione estimations in our experiments involved quan-titation of both GSHþGSSG,depletion of GSH cannot be due to the accumulation of GSSG thus ruling out the possibility that increased GSSG concentration might be affecting the g-GCL activity.Fig.4shows that BSO treated cells exhibit$50%decrease in g-GCL activity compared to the untreated cells.Interestingly, cells pretreated with R-lipoic acid upon incubation with BSO showed an increase in g-GCL activity compared to cells treated with BSO alone.These data suggest that R-lipoic acid might contribute to an increased GSH pool during BSO treatment due to the induction of g-GCL activity.However,when cells are treated with R-lipoic acid alone there is a signiﬁ-cant increase($1.6fold)in GSH levels without a concomitant increase in g-GCL activity suggesting that GSH levels are preserved by the direct anti-oxidant properties of R-lipoic acid and not due to increased g-GCL activity(Fig.2a).Decreases in mitochondrial NADH dehydrogenase(complex I)activity associated with GSH depletion also appear to be preserved via R-lipoic acid pretreatment(Fig.3).Taken together, these data suggest that GSH depletion,which may lead to oxidation of protein sulfhydryl residues in the mito-chondrial complex I important for normal functions484S.Bharath et al./NeuroToxicology23(2002)479–486resulting in profound effects on subsequent mitochon-drial performance,may be prevented by the direct action of R-lipoic acid.Our earlierﬁndings suggested that the activity of NADH dehydrogenase,the enzy-matic component of complex I,appears to be thiol-regulated(Jha et al.,2000).The preservation of mito-chondrial complex I activity via R-lipoic acid suggests that it protects against the oxidation of proteins by preventing the use of the cellular GSH pool or by increasing the de novo synthesis of cellular GSH in the presence of BSO-mediated oxidative stress.Whether such treatment would be effective in actually altering brain levels of GSH and thus providing beneﬁcial effects in vivo has not yet been demonstrated. However,our data suggest that maintaining thiol homeostasis may be critical for protecting dopami-nergic neurons of the SN against neurodegeneration and that antioxidants such as R-lipoic acid may be of therapeutic value in PD.It would be of interest to conduct additional experiments using intermediates of dopamine metabolism or MPTP to explore thera-peutic value of R-lipoic acid pretreatment in PD. Nevertheless,since GSH depletion is the earliest event in PD pathogenesis,our data clearly suggests that R-lipoic acid can prevent the deleterious effects that follow by preventing mitochondrial GSH depletion.ACKNOWLEDGEMENTSThis work was supported by a grant from the National Institute of Health(NIH,R01AG12141) to JKA and by a grant from the Ellison Foundation (SS-0422-99),the National Institute of Health (NIH,R01,AG17140),and the National Institute of Environmental Health Sciences Center(NIH,P30, ES01896)to BNA.We thank Dr.M.Jyothi Kumar for technical help and criticaldiscussionsandDr.Simon Melov,Buck Institute,for use of his double beamed spectrophotometer.REFERENCESAdams Jr,JD,Chang ML,Klaidman L.Parkinson’s disease-redox mechanisms.Curr Med Chem2001;8:809–14.Ames BN.Micronutrients prevent cancer and aging.Toxicol Lett 1999;103:5–18.Andersen JK,Mo JQ,Hom DG,Lee FY,Harnish P,Hamill RW, McNeill TH.Effect of buthionine sulfoximine,a synthesis inhibitor of the antioxidant glutathione,on murine nigrostriatal neurons.J Neurochem1996;67:2164–71.Beal MF.Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses.Ann Neurol1992;31:119–30.Burke RE.Parkinson’s disease.In:Koliatos,Ratan RR,editors. Cell death and disease of the nervous system.Totowa(NJ): Humana Press,1998.p.459–75.Bushtamante J,Lodge JK,Marcocci L,Tritschler HJ,Packer L, Rihn BH.Alpha-lipoic acid in liver metabolism and disease. Free Radic Biol Med1998;24:1023–39.Davey GP,Peuchen S,Clark JB.Energy thresholds in brain mitochondria:potential involvement in neurodegeneration.J Biol Chem1998;273:12753–7.Fuchs J,Packer L,Zimmer G.Lipoic acid in health and disease. New York:Marcel Dekker,1997.Greene L,Tischler A.Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.Proc Natl Acad Sci USA1976;73: 2424–8.Haas RH,Nasirian F,Nakano K,Ward D,Pay M,Hill R,Shults CW.Low platelet mitochondrial complex I and complex II/III activity in early untreated Parkinson’s disease.Ann Neurol 1995;37:714–22.Hagen TM,Ingersoll RT,Lykkesfeldt J,Liu J,Wehr CM,Vinarsky V,Barthlomew JC,Ames AB.(R)-alpha-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage,and increased metabolic rate. FASEB J1999;13:411–8.Hagen TM,Vinarsky V,Wehr CM,Ames BN.(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo. Antiox Redox Signal2000;2:473–83.Hager K,Marahrens A,Kenklies M,Riederer P,Munch G.Alpha-lipoic acid as a new treatment option for Alzheimer type dementia.Arch Gerontol Geriat2001;32:275–82.Hallman H,Lange J,Olson L,Stromberg I,Jonsson G. Neurochemical and histochemical characterization of neurotoxic effects of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse.J Neurochem1985;44: 117–27.Han D,Handelman G,Marcocci L,Sen CK,Roy S,Kobuchi H, Tritschler HJ,Flohe L,Packer L.Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization. Biofactors1997;6:321–38.Head KA.Natural therapies for ocular disorders,part two: cataracts and glaucoma.Alternative Med Rev2001;6:141–66. Hillered L,Chan PH.Effects of arachidonic acid on respiratory activities in isolated brain mitochondria.J Neurosci Res 1988;19:94–100.Huang CS,Chang LS,Anderson ME,Meister A.Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.J Biol Chem1993;268:19675–80. Jacob S,Henriksen EJ,Tritschler HJ,Augustin HJ,Dietz GJ. Improvement of insulin-stimulated glucose disposal in type2 diabetes after repeated parenteral administration of thioctic acid.Endocrinol Diabetes1996;104:284–8.Jenner P.Altered mitochondrial function,iron metabolism and glutathione levels in Parkinson’s disease.Acta Neurol Scand Suppl1993;146:6–13.Jha N,Andersen JK.Loss of glutathione(GSH)in Parkinson’s disease:how does GSH act to protect dopaminergic neurons of the substantia nigra?In:Pandalai SG,editor.Recent researchS.Bharath et al./NeuroToxicology23(2002)479–486485。

使用芳香化酶抑制剂的乳腺癌患者维生素D水平变化的相关因素分析

7
0. 19±1.77a
0. 030
<0. 001
注：与使用AI组比较』P<0.05。
中国骨质疏松杂志 2021 年 6 月第 27 卷第 6 期 Chin J Osteoporos, June 2021,Vol 27, No.6
815
表2使用AI和未使用AI组的乳腺癌患者25(OH)D3缺乏率比较(％)
Analysis the related factors of vitamin D level changes in breast cancer patients treated with AI
HUANG Wenjuan, FAN Fangfang, HU Lan, ZHAO Shengjun* Department of Clinical Pharmacy, Traditional Chinese Medical Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830000, China * Corresponding author: ZHAO Shengjun, Email: 1519531677@
Table 2 Comparison of 25( OH) D3deficiency rates between using AI and not using AI breast cancer patients (%)
组别
使用AI组未使用AI组
P值
例数/ n
121 23
A组/ n
11 7
维生素D非正常组
表1使用AI和未使用AI组的乳腺癌患者25(OH)D3水平的比较(斤士s,^g/L)
Table 1 Comparison of 25( OH) D3 levels between using AI and not using AI breast cancer patients(无士s,Rg/L)

细胞蛇的研究进展

2007年,英国牛津大学的刘骥陇等在研究果蝇U 小体和P 小体(U 小体和P 小体是真核生物细胞质中的无膜细胞器)的功能关系时,用4种针对Cup (P 小体中的一种蛋白质)的抗体,对雌性果蝇的卵巢组织进行免疫组织化学染色,染色结果除了预期标记上的P 小体外,还标记出了长条形的丝状结构[1]。

这种结构的形状和数量与纤毛很相似,导致当时以为在果蝇中找到了有纤毛的新细胞类型。

但后来的一系列实验表明,该结构与纤毛没有关系,于是将其命名为“细胞蛇”。

最初是抗Cup 抗体不纯产生假象,意外发现的细胞蛇,而采用亲和层析纯化后的抗Cup 抗体无法再DOI:10.16605/ki.1007-7847.2020.10.0258细胞蛇的研究进展收稿日期:2020-10-22;修回日期:2020-11-19;网络首发日期:2021-07-27基金项目:宁夏自然科学基金项目(2020AAC03179);国家自然科学基金资助项目(31560329)作者简介:李欣玲(1999—),女,广西贵港人,学生;*通信作者:俞晓丽(1984—),女,宁夏银川人,博士,副教授,主要从事干细胞与生殖生物学研究,E-mail:********************。

李欣玲,张樱馨,李进兰,潘文鑫,王彦凤,杨丽蓉,王通,俞晓丽*(宁夏医科大学生育力保持教育部重点实验室临床医学院基础医学院,中国宁夏银川750000)摘要:细胞蛇是近年来细胞生物学研究的热门方向之一,由于其在细胞的增殖、代谢和发育上具有一定的生物学功能,因此,对一些疾病如癌症等的临床诊断或治疗具有一定的指导意义。

细胞蛇是由三磷酸胞苷合成酶(cytidine triphosphate synthetase,CTPS)聚合而成的无膜细胞器,其形成过程及功能在不同类型的细胞中不尽相同。

例如:细胞蛇能促进癌细胞增殖,并使患者病情恶化;过表达的细胞蛇可抑制神经干细胞增殖,影响大脑皮层发育;在卵泡细胞中,细胞蛇相当于CTPS 的存储库,在卵子发生过程起到促进细胞增殖和代谢的作用。

乳腺癌的内分泌治疗进展PPT

绝经前激素受体阳性早期乳腺癌患者的内分泌治疗策略目前我国绝经前激素受体阳性早期乳腺癌患者辅助内分泌治疗，使用TAM 5~10年是标准方案。联合卵巢功能抑制在小于35岁的人群中相比单用TAM能明显获益，但辅助化疗后激素水平恢复到绝经前水平很难作为一个因素来评价是否应联合卵巢功能抑制治疗，因为还与化疗的方案、疗程及监测的时间有关。大于等于4个淋巴结转移是支持联合卵巢功能抑制治疗的重要考虑因素。其次，如果有1～3个淋巴结转移、组织学3级等其他多个危险因素，也可考虑联合卵巢功能抑制治疗。多基因检测在国内很少开展，如显示不良预后，也可支持联合卵巢功能抑制治疗。根据目前的研究结果，建议卵巢功能抑制治疗的时间为5年。对于一部分危险程度较低的患者，也可以考虑治疗2~3年。使用他莫昔芬的患者，治疗期间注意避孕，并每半年至1年行1次妇科检查，通过B超检查了解子宫内膜厚度。服用他莫昔芬5年后，患者仍处于绝经前状态，部分患者(如高危复发)可考虑延长服用至10年。
内分泌治疗与其他辅助治疗的次序辅助内分泌治疗与化疗同时应用可能会降低疗效。一般在化疗之后使用，但可以和放射治疗以及曲妥珠单抗治疗同时应用。
卵巢去势推荐用于下列绝经前患者： ① 高风险且化疗后未导致闭经的患者，可同时与他莫昔芬联合应用；卵巢去势后也可考虑与第三代芳香化酶抑制剂联合应用(TEXT与SOFT 联合分析提示卵巢去势联合第三代芳香化酶抑制剂优于卵巢去势联合三苯氧胺)；② 不愿意接受辅助化疗的中度风险患者，可同时与他莫昔芬联合应用；③ 对他莫昔芬有禁忌者。卵巢去势有手术切除卵巢、卵巢放射及药物去势。若采用药物性卵巢去势，目前推荐的治疗时间是2~5年。
SERM作用机制
选择性雌激素受体调节剂（ SERM ）如：三苯氧胺、托瑞米芬、雷洛昔芬，可竞争性与ER结合，结合后仍能形成二聚体，并与ERE结合。转录活性仅保留了部分其产生对抗雌激素作用还是类雌激素样作用取决于不同组织内的共激活因子或共抑制因子的状态

Cationic 17 α-substituted-estradiol derivatives u

专利名称：Cationic 17 α-substituted-estradiolderivatives useful as anti-cancer agent发明人：Surendar Reddy Bathula,Rajkumar Banerjee 申请号：US12294229申请日：20071231公开号：US08012952B2公开日：20110906专利内容由知识产权出版社提供摘要：The present invention provides a novel series of cationic, lipid-based, 17α-substituted-estradiol derivatives. The present invention further provides a process for the preparation of a novel series of 17α-substituted-estradiol derivatives. The invention also provides information about highly selective anticancer activities of these molecules in estrogen responsive cell lines. The compound elicits high level of toxicity to gynecological cancer cell lines such as MCF-7, T47D (estrogen receptor positive cell lines), MDA-MB-468 (estrogen receptor knock-out cell line), HeIa (cervical cancer). The present class of cationic lipid-based, estradiol derivatives is likely to find specific use in developing target specifically deliverable anticancer drugs for the treatment of gynecological cancers that are most prevalent in women population irrespective of ethnicity.申请人：Surendar Reddy Bathula,Rajkumar Banerjee地址：Andhra Pradesh IN,Andhra Pradesh IN国籍：IN,IN代理机构：DLA Piper LLP (US)更多信息请下载全文后查看。

17β-雌二醇对慢性低氧性肺动脉高压雌性大鼠模型的治疗作用

17β-雌二醇对慢性低氧性肺动脉高压雌性大鼠模型的治疗作用邱逸超;刘智超;李露;刘欣媛;韦建缘;尹雪冬;李美婵;王健;陈豫钦【摘要】Objective To investigate the efficacy of 17β-estradiol on chronic hypoxic pulmonary hypertension in female rats.Methods Totally 32 female SD rats (250-300 g) were randomized into normoxia group (Group A), nor-moxia+17β-estradiol group (Group B), hypoxia group (Group C) and hypoxia+17β-estradiolgroup (Group D) by the random number table.The rats in Group A and B were fed in normoxic cabin , while those in Group C and D were put into the atmospheric hypoxic cabin .17β-estradiol [100μg/(kg· d)] were intraperitoneally infused into rats in Group B and D since the first day after experiment , and placebo sesame oil were given in Group A and C .After successive feed-ing for 28d, mean right ventricular pressure (mRVP), right ventricular systolic pressure (RVSP) and right ventricle hy-pertrophy index RV/( LV+S) were measured by right cardiac manometric method .Lung tissue sections were constructed . The ratios of wall thickness and wall area to pulmonary arteries were quantified by using the Image Pro Plus 6.0 software. Results The hypoxia significantly induced the increases of mRVP , RVSP and RV/(LV+S).Nevertheless, the increas-ing levels of mRVP, RVSP and RV/(LV+S) were obviously decreased after intervention of 17β-estradiol.The RVSP and mRVP in Group C [(33.4 ±2.4), (15.6 ±0.8)mmHg] were significantly higher than those in Group A [(27.7 ± 0.9), (11.5 ±0.9)mmHg, P＜0.05].The RVSP and mRVP in Group D [(27.1±4.9), (13.0 ±1.7)mmHg] were signifi cantly lower than those in Group C (P ＜0.05).The RV/(LV+S) in Group C (0.39 ±0.02) was significantly higher than those in Group A and Group B (0.24 ±0.06 and 0.28 ±0.04, respectively, P＜0.05).The RV/(LV+S) in Group D (0.35 ±0.03) was significantly lower tha n that in Group C (P＜0.05).The wall thickness/artery radius (WT%) in Group C was significantly higher than that in Group A [(10.0 ±0.7)%vs.(33.2±4.0)%, P＜0.05].In Group D, WT%[(16.6 ±2.4)%] was significantly lower than that in Group C (P＜0.05).The wall area/total vascu-lar area (WA%) in Group C was significantly higher than that in Group A [(59.16±5.9)%vs.(17.91 ±2.5)%, P＜0.05].In Group D WA%[(26.30 ±2.9)%] was significantly lower than that in Group C (P＜0.05).Compared with Group A and B, Group C had increased thickening of small arterial wall and smooth muscle layer , narrowing lumen and much lymphocytes and neutrophil infiltration in peripheral vascular ; which were alleviated in GroupD .Conclusion 17β-estradiol markedly reduces RVSP , mRVP, RV/(LV+S) and pulmonary vascular remodeling in chronic hypoxia induced pulmonary hypertension female rats.%目的研究腹腔注射17-β雌二醇对慢性低氧性肺动脉高压雌性大鼠模型的疗效及安全性.方法 32只SD雌性大鼠随机分为常氧组(A 组)、常氧+17β-雌二醇干预组(B组)、低氧组(C组)、低氧+17β-雌二醇干预组(D 组). A组、B组置于SPF级动物房内饲养,而C组、D组置于全自动常压低氧箱中,持续低氧[氧浓度为(10.0 ±0.5)%]28 d.从实验第1天开始,B组、D组按照100μg/(kg· d)的剂量腹腔注射17β-雌二醇;A组、C组腹腔注射相同体积的麻油. 28 d 后,测定大鼠右心室收缩压( RVSP)及平均右心室压(mRVP);取心脏测右心肥厚指数;取肺组织包埋切片行 HE 染色观察,测定管壁厚度占外径的百分比(WT%)和管壁面积占血管总面积的百分比(WA%),观察肺小动脉血管形态组织病理学变化.结果慢性低氧明显诱导了雌性 RVSP、mRVP 和右心肥厚指数的升高,经过17β-雌二醇干预后,这种升高显著降低(P＜0.05). C组大鼠 RVSP 和 mRVP 分别为(33.4 ±2.4) mmHg 和(15.6 ±0.8) mmHg,显著高于 A 组[(27.7 ±0.9)mmHg、(11.5±0.9)mmHg](P＜0.05).与C组相比,D组RVSP和mRVP显著降低[(27.1 ±4.9)mmHg、(13.0 ±1.7)mmHg](P＜0.05). A组和B组的右心肥厚指数为0.24 ±0.06和0.28 ±0.04;与A组比较,C 组右心肥厚指数明显较高(0.39 ±0.02)(P＜0.05);与C 组相比,D组的右心肥厚指数(0.35 ± 0.03)较低(P＜0.05).与A组比较,C 组的 WT%明显较高[(33.2 ±4.0)%](P ＜0.05);D 组的 WT%为(16.6 ±2.4)%,与C 组比较差异有统计学意义(P＜0.05). C组的WA%由(17.91 ±2.5)%增加至(59.16 ±5.9)%(P＜0.05);D组的WA%为(26.30 ±2.9)%,与C组比较差异有统计学意义(P＜0.05).与A组和B组对比,C组肺小动脉管壁增生增厚,管腔变窄明显;但D组上述形态学改变较C组减轻.结论17β-雌二醇能有效降低慢性低氧性肺动脉高压雌性大鼠模型的 RVSP、改善右心室肥厚,对慢性低氧中的肺血管的重塑具有一定的改善作用,对低氧引起慢性肺动脉高压雌性大鼠模型具有较好的疗效.【期刊名称】《广东医学》【年(卷),期】2018(039)010【总页数】4页(P1445-1448)【关键词】高血压,肺性;缺氧;17β-雌二醇;模型,动物【作者】邱逸超;刘智超;李露;刘欣媛;韦建缘;尹雪冬;李美婵;王健;陈豫钦【作者单位】广州医科大学第一临床学院广东广州 510120;广州医科大学第一临床学院广东广州 510120;广州医科大学第一临床学院广东广州 510120;广州医科大学第一临床学院广东广州 510120;广州医科大学第三临床学院广东广州510120;广州医科大学第三临床学院广东广州510120;呼吸疾病国家重点实验室、广州医科大学附属第一医院广州呼吸健康研究院广东广州 510120;呼吸疾病国家重点实验室、广州医科大学附属第一医院广州呼吸健康研究院广东广州 510120;呼吸疾病国家重点实验室、广州医科大学附属第一医院广州呼吸健康研究院广东广州 510120【正文语种】中文肺动脉高压疾病危害大，临床治疗药物及手段有限。

Estradiol 17-(β-D-Glucuronide) (sodium salt) 15087-02-2 GlpBio

Product Data SheetProduct Name:Estradiol 17-(β-D-Glucuronide) (sodium salt)Cat. No.:GC10964Chemical PropertiesCas No.15087-02-2ChemicalName(17β)-3-hydroxyestra-1,3,5(10)-trien-17-yl β-D-glucopyranosiduronic acid, monosodium saltCanonical SMILES [H][C@@]12CCC3=CC(O)=CC=C3[C@@]1([H])CC[C@@]4(C)[C@@]2([H])CC[C@@H]4O[C@@]5([H])[ C@H](O)[C@@H](O)[C@H](O)[C@@H](C([O-])=O)O5.[Na+]Formula C24H31O8 • Na M.Wt470.5Solubility ≤20mg/ml in DMSO;10mg/ml indimethyl formamideStorage Store at -20°CGeneral tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.Shipping Condition Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request.StructureBackground Km: 75 μMProduct Data SheetEstradiol 17-(β-D-Glucuronide) is a substrate of the multidrug resistance protein 2 (MRP2).MRP2 is a member of the superfamily of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes. MRP2 is a member of the MRP subfamily that is involved in multi-drug resistance. MRP2 is expressed in the apical side of the hepatocyte and functions in biliary transport.In vitro: Estradiol 17-(β-D-Glucuronide) was identiﬁed as an ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, and was found to be readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells. The involvement of MRP was conﬁrmed by demonstrating that transport was completely inhibited by a monoclonal antibody speciﬁc for an intracellular conformational epitope of the protein [1].In vivo: Animal study found that estradiol 17-(β-D-Glucuronide) could induce an immediate, profound and reversible inhibition of bile ﬂow after its i.v. administration to the rat. Moreover, the cholestasis degree was found to be dose-dependent in the range of 8.5 to 21 mumol/kg i.v. A dose of 11 mumol/kg i.v. was able to inhibit bile ﬂow and bile acid secretory rate 65 to 70% within 15 to 30 min of its administration. In addition, the bile ﬂow and bile acid secretion returned to near control levels within 3 hours [2].Clinical trial: So far, no clinical study has been conducted.References:[1] Loe, D. W.,Almquist, K.C.,Cole, S.P., et al. ATP-dependent 17β-estradiol 17-(β-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition by cholestatic steroids. The Journal of Biological Chemisty 271(16), 9683-9689 (1996).[2] Meyers M, Slikker W, Pascoe G, Vore M. Characterization of cholestasis induced by estradiol-17 beta-D-glucuronide in the rat. J Pharmacol Exp Ther. 1980 Jul;214(1):87-93.。