Oxidative stress inbibits distant metastasis by human melanoma cells

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Effect of the Lycium barbarum polysaccharides

Journal of Ethnopharmacology111(2007)504–511Effect of the Lycium barbarum polysaccharideson age-related oxidative stress in aged miceX.M.Li a,∗,Y.L.Ma b,X.J.Liu ca School of Food Science and Technology of the XingJiang Agriculture University,Urumqili City,XinJiang830000,PR Chinab School of Traditional Chinese Medicine of the NanZhou University,NanZhou City,GanSu720000,PR Chinac Department of Chinese Herb Medicine of the XingJiang University,Urumqili City,XinJiang830000,PR ChinaReceived7October2006;received in revised form2December2006;accepted14December2006Available online28December2006AbstractOxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various physiological function disorders. Consequently,the concept of anti-age by antioxidants has been developed.Lycium barbarum fruits have been used as a traditional Chinese herbal medicine and the data obtained in in vitro models have clearly established the antioxidant potency of the polysaccharides isolated from the fruits.In the present study,the age-dependent changes in the antioxidant enzyme activity,immune function and lipid peroxidation product were investigated and effect of Lycium barbarum polysaccharides on age-induced oxidative stress in different organs of aged mice was checked.Lycium barbarum polysaccharides(200,350and500mg/kg b.w.in physiological saline)were orally administrated to aged mice over a period of30days.Aged mice receiving vitamin C served as positive control.Enzymatic and non-enzymatic antioxidants,lipid peroxides in serum and tested organs,and immune function were measured.Result showed that increased endogenous lipid peroxidation,and decreased antioxidant activities,as assessed by superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and total antioxidant capacity(TAOC),and immune function were observed in aged mice and restored to normal levels in the polysaccharides-treated groups.Antioxidant activities of Lycium barbarum polysaccharides can be compable with normal antioxidant,vitamin C.Moreover,addition of vitamin C to the polysaccharides further increased the in vivo antioxidant activity of the latter.It is concluded that the Lycium barbarum polysaccharides can be used in compensating the decline in TAOC,immune function and the activities of antioxidant enzymes and thereby reduces the risks of lipid peroxidation accelerated by age-induced free radical.©2007Elsevier Ireland Ltd.All rights reserved.Keywords:Oxidative stress;Vitamin C;Lycium barbarum polysaccharides;Antioxidant;Lipid peroxidation;Anti-aging;Superoxide anions1.IntroductionA major characteristic of an aging organism is its progres-sive functional decline,including a loss of adaptive responses to stresses,with the passage of time(Ian and Grotewiel,2006). One currently major cause of aging is the concept of oxidative stress as a root of aging(Golden and Melov,2001).Oxida-tive stress is described generally as a condition under which increased production of free radicals,reactive species(including singlet oxygen and reactive lipid peroxidation products,such as reactive aldehydes and peroxides),and oxidant-related reactions occur that result in damage.∗Corresponding author.Tel.:+869915667541.E-mail address:xj.goodli@(X.M.Li).Current studies suggest that development of anti-aging drugs from Chinese medicinal herbs may be one of the possible inter-ventions(Chang,2001;Bastianetto and Quirion,2002;Lei et al.,2003).Oriental herbal medicine has been widely investi-gated for drug development because it has fewer side effects (Wong et al.,1994).Lycium barbarum belongs to the plant fam-ily Solanaceae.Red-colored fruits of Lycium barbarum have been used as a traditional Chinese herbal medicine for thousands of years(Gao et al.,2000).The earliest known Chinese medici-nal monograph documented medicinal use of Lycium barbarum around2300years ago.Lycium barbarum fruits have a large variety of biological activities and pharmacological functions and play an important role in preventing and treating various chronic diseases,such as diabetes,hyperlipidemia,cancer,hep-atitis,hypo-immunity function,thrombosis,and male infertility (Gao et al.,2000;Li,2001).It is well recognized that freeX.M.Li et al./Journal of Ethnopharmacology111(2007)504–511505radical scavengers or antioxidants plays a important role in slow-ing down biological aging(Andr`e s et al.,2006;Linnane and Eastwood,2006).The evidence suggests that Lycium barbarum is effective to be an anti-aging agent as well as nourishment of eyes,livers and kidneys.The anti-aging property of Lycium barbarum is found in the polysaccharides isolated from the red-colored fruits and has been investigated in different models(Qi et al.,2001;Peng et al.,2001;Wang et al.,2002;Gan et al., 2003,2004;Zhang et al.,2005).For example,extracts of Lycium barbarum have anti-decrepit effect in brain and heart tissues in mice by increasing the activity of superoxide dismutase(SOD) (Xu and Fang,2000).The extracts can still prolongs the life span of Drosophila(Xu,2003).Polysaccharides isolated from Lycium barbarum fruits exhibit anti-aging function in fruitﬂies and mice(Wang et al.,2002).Although numerous studies have been published on humans and animals examining the health aspects of Lycium barbarum polysaccharides,to our knowl-edge,there have been scarce studies to investigate its beneﬁcial effects on health from the aspect of its antioxidant activity in vivo.Therefore,in the present study,we investigated age-dependent changes in the activity of antioxidant enzymes and the immune function in the mice studied and assess the regulatory effects of polysaccharides isolated from Lycium barbarum fruits on oxidative stress in aged mice to improve the understanding of the health beneﬁts of these polysaccharides.2.Methods and materials2.1.Preparation of polysaccharidesFruits of Lycium barbarum,family solanacae,originated from china were purchased from JingHe county herb market (Xinjiang,China),and identiﬁed by Professor D.S.Chen,School of Traditional Chinese Medicine,Xinjiang University.V oucher specimens(HYT-PM040008)were preserved in XinJiang Nat-ural Product Research Institute.Polysaccharides from Lycium barbarum was prepared by the method of Luo et al.(2004).The dried fruit samples(100g) were ground toﬁne powder and put in1.5l of boiling water and decocted for2h by a traditional method for Chinese medici-nal herbs.The decoction was left to cool at room temperature,ﬁltered and then freeze-dried to obtain crude polysaccharides. The dried crude polysaccharides were reﬂuxed three times to remove lipids with150ml of chloroform:methanol solvent (2:1)(v/v).Afterﬁltering the residues were air-dried.The result product was extracted three times in300ml of hot water (90◦C)and thenﬁltered.The combinedﬁltrate was precipi-tated using150ml of95%ethanol,100%ethanol and acetone, respectively.Afterﬁltering and centrifuging,the precipitate was collected and vacuum-dried,giving desired polysaccharides (13g).The content of the polysaccharides was measured by phenol-sulfuric method(Masuko et al.,2005).Result showed that 2.2.Determination of in vivo antioxidant activity of the polysaccharides2.2.1.Animals grouping and treatingSixty20-month-old,body weight28–40g,aged Kunming mice and ten3-month-old,body weight19–26g,young Kun-ming mice were provided by Laboratory Animal breeding Center attached to our institute.The animals were kept under controlled conditions(temperature:23±2◦C;humidity: 55±5%;14h light–10h dark cycle)using an isolator caging system(Niki Shoji,Co.,Tokyo)and allowed free access to standard laboratory pellet diet and water throughout the exper-imental period.All experimental animals were overseen and approved by the Animal Care and Use Committee of XinJiang Medical University before and during experiments.Aged Kunming mice were randomly divided into six groups (10for each):Group II(the aged control),Group III,Group IV,Group V,Group VI and Group VII.Young Kunming mice (Group I)served as normal control.The polysaccharides and vitamin C were administered orally to test animals using vehicle solution(physiological saline)by using a gastric gavage. Group I:Normal control mice were maintained on standard laboratory pellet diet and water ad libitum,withoutadministering medicine for30consecutive days. Group II:Aged control mice were maintained on standard laboratory pellet diet and water ad libitum,withoutadministering medicine for30consecutive days. Group III:Aged mice received polysaccharides(200mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group IV:Aged mice received polysaccharides(350mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group V:Aged mice received polysaccharides(500mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group VI:Aged mice received medicine(polysaccharides plus vitamin C;1/1)(500mg/kg b.w.)in appropri-ate volumes of physiological saline by using gastricgavage and allowed free access to standard labora-tory pellet diet and water for30consecutive days. Group VII:Aged mice received vitamin C(500mg/kg b.w.) in appropriate volumes of physiological saline byusing gastric gavage and allowed free access tostandard laboratory pellet diet and water for30consecutive days.506X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511were harvested,kept at−20◦C until analyzed.Blood sam-ples were centrifuged at4000rpm for3min at4◦C and the serum was separated.The serum MDA level was measured. The organs(including liver,heart,brain,kidney and lung)were removed,weighed and homogenized immediately with DY89-II homogenizer(NingBo Scientz Biotechnology Co.,Ltd.)ﬁtted with teﬂon plunger,in ice chilled10%KCl solution(10ml/g of tissue).The suspension was centrifuged at671×g at4◦C for10min and clear supernatant was used for the following estimations of activity of SOD,CAT,GSH-Px,TAOC,and levels of MDA by spectrophotometric methods.Spleen and thymus were removed and kept frozen at−80◦C until mea-surement.2.2.2.Analytical methodsSuperoxide dismutase activity was measured according to the method described by Misra and Fridovich(1972)based on the inhibition of auto-oxidation of epinephrine by SOD at480nm in a LKB Ultraspec-2spectrophotometer.Tissue homogenate (0.5ml)was diluted to1.5ml with distilled water,and250␮l of chilled ethanol and100␮l of chilled chloroform were added.The mixture was shaken and centrifuged.The supernatant was used for the assay of enzyme activity.1.5ml of the supernatant was added to1.5ml of0.1mol/l carbonate–bicarbonate buffer,pH 10.2,containing0.3mmol/l EDTA.The contents were mixed, and the reaction was initiated by adding200␮l of epinephrine (pH3.0,3mmol/l)to the buffered reaction mixture.The change in optical density per minute was measured at480nm.The enzyme activity was expressed as unit per milligram of pro-tein,where1U is deﬁned as the enzyme concentration required to inhibit50%of epinephrine auto-oxidation in1min under the assay conditions.The assay of Beers and Sizer(1952)was used to measure CAT.Substrate solution for CAT was59mM H2O2in50mM potassium phosphate buffer at pH7.0.Assays were initiated by the addition of0.1ml of supernatant to1.9ml of deionized water and1ml of substrate solution.The disappearance of H2O2 was measured as the decrease in absorbance at240nm.Catalase activity was expressed as U/mg protein(1U is the amount of enzyme that utilizes1␮mol of hydrogen peroxide/min).The GS-Px activity of the supernatant was determined spectrophotometrically at423nm.The reaction mixture was composed of GSH,distilled water,and the supernatant.The reac-tion was stopped by adding trichloroacetic acid.The content of residual GSH was then measured using5-5 -dithiobis-(2-nitrobenzoic acid)(DTNB).One unit of GS-Px activity was deﬁned as1␮mol/l GSH consumption per minute(Liu and Ng, 2000).Lipid peroxidation(LPO)was measured by high performance liquid chromatography(Shimadzu LC10A,Shimadzu,Kyoto, Japan)as described previously(Nielsen et al.,1997).The pho-todiode array detector(Shimadzu SPD-M10A VP)and a C18 column(4.6␮m×25.5␮m Shimpack HRC-ODS)were used for assay.After pretreatment with thiobarbituric acid,the sam-ple was injected and an isocratic elution was carried out with at a wavelength of532nm.Peak authenticity was conﬁrmed by use of pure1,1,3,3-tetraethoxipropane standards.The malondi-aldehyde(MDA)content of the samples was expressed as nmol MDA formed/mg protein.Total antioxidant capacity(TAOC)was measured on an Olympus AU-600analyzer using the TAOC kit(Medikon SA, Gerakas,Greece)as described previously(Kampa et al.,2002). Brieﬂy,antioxidants in the sample inhibit the bleaching of crocin from2,2-azobis-(2-amidinopropane)dihydrochloride(ABAP) to a degree that is proportional to their concentration.The assay was performed at37◦C in the following steps:2␮l of sample, calibrator or control were mixed with250␮l of crocin reagent (R1)and incubated for160s.Subsequently,250␮l of ABAP (R2)were added and the decrease in absorbance at450nm was measured26s later.Values of TAOC were expressed as U/mg protein.Lipofuscin(LPF)contents were determined by the method of Vernet et al.(1988)and Hill and Womersley(1991).For lipofus-cin measurement,0.5ml of tissue homogenate was suspended in3ml of isopropanol and2ml of chloroform.This was allowed to stand for30min and centrifuged at1800×g in a refrigerated centrifuge.Theﬂuorescence was measured using a spectroﬂu-orimeter with extinction at360nm and emission at440nm. Lipofuscin concentrations were expressed as␮g/g tissue.The phagocytic activity of neutrophils in whole blood was conducted as per the method described by Panasiuk et al.(2005). Brieﬂy,standard strain of Staphylococcus aureus was procured from the Division of Standardization(IVRI).Eighteen hours culture was opsonised with pooled mice serum in an incuba-tor for1h.An equal volume(500␮l)of PMNs and500␮l opsonised bacterial suspension was incubated at37◦C for half an hour,maintaining PMNs and bacteria at a1:5ratio.Thereafter, it was stained with500␮l of Acridine orange stain(0.015%, Sigma,St.Louis,MO,USA),vortexed and centrifuged at4◦C, 13000rpm to obtain cell pellet.Finally,500␮l crystal violet was added and centrifuged as above.The pellet was resus-pended in cold sterile PBS(500␮l)and wet mount seen under ultraviolet source with excitationﬁlter of530nm.Phagocytic activity,expressed by the percentage of phagocytosed neutrophil in100cells and phagocytic index,determined on the unit of Staphylococci ingested by single PMNs,was counted in100 cells.The spleen and thymus of the mice were also removed and weighed to obtain the index of the spleen and thymus.The thy-mus and spleen indices were assayed according to the method of Zhang et al.(2003)and calculated according to the follow-ing formula:Thymus or spleen index=weight of thymus or spleen/body weight×100.2.3.Statistical analysesAll data in table are expressed as mean±S.D.(n=10) and differences between groups were assessed by analysis of variance(ANOV A)and Student’s t-test.Differences were con-sidered to be statistically signiﬁcant if P<0.05.All statisticalX.M.Li et al./Journal of Ethnopharmacology111(2007)504–5115073.Results3.1.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in lungs in aged mice As shown in Table1,there was signiﬁcant difference in SOD activities,MDA level,TAOC observed in lung between the aged control and young mice control(P<0.05)but not in GSH-Px, and CAT activity.Declined antioxidant enzymes activity(SOD, CAT activity,TAOC)or increased lipid peroxidation product (MDA)in aged tissues(groups III–VII)were signiﬁcantly ele-vated or reduced with administration of polysaccharides and vitamin C in a dose-dependent manner.The antioxidant activity of polysaccharides is stronger than that of vitamin C at identical dose of500mg/kg b.w.3.2.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in livers in aged mice As was shown in Table1,signiﬁcantly decreased antioxidant enzymes activity(SOD,GSH-Px,CAT,TAOC)and increased MDA level were observed in livers in aged control mice compared with normal control(P<0.01).Administration of polysaccharides and vitamin C dose-dependently increased the activity of antioxidant enzymes,reduced the level of MDA in livers in aged mice(groups III–VII).Moreover,the inhibition by polysaccharide administration of age-induced oxidation is stronger than that of vitamin C at identical dose of500mg/kgb.w.3.3.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in hearts in aged miceA marked increase in MDA production and decrease of antioxidant enzymes activity(SOD,GSH-Px,CAT,TAOC), were observed in hearts of aged control mice(Table1)when compared with normal control(P<0.01).Polysaccharides and vitamin C treatment signiﬁcantly inhibited the formation of MDA in mice hearts and raised antioxidant enzymes activity in a dose-dependent manner(groups III–VII).Likewise,polysac-charides exhibited stronger antioxidation effects than vitamin C at identical dose of500mg/kg b.w.Table1Effect of polysaccharides on activities of SOD(NU/mg protein),CAT(U/mg protein),GSH-Px(U/mg protein),TAOC(U/mg protein)and levels of MDA(nmol/mg protein or/ml serum)in tested organs in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII LungSOD11.41±1.1310.18±0.77c9.98±0.58b12.98±0.60b13.99±1.21b15.52±0.78b11.59±1.64a CAT 4.82±0.57 4.52±0.47 4.46±0.46 4.68±0.64 5.05±0.56a 5.47±0.61b 4.74±0.39 GSH-Px 3.11±0.61 2.77±0.39 3.98±0.47b 4.43±0.33b 5.92±0.53b 6.21±0.65b 3.91±0.43b TAOC 1.13±0.090.96±0.06c 1.04±0.14 1.32±0.14b 1.49±0.13b 1.71±0.09b 1.30±0.13b MDA 2.34±0.49 2.85±0.34c 2.37±0.23b 2.20±0.27b 2.03±0.24b 1.87±0.21b 2.51±0.17a LiverSOD8.70±0.677.58±0.66d8.45±0.38b9.45±1.15b15.88±1.06b18.17±0.94b14.02±0.97b CAT 2.14±0.20 1.65±0.19d 1.80±0.11a 1.92±0.15b 2.15±0.17b 2.86±0.40b 1.86±0.16a GSH-Px10.91±0.928.78±0.81d10.41±0.81b12.69±1.18b14.24±1.07b17.62±1.53b10.97±0.84b TAOC 2.01±0.190.84±0.11d 1.16±0.19b 1.36±0.13b 1.87±0.28b 2.44±0.41b 1.48±0.16b MDA13.46±0.9615.64±0.64d14.53±0.66b12.57±1.11b10.56±0.75b8.75±0.41b9.83±0.73b HeartSOD16.44±0.8314.53±0.94d15.12±0.7216.58±0.55b22.58±0.91b28.43±0.69b16.91±0.68b CAT 2.25±0.32 1.55±0.26d 1.58±0.21 1.65±0.21 1.86±0.19b 1.91±0.20b 1.65±0.27 GSH-Px9.91±1.228.84±0.53d9.54±0.23b10.38±0.25b10.97±0.51b11.93±0.48b10.52±0.93b TAOC0.98±0.170.71±0.09d0.85±0.11b0.97±0.08b 1.31±0.13b 1.77±0.08b 1.06±0.12b MDA 3.97±0.29 5.13±0.30d 4.67±0.17b 3.96±0.21b 3.57±0.24b 2.95±0.12b 4.69±0.12b BrainSOD19.11±0.9717.8±0.51d18.54±1.0520.58±1.32b23.89±1.35b27.82±1.12b20.63±0.86b CAT 4.41±0.37 4.12±0.24 4.23±0.45 4.95±0.11b 5.21±0.27b 5.76±0.31b 4.31±0.09b GSH-Px 4.30±0.50 2.95±0.43d 3.35±0.32 4.41±0.52b 6.03±0.75b8.16±0.48b 5.73±0.33b TAOC0.92±0.230.70±0.08d0.76±0.17 1.84±0.37b 2.33±0.11b 2.89±0.19b 2.04±0.09b MDA9.74±1.3811.28±1.23c10.71±0.888.27±1.51b 6.56±0.85b 4.27±0.16b7.95±0.63b SerumMDA17.34±2.1232.49±2.97d28.78±2.04b23.83±1.77b19.56±1.83b16.63±1.55b24.82±2.79b The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.a P<0.05,compared with aged control group(II).508X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511Table2Effect of polysaccharides on thymus and spleen index in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII Thymus index0.385±0.0340.263±0.027c0.273±0.0250.336±0.040b0.457±0.043b0.572±0.063b0.431±0.034b Spleen index0.533±0.0520.452±0.044c0.486±0.0820.512±0.068a0.550±0.080b0.591±0.076b0.512±0.042b The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.Thymus or spleen index=weight of thymus or spleen/body weight×100.a P<0.05,compared with aged control group(II).b P<0.01,compared with aged control group(II).c P<0.01,compared with normal group(I).3.4.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in brains in aged mice Data on age-induced changes in brains are summarised in Table1;there was only a slight change in CAT activity (P>0.05),but signiﬁcant decreases in SOD,GSH-Px activity, and TAOC,and an increase in MDA level with age compared with control young animals(P<0.05,P<0.01).Administration of polysaccharides and vitamin C dose-dependently elevated these antioxidant enzymes activity and reduced MDA level in brains(groups III–VII).Antioxidant activity of the polysaccha-rides in vivo was better than vitamin C at500mg/kg b.w.3.5.Effect of the Lycium barbarum polysaccharides on thymus and spleen index in aged miceAs was shown in Table2,signiﬁcantly decreased thymus and spleen weight were observed with age(P<0.01)in compari-son with normal control.Administration of polysaccharides and vitamin C are seen to have remarkable effects on increasing the two indices in immune organ in aged mice in a dose-dependent manner(groups III–VII).The reversal of age-induced decreased thymus and spleen weight by polysaccharides administration is stronger than that by vitamin C at identical dose of500mg/kgb.w.3.6.Effect of the Lycium barbarum polysaccharides on macrophage function in aged miceLikewise,in the present study we observed that the tested indices(phagocytic index and phagocytic activity)markedly decreased with age(Table3)(P<0.01)in comparison with nor-mal control.Supplementation of polysaccharides and vitamin C both signiﬁcantly raised the two indices in a dose-independent manner in aged mice(groups III–VII).In terms of the effect on phagocytic indices,polysaccharides administration was basically consistent with vitamin C of identical given dose but stronger than the latter on stimulating phagocytic activity.3.7.Effect of the Lycium barbarum polysaccharides onLPF level in tested organs in aged miceTable4represents the effect of polysaccharides and vitamin C on the levels of LPF in different tested organs in control and experimental animals.The LPF level in aged mice is markedly higher than that in young mice(P<0.01).A signiﬁcant reduc-tion(P<0.01)was found in the levels of LPF in all experiment animals groups(groups V–VII),when compared with aged con-trol.Moreover,the level was dose-independently decreased in polysaccharides-treated animals(groups III–VI).It was found in the present study that polysaccharides administration was still more effective in reducing LPF level than vitamin C at the identical dose.3.8.Effect of the Lycium barbarum polysaccharides on serum MDA level in aged miceEffect of the Lycium barbarum polysaccharides on serum MDA level in aged mice was shown in Table1.Signiﬁcant differ-ences in serum MDA level were detected between normal control and aged control groups(P<0.05).Data from polysaccharides treatment were pooled and compared to the aged control.Result indicated that polysaccharides treatment signiﬁcantly decreased serum MDA level(P<0.05)compared with aged control.Table3Effect of polysaccharides on macrophage function in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII Phagocytic index(k) 3.32±0.44 1.93±0.25d 2.17±0.22 2.65±0.43b 2.87±0.58b 3.12±0.45b 3.18±0.33b Phagocytic activity(α)87.31±5.6572.06±2.98c73.83±4.3279.97±7.1181.42±7.38a86.43±8.39a76.51±7.39 The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.Phagocytic activity,expressed by the percentage of phagocytosed neutrophil in100cells and phagocytic index,determined on the unit of Staphylococci ingested by single PMNs,was counted in100cells.a P<0.05,compared with aged control group(II).X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511509 Table4Effect of polysaccharides on LPF level(␮g/g tissue)in tested organs in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VIIBrain0.224±0.0270.440±0.041b0.434±0.0360.358±0.051a0.312±0.031a0.285±0.027a0.398±0.022a Heart0.115±0.0090.187±0.014b0.180±0.0240.174±0.011a0.160±0.012a0.133±0.013a0.184±0.023 Liver0.245±0.0190.444±0.036b0.360±0.019a0.351±0.014a0.330±0.011a0.284±0.027a0.348±0.019a Kidney0.206±0.0110.382±0.023b0.371±0.0250.344±0.019a0.305±0.019a0.284±0.013a0.369±0.024The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.a P<0.01,compared with aged control group(II).b P<0.01,compared with normal group(I).Moreover,the compound of polysaccharides plus vitamin C exhibited stronger antioxidant activity than either of the two antioxidants.4.DiscussionAging is a progressive deterioration of physiological function that impairs the ability of an organism to maintain homeostasis and consequently increases the organism’s susceptibility to dis-ease and death(Nohl and Hegner,1978).Nearly all organisms manifest functional declines as a result of aging.It is widely accepted that disorganizing free radical reactions linked to oxy-gen metabolism or“oxidative stress”(Chance et al.,1979;Sies, 1986;Gutteridge,1987)play an important role not only in normal aging(Harman,1956;Lesser,2006)but also in many age-related degenerative processes(Harman,1981).Oxidation of lipids produces lipid peroxides that can reduce membraneﬂuidity,inactivate membrane-bound proteins and decompose into cytotoxic aldehydes such as malondialdehyde or hydroxynonenal(Richter,1987).Accumulation of hydrox-ynonenal increases with age in several Drosophila tissues (Zhang and Xu,2006)and the level of malondialdehyde and hydroxynonenal-conjugated collagen protein increases with age in rat tissue(Noberasco et al.,1991).We have also observed an increase in the levels of MDA,a marker of lipid peroxidation in the test organs of aged mice.Hence,lipid oxidation is closely associated to aging.On the contrary,LBP treatment demon-strated decreased level of lipid peroxides and this could be in part due to reduced formation of lipid peroxides from age-dependent free radicals.A vast number of evidence implicates that aging is associated with a decrease in antioxidant status and that age-dependent increases in lipid peroxidation are a consequence of dimin-ished antioxidant protection(Schuessel et al.,2006;Alvarado et al.,2006),being in agreement with our current study.The major antioxidant enzymes,including SOD,GPX and CAT, are regarded as theﬁrst line of the antioxidant defense sys-tem against reactive oxygen species generated in vivo during oxidative stress.SOD dismutates superoxide radicals to form hydrogen peroxide,which in turn is decomposed to water and oxygen by GSH-Px and CAT,thereby preventing the formation of hydroxyl radicals(Yao et al.,2005).Therefore,these enzymes act cooperatively at different sites in the metabolic pathway of in various organs.For example,some previous reports(Y¨u ksel and Asma,2006;Miquel et al.,2006)have shown that some of the antioxidant enzymes in important organs such as liver, heart,kidney,and brain,were decreased with aging,whereas, other investigators have indicated no alteration or increased activities in the antioxidant enzymes(Rolo and Palmeira,2006; Masztalerz et al.,2006).The differences among those data might be,in part,due to differences in the animal’s sex,strain,and age used,assay method,the enzyme property examined,and/or experimental conditions used.In our study along with increased lipid peroxidation,age-induced oxidative injury was found to reduce the total antioxidant capacity(TAOC),which reﬂects the non-enzymatic antioxidant defense system,as well as antiox-idant enzyme levels(SOD,CAT,GSH-Px)in test organs of aged mice and this observation concurs with earlierﬁndings (Kogan et al.,2005).Due to depletion in antioxidant levels, the free radicals are not neutralized and aged organs show enhanced susceptibility to lipid peroxidation.The observation that LBP treatment signiﬁcantly restores the marker enzymes activity of aged mice compared to aged control suggests the reversal by these drugs administration of age-induced oxida-tion.The enhanced activity of SOD,CAT and GSH-Px and increased TAOC in the aging animals can be very effective in scavenging the various types of oxygen free radicals and their products.So the inhibitory effect of the Lycium barbarum polysaccharides on lipid peroxidation might be,at least in part, attributed to its inﬂuence on the antioxidant enzymes and non-enzymatic ind et al.(1990)have reported that,in general,the age-related changes in the activities of SOD,GSH-Px,and CAT were paralleled by a similar change in the relative level of the mRNA expressions coding for these enzymes in brain,hepatocytes,and kidney.As for lung,Gomi and Matsuo (2002)have shown that aging decreased the mRNA expres-sions of SOD and GSH-Px but did not change CAT.Their study also revealed the discrepancy between the activity and mRNA expression of either SOD or GSH-Px.Thus,theseﬁndings, including the currentﬁndings,may suggest that the activities of antioxidant enzymes in aged tissues could be controlled by translational process and/or post-translational process,but not by transcriptional process.Future investigation,however,will be required to determine additional mechanisms.It is possible that the effect of the Lycium barbarum polysaccharides on SOD, CAT and GSH-Px was associated with its effect on translational。

Antioxidative and ACE inhibitory activities of protein hydrolysates from the muscle of brownstripe r

Process Biochemistry 46(2011)318–327Contents lists available at ScienceDirectProcessBiochemistryj 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 /p r o c b ioAntioxidative and ACE inhibitory activities of protein hydrolysates from themuscle of brownstripe red snapper prepared using pyloric caeca and commercial proteasesSutheera Khantaphant a ,Soottawat Benjakul a ,∗,Hideki Kishimura ba Department of Food Technology,Faculty of Agro-Industry,Prince of Songkla University,Hat Yai,Songkhla,90112,ThailandbLaboratory of Marine Products and Food Science,Research Faculty of Fisheries Science,Hokkaido University,Hakodate 041-8611,Japana r t i c l e i n f o Article history:Received 4June 2010Received in revised form 10September 2010Accepted 10September 2010Keywords:Protein hydrolysate Antioxidative activityGastrointestinal model system Oxidation model system Two-step hydrolysisa b s t r a c tProtein hydrolysates from the muscle of brownstripe red snapper (Lutjanus vitta )prepared using Alcalase or Flavourzyme as the ﬁrst step with 40%degree of hydrolysis (DH),followed by hydrolysis with pyloric caeca protease (PCP)as the second step for 2(HAP)and 1h (HFP),respectively,were prepared and determined for their antioxidative and angiotensin I-converting enzyme (ACE)inhibitory activities.HAP had the higher DPPH and ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP),while HFP showed the higher ferrous chelating activity and ACE inhibitory activity (p <0.05).Both HAP and HFP were able to retard lipid oxidations in lecithin–liposome and ␤-carotene–linoleic acid oxidation model systems in dose-dependent manner.HAP and HFP contained 87.36and 86.55%protein (wet basis),respectively with glutamic acid/glutamine as the major amino acids,followed by aspartic acid/asparagine,lysine,alanine and leucine,respectively.HFP showed a slightly greater efﬁciency in prevention of lipid oxidation in all systems tested.Antioxidative activities,except DPPH radical scavenging activity,of both HAP and HFP after being subjected to gastrointestinal tract model system (GIMs)increased,suggesting the enhancement of antioxidative activities of both hydrolysates after ingestion.©2010Elsevier Ltd.All rights reserved.1.IntroductionLipid oxidation is one of the major deteriorative processes in many types of foods,leading to the changes in food quality and nutritional value.Additionally,potentially toxic reaction products can be produced.Lipid oxidations have been known to be the major causes of many serious human diseases,such as cardiovascular dis-ease,cancer,and neurological disorders as well as the aging process [1,2].To prevent oxidative deterioration of foods and to provide protection against serious diseases,such as cancer and atheroscle-rosis [3,4],it is important to inhibit the oxidation of lipids and the formation of free radicals occurring in the foodstuff and living body.To tackle the problem,antioxidants,both natural and synthetic ones,have been used widely.Nevertheless,synthetic antioxidants have been suspected of being responsible for toxicity in the long term and their use in foodstuffs is restricted or prohibited in some countries [5–7].Therefore,there is a growing interest on natu-ral antioxidants,especially peptides derived from hydrolyzed food proteins.Protein hydrolysates from several ﬁsh species includ-ing round scad (Decapterus maruadsi )[8],yellow stripe trevally∗Corresponding author.Tel.:+6674286334;fax:+6674558866.E-mail address:soottawat.b@psu.ac.th (S.Benjakul).(Selaroides leptolepis )[9],Paciﬁc hake (Merluccius productus )[10],tilapia (Oreochromis niloticus )[11],silver carp (Hypophthalmichthys molitrix )[12]and smooth hound (Mustelus mustelus )[13]have been reported to possess antioxidative activities.Hypertension has been considered as the most common seri-ous chronic health problem [14].Since angiotensin I-converting enzyme (ACE)(EC 3.4.15.1)is physiologically important in raising blood pressure,the inhibition of ACE activity can lead to an over-all antihypertensive effect.The synthetic ACE inhibitors are now widely used as pro-drugs but these synthetic ACE inhibitors can cause many signiﬁcant undesirable side effects [14,15].Therefore,the natural safe compounds are desirable for prevention of hyper-tension instead of the synthetic counterpart.Among those,food protein derived peptides are promising natural products exhibiting ACE inhibitory activities.Protein hydrolysates with antihyperten-sive activity have also produced from sardinelle (Sardina pilchardus )by-products [16],tuna cooking juice [17],salmon protein [18]and tilapia [19].In Thailand,brownstripe red snapper (Lutjanus vitta )is one of the raw materials for surimi production [20].Besides being pro-duced into surimi,this species and its viscera,especially pyloric caeca,can be used as raw material for production of protein hydrolysate and as the source of proteases,respectively.Produc-tion of ﬁsh protein hydrolysates with bioactivity can pave the way1359-5113/$–see front matter ©2010Elsevier Ltd.All rights reserved.doi:10.1016/j.procbio.2010.09.005S.Khantaphant et al./Process Biochemistry46(2011)318–327319for full utilization of these species.Many factors affect the bioac-tivity of protein hydrolysates,e.g.type of proteases[9],steps of hydrolysis[21],etc.This work aims to study antioxidative and ACE inhibitory activ-ities of protein hydrolysate from brownstripe red snapper muscle prepared using its pyloric caeca protease in combination with commercial proteases via2-step hydrolysis and to investigate the bioactivities of selected hydrolysate after digestion in gastrointesti-nal tract model system.2.Materials and methods2.1.Enzymes and chemicalsAlcalase2.4L(E.C.3.4.21.62)(2.4AU/g)and Flavourzyme500L(E.C.3.4.21.77) (500LAPU/g)were provided by Novozyme(Bagsvaerd,Denmark).2,2 -Azinobis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),1,1-diphenyl-2-picrylhydrazyl (DPPH),2,4,6-trinitrobenzenesulfonic acid(TNBS),1,1,3,3-tetramethoxypropane, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4 ,4 -disulfonic acid(ferrozine),2,4,6-tripyridyl-triazine(TPTZ),l-␣-phosphatidylcholine(lecithin)and linoleic acid were purchased from Sigma(St.Louis,MO,USA).Thiobarbituric acid(TBA),potassium persulfate,␤-carotene and Tween40were obtained from Fluka(Buchs,Switzer-land).Tris(hydroxymethyl)aminomethane(Tris–HCl)was procured from Merck (Darmstadt,Germany)and sodium sulﬁte was obtained from Riedel-deHaën(Seelze, Germany).All chemicals were of analytical grade.2.2.Preparation of pretreatedﬁsh minceBrownstripe red snapper,stored in ice and off-loaded approximately24–36h after capture,were purchased from a dock in Songkhla province,Thailand.Fish were transported in ice with theﬁsh/ice ratio of1:2(w/w)to the Department of Food Technology,Prince of Songkla University within1h.Upon arrival,wholeﬁsh were washed and onlyﬂesh was separated manually.Flesh was minced to uniformity using Moulinex AY46blender(Group SEB,Lyon,France)and phospholipid mem-brane was removed by further homogenizing with nine volumes of cold8mmol L−1 CaCl2solution containing5mmol L−1citric acid[22]using an IKA Labortechnik homogenizer(Selangor,Malaysia)at a speed of11000rpm for2min.After contin-uous stirring for60min at4◦C,the sample was centrifuged at4000×g for15min at4◦C using a Beckman Coulter centrifuge Model Avant J-E(Beckman Coulter,Inc., Fullerton,CA,USA).Thereafter,the pellet was washed by homogenizing withﬁve volumes of cold distilled water using a homogenizer at a speed of11000rpm for 2min,followed by stirring at4◦C for15min prior to centrifuging at9600×g for 10min at4◦C.The washing process was repeated twice.Pretreated mince obtained was kept in polyethylene bags and placed in ice until use,but not longer than2h.2.3.Preparation of proteases from pyloric caecaPyloric caeca from brownstripe red snapper was collected and powdered in liq-uid nitrogen.Thereafter,the pyloric caeca extract was prepared according to the method of Khantaphant and Benjakul[20].Pyloric caeca powder was suspended in ten volumes of extraction buffer(50mmol L−1Tris–HCl buffer,pH8.0,contain-ing10mmol L−1CaCl2.The mixture was homogenized at11000rpm for2min.The homogenate was continuously stirred for30min at4◦C and centrifuged at8000×g for30min at4◦C.The supernatant wasﬁltered through a Whatmanﬁlter paper No.1(Schleicher&Schuell,Maidstone,England).Theﬁltrate obtained was further subjected to40–60%saturation ammonium sulfate precipitation.After stirring at 4◦C for30min,the mixture was centrifuged at8000×g for30min at4◦C and the pellet obtained was dissolved in50mmol L−1Tris–HCl buffer,pH8.0followed by dialysis against20volumes of the extraction buffer overnight at4◦C with three changes of dialysis buffer.The dialysate was kept in ice and referred to as‘pyloric caeca protease;PCP’.The proteolytic activity of PCP was determined using casein as a substrate under optimal condition(60◦C and pH8.5)2.4.Preparation of protein hydrolysate from brownstripe red snapper2.4.1.One-step hydrolysis using different single proteasesPretreated brownstripe red snapper mince with a protein content of95.42%pro-tein(dry basis)determined by Kjeldahl method[23]was mixed with50mmol L−1 Tris–HCl buffer with pH of7.0,8.0and8.5for hydrolysis by Flavourzyme,Alcalase and PCP,respectively,to obtain aﬁnal protein concentration of20g protein L−1.The mixtures were homogenized at a speed of11,000rpm for1min and the pH was rechecked and readjusted using1mol L−1NaOH or1mol L−1HCl.The homogenates were pre-incubated for10min at50◦C for Alcalase and Flavourzyme and at60◦C for PCP[20].To start the hydrolysis,the different levels of enzymes were added into the mixture to obtain the desirable degree of hydrolysis(DH)of20,30and 40%following the method of Benjakul and Morrissey[24].After2h of hydrolysis, the reactions were inactivated by placing the mixture in boiling water for10min. Thereafter,the mixture was centrifuged at2000×g at4◦C for10min.The super-natant was collected and referred to as protein hydrolysate prepared using Alcalase (HA),Flavourzyme(HF)or PCP(HP).All protein hydrolysates were determined for antioxidative activities.2.4.2.Two-step hydrolysis using different proteasesAfter2h of theﬁrst hydrolysis,the mixtures with DH of40%,which had the high-est antioxidative activities,were heated for10min in boiling water and adjusted to the desirable pH using1mol L−1NaOH or1mol L−1HCl for proteases used for the second step.Those proteases included Alcalase,Flavourzyme and PCP.The same amount of proteases used in theﬁrst step was added into the pre-incubated mix-ture with optimal temperature of each protease.Reaction was conducted for1,2, 3and5h.At the time designated,the reaction was terminated by submerging the mixture in boiling water for10min.The mixture was then centrifuged at2000×g at4◦C for10min and the supernatant was collected and adjusted to neutral pH. The neutral solution was referred to as‘protein hydrolysates’and lyophilized to obtain hydrolysate powder.The hydrolysates obtained from HP with further hydrol-ysis using Alcalase(HPA)or Flavourzyme(HPF),hydrolysate from HA using PCP or Flavourzyme for the second step(HAP and HAF)and hydrolysate from HF using PCP or Alcalase for the second step(HFP and HFA)were determined for their antioxida-tive and ACE inhibitory activities.2.5.Determination of antioxidative activities2.5.1.DPPH radical scavenging activityDPPH radical scavenging activity was determined according to the method of Blois[25]with a slight modiﬁcation.Sample solution(1.5mL)was added with1.5mL of0.1mmol L−1DPPH in950mL L−1ethanol.The mixture was allowed to stand for 30min in dark at room temperature.The resulting solution was measured at517nm. The control was prepared in the same manner except that distilled water was used instead of the sample.The DPPH radical scavenging activity was calculated from Trolox standard curve(0–60␮mol L−1)and expressed as␮mol Trolox equivalents (TE)g−1protein.2.5.2.ABTS radical scavenging activityABTS radical scavenging activity was determined as described by Re et al.[26]. ABTS radical(ABTS•+)was produced by reacting ABTS stock solution(7.4mmol L−1 ABTS)with2.6mmol L−1potassium persulfate at the ratio of1:1(v/v).The mixture was allowed to react in the dark for12h at room temperature.Prior to assay,the ABTS•+solution was diluted with methanol to obtain an absorbance of1.1(±0.02) at734nm.To initiate the reaction,150␮L of sample was mixed with2.85mL of ABTS•+solution.The mixture was incubated at room temperature for2h in dark. The absorbance was then read at734nm.A Trolox standard curve(0–200␮mol L−1) was prepared.Distilled water was used instead of the sample and prepared in the same manner to obtain the control.ABTS radical scavenging activity was expressed as␮mol TE g−1protein.2.5.3.Ferric reducing antioxidant power(FRAP)The ability of samples to reduce ferric ion(Fe3+)was evaluated by the method of Benzie and Strain[27].FRAP reagent(a freshly prepared mixture of 10mmol L−1TPTZ solution in40mmol L−1HCl,20mmol L−1FeCl3.6H2O solution and300mmol L−1acetate buffer,pH3.6(1:1:10,v/v/v))(2.85mL)was incubated at 37◦C for30min prior to mixing with150␮L of sample.The reaction mixture was allowed to stand in dark for30min at room temperature.Absorbance at593nm was read and FRAP was calculated from the Trolox standard curve(0–60␮mol L−1)and expressed as␮mol TE g−1protein.The control was prepared in the same manner except that distilled water was used instead of the sample.2.5.4.Ferrous chelating activityChelating activity of samples towards ferrous ion(Fe2+)was measured by the method of Benjakul et al.[28]with a slight modiﬁcation.Sample(200␮L)was mixed with800␮L of distilled water.Thereafter,0.1mL of2mmol L−1FeCl2and0.2mL of 5mmol L−1ferrozine were added.The mixture was allowed to react for20min at room temperature.The absorbance was then read at562nm.The standard curve of EDTA(0–1.0mmol L−1)was prepared.The control was prepared in the same manner except that distilled water was used instead of the sample.Ferrous chelating activity was expressed as␮mol EDTA equivalents g−1protein.2.6.Determination of ACE inhibitory activityThe angiotensin I-converting enzyme inhibitory activity was determined as described by Hayakari et al.[29]with slight modiﬁcations.Sample(0.3mL)was incubated with725unit ACE L−1(50␮L)at37◦C for5min.Thereafter,the incubated mixture was added into the assay mixture(0.2mL)which was100mmol L−1K2HPO4 buffer(pH8.3)containing600mmol L−1NaCl and3mmol L−1hippuryl-l-histidyl-l-leucine(HHL).The mixture was incubated at37◦C for15min.To terminate the reaction,1.5mL of30g L−12,4,6-trichloro-1,3,5-triazine(dissolved in dioxane)and 3mL of0.2mol L−1K2HPO4buffer(pH8.3)were added and mixed thoroughly.The mixture was left for10min until the solution was clear and the absorbance at382nm was determined.Sample blank was prepared in the same manner except that HHL was added after the reaction mixture was terminated.The control was prepared320S.Khantaphant et al./Process Biochemistry46(2011)318–327by using distilled water instead of the sample,whereas the control blank was donein the same manner with the control but HHL was added after the termination.%inhibition of ACE was determined using the following equation:%Inhibition=A C−A SA C×100where A C=A control−A control blank and A S=A sample−A sample blank.2.7.Determination of antioxidative activity in different model systems2.7.1.ˇ-Carotene linoleic acid emulsion model systemThe antioxidative activity of the sample in the␤-carotene linoleic acid emulsion model system was determined as described by Binsan et al.[30].␤-Carotene(1mg) was dissolved in10mL of chloroform.Thereafter,the solution(3mL)was added to 20mg linoleic acid and200mg Tween40.Chloroform was then removed by purg-ing with nitrogen.Fifty milliliters of oxygenated distilled water were added to the ␤-carotene emulsion and mixed well.Hydrolysate(200␮L)was then mixed with 3mL of oxygenated␤-carotene emulsion to obtain theﬁnal concentrations of500 and1000ppm.The oxidation of␤-carotene emulsion was monitored spectrophoto-metrically at470nm after0,10,20,3040,60,90and120min of incubation at50◦C in dark.BHT at levels of100ppm was also used.The control was prepared by using distilled water instead of hydrolysate in the assay system.2.7.2.Lecithin liposome model systemThe antioxidative activity of protein hydrolysates in a lecithin liposome system was determined according to the method of Frankel et al.[31]slightly modiﬁed by Thiansilakul et al.[8].Lecithin liposome system was prepared by suspending lecithin in deionized water at a concentration of8g L−1.The mixture was stirred with a glass rod followed by soniﬁcation for30min in a sonicating bath(Elma Model S30H, Singen,Germany).Protein hydrolysate(3mL)was added to the lecithin liposome system(15mL)to obtain aﬁnal concentration of1000ppm.The mixture was son-icated for2min.To initiate the reaction,20mL of0.15mol L−1cupric acetate were added.The mixture was shaken in the dark at120rpm using a shaker(Heidolph Model Unimax1010,Schwabach,Germany)at37◦C.The systems containing25or 100ppm BHT were also prepared.The control was prepared in the same manner, except that distilled water was used instead of sample.Oxidation in lecithin lipo-some systems was monitored at6h intervals by determining the formation of TBARS and conjugated dienes.2.8.Determination of thiobarbituric acid reactive substances(TBARS)Thiobarbituric acid reactive substances(TBARS)were determined as described by Buege and Aust[32]with a slight modiﬁcation.Sample was homogenized with TBARS solution(3.75g L−1TBA,150g L−1TCA and0.25mol L−1HCl)with a ratio of 1:4(w/v).The mixture was heated in boiling water for10min to develop the pink color.Then the mixture was cooled with running water and centrifuged at5000×g for10min at room temperature using Hettich centrifuge(Hettich Model MIKRO-20,Tuttlingen,Germany).The supernatant was collected and measured at532nm using a UV-1800Spectrophotometer(Shimadzu,Kyoto,Japan).TBARS was calcu-lated from a standard curve of malonaldehyde(MDA)(0–10mg L−1)and expressed as mg MDA kg−1sample.2.9.Determination of conjugated dieneConjugated diene formed in the sample was measured according to the method of Frankel et al.[31].Sample(0.1mL)was dissolved in methanol(5.0mL)and con-jugated dienes were measured as the increase in absorbance at234nm.2.10.Preparation of gastrointestinal tract model system(GIMs)Gastrointestinal tract model system was prepared according to the method of Lo et al.[33]with slight modiﬁcation.Hydrolysate powder was dissolved in distilled water to obtain a concentration of50g protein L−1.The solution was adjusted to pH 2.0with1mol L−1HCl and pepsin dissolved in0.1mol L−1HCL was added to obtain theﬁnal concentration of40g pepsin kg−1protein.The mixture was incubated at 37◦C for1h with continuous shaking(Memmert Model SV1422,Schwabach,Ger-many).Thereafter,the pH of reaction mixture was raised to5.3with1mol L−1NaOH before adding20g pancreatin kg−1protein and the pH of mixture was adjusted to 7.5with1mol L−1NaOH.The mixture was incubated at37◦C for3h with continuous shaking.The digestion was terminated by submerging the mixture in boiling water for10min.During digestion,the mixture was randomly taken and determined for antioxidative activities at0,20,40,60,80,100,120,150,180,210and240min.2.11.Proximate analysisHAP and HFP were determined for protein,fat,ash and moisture contents according to the methods of AOAC[23]with the analytical number of992.15,991.36, 942.05and950.46,respectively.2.12.Amino acid analysisHAP and HFP were hydrolyzed under reduced pressure in4.0M methanesul-fonic acid containing0.2%(v/v)3-2(2-aminoethyl)indole at115◦C for24h.The hydrolysates were neutralized with3.5M NaOH and diluted with0.2M citrate buffer (pH2.2).An aliquot of0.4mL was applied to an amino acid analyzer(MLC-703;Atto Co.,Tokyo,Japan).2.13.Determination of protein concentrationProtein concentration was measured by the method of Lowry et al.[34]using bovine serum albumin as a standard.2.14.Statistical analysisExperiments were run in triplicate.All data were subjected to analysis of vari-ance(ANOVA)and differences between means were evaluated by Duncan’s Multiple Range Test.For pair comparison,T-test was used[35].SPSS Statistic Program(Ver-sion10.0)(SPSS Inc,Chicago,IL,USA)was used for data analysis.3.Results and discussion3.1.Antioxidative activities of brownstripe red snapper protein hydrolysates prepared with single step of hydrolysis usingdifferent proteasesProtein hydrolysates from brownstripe red snapper muscle prepared using protease from pyloric caeca of brownstripe red snapper,Alcalase and Flavourzyme referred to as HP,HA and HF, respectively,with different DH(20,30and40%DH)showed varying antioxidative activities(Fig.1).3.1.1.DPPH radical scavenging activityThe DPPH radical scavenging activity of HA increased with increasing DH(p<0.05)(Fig.1(a)).HF showed the similar activity at all DH used(p>0.05),whereas HP showed no further increase in activity when DH was higher than30%(p>0.05).At all designated DH,HA and HF showed the higher activity than did HP(p<0.05). The result suggested that the peptides in different hydrolysates might be different in term of chain length and amino acid sequence, which contributed to varying capabilities of scavenging DPPH rad-icals.The increase in DPPH radical scavenging activity of HA was in agreement with Thiansilakul et al.[8]who reported the increases in DPPH radical scavenging activity as the DH of the hydrolysate from round scad muscle protein prepared using Flavourzyme and Alcalase increased.On the other hand,Klompong et al.[9]found that DPPH radical scavenging activity of protein hydrolysate pre-pared from the muscle of yellow stripe trevally using Flavourzyme and Alcalase decreased when DH increased.Invert correlation between DH and DPPH radical scavenging activity was obtained for protein hydrolysates prepared from alkaline-aided channel catﬁsh protein isolates using Protamex[36].You et al.[37]reported that loach protein hydrolysate showed the greater DPPH radical scav-enging activity when DH increased.DPPH is a stable free radical and can be scavenged with a proton-donating substance,such as an antioxidant[25].Therefore,protein hydrolysates from brown-stripe red snapper muscle more likely contained peptides acting as hydrogen donors,thereby scavenging free radicals by converting them into more stable products.3.1.2.ABTS radical scavenging activityIn general,ABTS radical scavenging activities of protein hydrolysates increased as DH increased(p<0.05)(Fig.1(b)).The highest activity was observed in HP and HA with40%DH(p<0.05). However,no difference in activity was found in HF with all DH used(p>0.05).Among all hydrolysates,HA had the lowest activity for all DH tested(p<0.05).At DH of20%,HF showed higher ABTS radical scavenging activity than did HP(p<0.05).Conversely,HPS.Khantaphant et al./Process Biochemistry46(2011)318–327321Fig.1.Antioxidative activities of protein hydrolysate from brownstripe red snapper muscle prepared using pyloric caeca protease(PCP)from brownstripe red snapper (HP),Alcalase(HA)and Flavourzyme(HF)with different DHs.Bars represent the standard deviation(n=3).Different capital letters within the same enzyme used indicate signiﬁcant differences(p<0.05).Different letters within the same DH indicate signiﬁcant differences(p<0.05).had the highest activity when DH of40%was used(p<0.05).Pro-tein hydrolysate from alkali-solubilized tilapia protein prepared using various proteases showed a sharp increase in ABTS radical scavenging activity when DH increased from18to23%[11].Loach protein hydrolysate showed a similar result,in which ABTS radical scavenging activity increased with increasing DH[37].For both DPPH and ABTS assays,HF showed no differences in activities with all DH tested(p>0.05).ABTS radical assay is used for determining the antioxidative activity,in which the rad-ical is quenched to form ABTS-radical complex[26].Generally, all hydrolysates contained peptides,which were able to scav-enge ABTS radicals,leading to the termination of radical chain reaction.3.1.3.Ferric reducing antioxidant power(FRAP)Ferric reducing antioxidant power(FRAP)measures the reduc-ing ability against ferric ion(Fe3+),indicating the ability of hydrolysates to transfer an electron to the free radical[27].FRAP of different hydrolysates with varying DH is depicted in Fig.1(c). An increase in FRAP was observed in all hydrolysates when DH increased(p<0.05).FRAP of HA was generally higher than those of HP and HF at all DHs tested(p<0.05).The result suggested that HA had the greater reducing power than did others,leading to the greater efﬁcacy in prevention and retardation of propagation in lipid oxidation.However,protein hydrolysates prepared from alkaline-aided channel catﬁsh protein isolates showed the decrease in reducing power with increasing DH[36].Raghavan et al.[11] reported that alkaline-solubilized tilapia protein hydrolysate pre-pared using Flavourzyme showed the increase in reducing ferric ion when DH increased.The hydrolysis most likely increased the reducing power,especially when the cleavage of peptides increased as indicated by the increase in DH.The result indicated that pep-tides generated from the hydrolysis by different proteases had the different capacities of providing electron to the radicals.3.1.4.Ferrous chelating activityFerrous chelating activities of hydrolysates prepared using dif-ferent proteases with different DHs are shown in Fig.1(d).Chelating activity against Fe2+of HP slightly increased when DH increased up to30%(p<0.05).For HA and HF,the decreases in ferrous chelating activities were found as DH increased(p<0.05).Ferrous ion(Fe2+) is the most powerful pro-oxidant among metal ions[38],leading to the initiation and acceleration of lipid oxidation by interaction with hydrogen peroxide in a Fenton reaction to produce the reactive oxy-gen species,hydroxyl free radical(OH•)[39].Therefore chelation of metal ions by peptides in hydrolysates could retard the oxida-tive reaction.The result indicated that a higher DH rendered HA and HF with lower metal-chelating activities.The shorter chain of peptides might lose their ability to form the complex with Fe2+. At DH of20%,HF showed the highest chelating activity(p<0.05), followed by HA and HP,respectively.Peptides in HF could effec-tively chelate the Fe2+,leading to the retardation of initiation stage. The result indicated that the limited hydrolysis of muscle protein resulted in the enhanced ferrous chelating activity,compared with the excessive hydrolysis.The higher chelating activity of HP was coincidental with the higher DPPH and ABTS radical scavenging activity and FRAP,as the DH increased.Fe2+chelating activity of round scad protein hydrolysate prepared using Alcalase showed the increase in chelating activity with increasing DH,but those treated with Flavourzyme showed no difference in activity at all DH tested[8].With the same enzymes used,chelating activity of pro-tein hydrolysate prepared from the muscle of yellow stripe trevally322S.Khantaphant et al./Process Biochemistry46(2011)318–327using Flavourzyme and Alcalase increased with increasing DH[9]. Higher ferrous chelating activity was reported for hydrolysate of sil-ver carp using Alcalase and Flavourzyme when DH increased[12]. Apart from Fe,other transition metals,such as Cu and Co can affect the rate of lipid oxidation and decomposition of hydroperoxide. Theodore et al.[36]reported that Cu2+chelating activity of catﬁsh protein hydrolysate increased with increasing DH.Some proteins and peptides can chelate metal ions like Fe2+due to the presence of carboxyl and amino groups in the side chains of acidic and basic amino acids[10,40].Alcalase is endopeptidase capable of hydrolyzing proteins with broad speciﬁcity for peptide bonds and prefers for the uncharged residue,whereas Flavourzyme is a mixture of endo-and exopeptidase enzyme,which can produce both amino acids and peptides[41].Hydrolysates showing differ-ent antioxidative activities might be attributed to the differences in the exposed side chains of peptides as governed by the speciﬁcity of different proteases towards peptide bonds in the proteins[42]. DH also greatly inﬂuenced the peptide chain length.The higher DH,the more cleavage of peptide chains took place.Peptides with various sizes and compositions had different capacities of scaveng-ing or quenching free radicals[8,9].PCP,Alcalase and Flavourzyme more likely cleaved the peptide bonds in brownstripe red snap-per muscle at different positions,resulting in the different products with varying antioxidative activities.With40%DH,all hydrolysates functioned more effectively as primary antioxidant,whereas the secondary antioxidative activity,chelating ability,was lowered.To enhance the antioxidative activity of peptides,especially as the pri-mary antioxidant,the hydrolysate with40%DH was prepared for theﬁrst step of hydrolysis.3.2.Antioxidative and ACE inhibitory activities of brownstripered snapper protein hydrolysate prepared with two-stephydrolysis using different proteases3.2.1.DPPH radical scavenging activityDPPH radical scavenging activity of protein hydrolysates with various hydrolysis times in the second step of hydrolysis using another protease is shown in Fig.2(a).For HAF,HAP,HFA and HFP, the activities increased with increasing time up to2h(p<0.05). Thereafter,the gradual decrease was observed when hydrolysis times were3and5h(p<0.05).No changes in DPPH radical scav-enging activity were observed for HPF,while HPA showed the maximal activity at3h of hydrolysis(p<0.05).The results sug-gested that scavenging activity against DPPH radical was enhanced by additional hydrolysis using another enzyme with an appropriate time.Generally,hydrolysates prepared by further hydrolyzing the hydrolysate obtained from theﬁrst step of hydrolysis with another protease for2h had a greater scavenging ability against DPPH rad-ical.3.2.2.ABTS radical scavenging activityABTS radical scavenging activity of protein hydrolysates pre-pared by two-step hydrolysis is shown in Fig.2(b).Only HAF and HAP showed the increases in ABTS radical scavenging activity,com-pared with their parent counterparts,HA.HAP with the hydrolysis time of2h for the second step had the highest activity(p<0.05). This suggested the enhancement of ABTS radical scavenging activ-ity when PCP was used for further hydrolysis of HA.However,the further hydrolysis of HF with another protease had no impact on the increases in activity(p>0.05).For HP,the decrease in activities was obtained when the second step of hydrolysis was performed (p<0.05).The results suggested that the second step of hydrolysis could slightly increase ABTS radical scavenging activity,depending on theﬁrst hydrolysate as well as the types of protease used for the second step.It was noted that the abilities of scavenging ABTS and DPPH radicals by hydrolysates were different.This might be due to the differences in ability of scavenging the different radicals,ABTS and DPPH,by the same peptide.3.2.3.Ferric reducing antioxidant power(FRAP)Fig.2(c)shows FRAP of protein hydrolysates prepared using two-step hydrolysis.The increases in FRAP were observed for all hydrolysates,when the second step of hydrolysis was applied.For HF and HP,when Alcalase was used for the second step of hydroly-sis,the continuous increases in FRAP were observed(p<0.05).For other hydrolysates,the hydrolysis time was the factor affecting the FRAP of resulting hydrolysate,depending on theﬁrst hydrolysate and the types of protease used for the second step of hydrolysis.The increase in FRAP with increasing hydrolysis time was coincidental with the increase in DPPH radical scavenging activity(Fig.2(a)). For HAF,HAP and HPF,the activities were increased with increas-ing hydrolysis time up to2h(p<0.05).For HFP,the hydrolysis time more than1h had the negative effect on FRAP(p<0.05).Gener-ally,further hydrolysis with another protease led to the increase in FRAP,but the activities of resulting hydrolysate were governed by hydrolysis time.3.2.4.Ferrous chelating activityFerrous chelating activities of hydrolysates prepared by two-step hydrolysis are shown in Fig.2(d).HFA and HPA showed the marked decrease in chelating activities when the second hydroly-sis was conducted(p<0.05).Alcalase used in the second step might generate peptides with the lower ability in Fe2+chelating.The abil-ities of HAF,HAP,HFP and HPF in chelating Fe2+ion were more pronounced,compared with their parent hydrolysate counterparts. The increases were observed when a certain time of the second hydrolysis was used.Among all protein hydrolysates,HFP showed the highest ferrous chelating activity,especially when the second step hydrolysis time of1h was used(p<0.05).3.3.ACE inhibitory activityThe inhibitory effect of all hydrolysates prepared using two-step hydrolysis against angiotensin I-convering enzyme(ACE)was determined as shown in Fig.2(e).For theﬁrst hydrolysate prepared using the different single proteases(0h),HF showed the highest ACE inhibition(11.44%)(p<0.05).Flavourzyme might produce the peptides with ACE inhibitory activity.Raghavan and Kristinsson [19]used Cryotin and Flavourzyme for hydrolysis of tilapia protein and found the higher ACE inhibition by hydrolysate prepared using Flavourzyme.When the second step of hydrolysis using another protease was performed,HFA and HFP showed the higher ACE inhi-bition when hydrolysis time of2and3h was used,respectively.HAP also showed high ACE inhibitory activity.Wu et al.[43]reported that shark meat treated with enzyme showed the higher ACE inhi-bition than that of untreated one.ACE inhibition by tilapia protein hydrolysate was reported,especially with increasing DH[19].Fur-thermore,smaller peptides are the better ACE inhibitors than the larger counterpart[19].The hydrolysis might release ACE inhibitory peptides in hydrolysate[43].Hydrolysates from muscle of differ-entﬁsh have been reported to possess ACE inhibitory activity,e.g. hydrolysates from tilapia protein[19],freshwater clam muscle[44], shark meat[43],Atlantic salmon,Coho salmon,Alaska pollack and southern blue whiting muscle[45].From the results,the use of Alcalase for theﬁrst hydrolysis and the use of PCP in the second hydrolysis for2h(HAP)yielded the resulting hydrolysate with the higher DPPH and ABTS radical scavenging activities and FRAP,compared to other hydrolysates (p<0.05).However HF hydrolyzed with PCP in the second hydrol-ysis step for1h(HFP)showed much higher chelating activity, compared with others(p<0.05).Therefore,HFP and HAP prepared with1and2h for the second step of hydrolysis,respectively,were。

银杏肽锌螯合物的制备、体外消化及抗氧化活性分析

郑义，李诗颖，李闯，等. 银杏肽锌螯合物的制备、体外消化及抗氧化活性分析[J]. 食品工业科技，2023，44（17）：420−427. doi:10.13386/j.issn1002-0306.2022110135ZHENG Yi, LI Shiying, LI Chuang, et al. Preparation, in Vitro Gastrointestinal Digestion and Antioxidant Activity of Ginkgo biloba Peptides-Zinc Chelate[J]. Science and Technology of Food Industry, 2023, 44(17): 420−427. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2022110135· 营养与保健 ·银杏肽锌螯合物的制备、体外消化及抗氧化活性分析郑　义1,2，李诗颖1，李　闯1，周小芮1，陈亚楠1，张秀芸1，张银雨1（1.徐州工程学院食品与生物工程学院，江苏徐州 221018；2.江苏省食品资源开发与质量安全重点建设实验室，江苏徐州 221018）摘　要：本文优化了银杏肽锌螯合物（Ginkgo biloba peptides-zinc chelate, Zn-GBP ）的制备工艺，分析了Zn-GBP 的体外消化特性及抗氧化活性。

采用单因素实验及响应面法优化了Zn-GBP 的制备工艺；采用体外模拟胃肠道消化测定了Zn-GBP 中锌离子的生物利用率；以DPPH 自由基清除能力、ABTS +自由基清除能力、还原能力为指标，评价了Zn-GBP 的体外抗氧化活性。

结果表明，Zn-GBP 的最佳制备工艺条件为：银杏肽与锌质量比3:1、螯合pH 8.2、螯合温度70 ℃、螯合时间2 h ；在此条件下，螯合率为49.23%±0.35% ，螯合物得率为42.34%±0.45%。

中国弯颈霉多糖抗氧化和抑制氧化应激活性研究

白明健，周颖，程昊，等. 中国弯颈霉多糖抗氧化和抑制氧化应激活性研究[J]. 食品工业科技，2024，45（2）：333−341. doi:10.13386/j.issn1002-0306.2023030199BAI Mingjian, ZHOU Ying, CHENG Hao, et al. Antioxidant and Oxidative Stress Inhibitory Activities of Tolypocladium sinense Polysaccharide[J]. Science and Technology of Food Industry, 2024, 45(2): 333−341. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023030199· 营养与保健 ·中国弯颈霉多糖抗氧化和抑制氧化应激活性研究白明健，周　颖，程　昊，边　聪，李名正，李　林*，张春晶*（齐齐哈尔医学院医学技术学院，黑龙江齐齐哈尔 161006）O −2摘　要：目的：探究中国弯颈霉菌丝体多糖（Tolypocladium sinense polysaccharide ，TSP ）的体外抗氧化活性和抑制过氧化氢诱导小鼠胰岛MIN6细胞氧化应激导致的细胞凋亡。

方法：采用热水浸提法提取中国弯颈霉菌丝体多糖，随后测定其超氧阴离子自由基（superoxide anion ，·）、羟自由基（hydroxy radical ，·OH ）、对1,1-二苯基苦基苯肼自由基（P-1,1-diphenylpicryl phenylhydrazine radical ，DPPH·）清除能力；采用200 μmol/L 过氧化氢（hydrogen peroxide ，H 2O 2）诱导小鼠胰岛MIN6细胞氧化应激，给予高剂量和低剂量TSP （0.625、0.156 mg/mL ）进行保护，MTT 法测定MIN6细胞生存率；倒置显微镜观察细胞形态；采用试剂盒测定培养基中乳酸脱氢酶（lactate dehydrogenase ，LDH ）水平，细胞内超氧化物歧化酶（superoxide dismutase ，SOD ）活性和丙二醛（malondialdehyde ，MDA ）含量；流式细胞术检测细胞凋亡；Western Blot 检测核因子E2相关因子2（nuclear factor E2-related factor2，Nrf-2）和磷酸化c-Jun 氨基末端激酶（phosphorylated c-jun N-terminal kinase ，pJNK ）相对表达量。

氧化应激与肝脏损伤

氧化应激与肝脏损伤氧化应激与肝脏损伤吴娜, 蔡光明, 何群吴娜,湖南中医药⼤学湖南省长沙市410208吴娜, 蔡光明,中国⼈民解放军302医院中药研究所北京市100039何群,湖南中医药⼤学药剂教研室湖南省长沙市410208作者贡献分布:本⽂选题, 参考⽂献检索及撰写由吴娜完成; 论⽂修改由蔡光明与何群完成.通讯作者:蔡光明, 100039, 北京市, 中国⼈民解放军302医院中药研究所.cgm1004@/doc/0f50be76a417866fb84a8e84.html电话: 010-********收稿⽇期: 2008-08-14 修回⽇期: 2008-09-21接受⽇期: 2008-09-22 在线出版⽇期: 2008-10-18Oxidative stress and hepatic injuryNa Wu, Guang-Ming Cai, Qun HeNa Wu,Hunan University of Traditional Chinese Medicine, Changsha 410208, Hunan Province, ChinaNa Wu, Guang-Ming Cai,Institute of Chinese Materia Medica, the 302th Hospital of Chinese PLA, Beijing 100039, China Qun He, Pharmaceutical Division, Hunan University of Traditional Chinese Medicine, Changsha 410208, Hunan Province, ChinaCorrespondence to:Guang-Ming Cai, Institute of Chinese Materia Medica, the 302th Hospital of Chinese PLA, Beijing 100039, China. cgm1004@/doc/0f50be76a417866fb84a8e84.htmlReceived: 2008-08-14 Revised: 2008-09-21Accepted:2008-09-22 Published online: 2008-10-18AbstractOxidative stress, initiated by reactive oxygen species, is the collective pathophysiological mechanism of many hepatopathies. Oxidative stress results in hepatic injury mainly by priminglipid peroxidation to change the function of biological membrane, covalent immobilization of biomacromolecules and destroying the enzyme activity considering cytokine (TNF-α and NF-κB) interaction. The role of oxidative stress in many hepatopathies such as fatty liver desease, viral hepatitis, hepatic fibrosis is innegligible.Key Words: Oxidative stress; Reactive oxygen species; Hepatic injury; Lipid peroxidation; CytokineWu N, Cai GM, He Q. Oxidative stress and hepatic injury. Shijie Huaren Xiaohua Zazhi 2008; 16(29): 3310-3315摘要活性氧⾃由基引发的氧化应激是多种肝病发病的共同病理⽣理基础. 氧化应激主要通过启动膜脂质过氧化改变⽣物膜功能、与⽣物⼤分⼦共价结合及破坏酶的活性等在细胞因⼦(如TNF-α、NF-κB)的共同作⽤下引起不同程度的肝损伤. 氧化应激在脂肪肝、病毒性肝炎、肝纤维化等肝病中可产⽣不容忽视的作⽤.关键词:氧化应激; 活性氧; 肝损伤; 脂质过氧化; 细胞因⼦吴娜, 蔡光明, 何群. 氧化应激与肝脏损伤. 世界华⼈消化杂志2008; 16(29): 3310-3315/doc/0f50be76a417866fb84a8e84.html /1009-3079/16/3310.asp0 引⾔胃⽣物体内的能量代谢将氧⽓作为有氧代谢过程中的电⼦接受体, 不可避免地产⽣活性氧(reactive oxygen species, ROS)⾃由基. ROS具有双重效应, 与某些⽣理活性物质的调控和炎症免疫过程密切相关, 但是过量的ROS容易导致氧化应激(oxidative stress, OS)状态[1]. 线粒体呼吸链复合体利⽤电⼦传递⽣产ATP, 是ROS的主要来源[2], 肝脏含有丰富的线粒体, 因此也是ROS 攻击的主要器官. OS可能是肝病的共同发病机制. 因此, 本⽂就近年来关于OS在不同肝损伤机制中的作⽤进⾏综述.1 氧化应激与脂肪性肝病脂肪性肝病指脂肪在肝细胞中的异常沉积, 可分为⾮酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD)和酒精性脂肪肝病(alcoholic fatty liver disease, AFLD). 在复杂的脂肪肝发病机制中OS起关键作⽤, ⽬前对脂肪肝发病机制⼴泛接受的理论是1998年Day et al[3]提出的“⼆次打击”学说. 第⼀次打击主要是胰岛素抵抗和脂肪代谢的失衡导致的脂肪在肝细胞中的沉积, 尤其是脂肪酸和⽢油三酯, 最终引起单纯性肝脂肪变性; 第⼆次打击为环境应激物(如饮⾷成分)及代谢应激物(如⾼⾎糖)主要通过对肝细胞线粒体的损伤产⽣OS, 在细胞因⼦等共同作⽤下, 引起脂肪性肝炎, 进⼀步形成脂肪性肝纤维化和脂肪性肝硬化.1.1 氧化应激与NAFLD随着肥胖及Ⅱ型糖尿病患病率的增加, NAFLD已成为当今医学领域的⼀个难题. NAFLD包含两种组织学损伤: 单纯性⾮酒精性脂肪肝(nonalcoholic fatty liver, NAFL)及⾮酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH). NAFLD的发病机制较复杂, 迄今尚未完全阐明, 过量的ROS使肝内抗氧化系统遭到严重破坏. ⾕胱⽢肽(glutathione, GSH)是细胞内重要的肽类抗氧化剂, 是诸多ROS清除酶的还原底物, 反应后⽣成具有潜在⾼细胞毒素的氧化型⾕胱⽢肽(oxidative glutathione, GSSG). Nobili et al[4]以GSSG/GSH的⽐值评估体内OS时发现NASH患者⾎液中GSSG增长了1.5倍, 导致GSSG/GSH发⽣显著性变化. 此外, 肝内其他重要的抗氧化物质, 如辅酶Q10, CuZn-SOD及过氧化氢酶(CAT)等的不断衰竭在NAFLD患者中也得到了证实[5].NASH患者的肝细胞以异常的线粒体为特征, 线粒体损伤是肝细胞损伤的重要原因之⼀, ⽽线粒体中过量游离脂肪酸(FFA)的氧化是导致线粒体损伤的重要原因: ⼀⽅⾯, FFA的β-氧化过程产⽣的ROS使线粒体膜发⽣脂质过氧化(lipid peroxidation, LPO),导致进⼀步的解偶联作⽤, 即抑制氧化磷酸化的作⽤, 同时也增强了线粒体的OS[6]. ROS的增多促使肿瘤坏死因⼦(tumor necrosis factor-α, TNF-α)的⽣成, 使其通过⼲预线粒体呼吸链并形成超氧阴离⼦(O2-)⽽加剧线粒体损伤, 并增加了线粒体膜的通透性[7]. 另⼀⽅⾯, ROS能导致mtDNA碱基的氧化, Seki et al[8]研究发现在17例NASH患者中, 有11例患者(64.7%)的肝细胞中表达了8-羟基鸟苷酸(8-OHdG)(mtDNA损伤的⼀种标志).ROS还能启动多种细胞因⼦, 如⽣长转化因⼦β(transforming growth factorβ, TGFβ)、⽩细胞介素-8(interleukin-8, IL-8)、NF-κB等. NF-κB、TGF-β、IL-8和LPO产物4-OH壬烯酮(4-hydroxynonenal, HNE)在⼀定程度上还可以导致中性粒细胞的浸润, 使促炎症反应加强, 最终导致肝细胞凋亡.OS与主要存在于肝实质细胞中的过氧化物酶体增殖物启动受体-α(peroxisome proliferator activated receptor alpha, PPARα)有关[9], PPARα主要对肝内脂肪酸氧化相关基因表达进⾏调控, 如脂酰辅酶A氧化酶(acyl-CoA oxidase, AOX)等[10].1.2 氧化应激与AFLD在AFLD的发病机制中OS同样受到格外关注. 细胞⾊素P450 2E1(CYP2E1)和NADPH氧化酶是⼄醇代谢过程中产⽣ROS的重要酶体系[11]. ⼄醇的代谢产物在CYP2E1及Fe3+参与下的氧化作⽤, 会产⽣⼤量的OS产物, 如OH·、O2-、H2O2等.ROS的增多将会引起细胞内ATP衰竭, 使线粒体氧化容量受损, 进⼀步影响⼄醛的氧化, 使⼄醛在肝脏中不断蓄积, 并可能通过抑制AMP激活的蛋⽩激酶AMPK和sirtuin 1蛋⽩活化肝脏的固醇调节组件结合蛋⽩-1(sterol regulatory element binding protein-1, SREBP-1)增加⽣脂酶基因的表达, 加速脂肪酸的合成[12]; ⼄醇的代谢受阻, 更易导致肠黏膜通透性增加, 肠源性内毒素可通过增强Kupffer细胞内毒素受体CD14和Toll样受体4的表达, 诱导Kupffer细胞中产⽣⼤量的以TNF-α为主的细胞因⼦, 加剧炎症反应, 进⼀步引起肝细胞的坏死、凋亡[13].在⼄醇介导的肝损伤中LPO产物衍⽣的抗原类引起的免疫反应可能起重要作⽤. 丙⼆醛(MDA), HNE可与⼈⾎清⽩蛋⽩结合(HSA), 分别形成MDA-HSA和HNE-HSA, 引起免疫反应[14]. 此外, MDA还能与⼄醛⽣成加合物MAA, 同样具有很强的致免疫特性[15].另外, PPARα也参与了⼄醇介导的肝损伤过程, 是其发病机制的重要原因之⼀, PPARα基因的失活将导致肝脏⼤量的脂质聚集,使脂质代谢机制紊乱[16]图1 氧化应激在NAFLD/AFLD中的损伤机制2 氧化应激与药物性肝病随着临床药物和⾮处⽅药种类的增多及联合⽤药的⼴泛应⽤, 药源性肝损害的发⽣率逐年增⾼. 根据2007年美国疾控中⼼的最新调查数据显⽰每年⼤约有由药物引起的1600例急性肝功能衰竭的案例[17]. 引起肝损害的常见药物有对⼄酰氨基酚(解热镇痛抗炎药)、阿莫西林(青霉素类抗⽣素)、胺碘酮(抗⼼律失常药), 他莫西芬(抗癌药), 司他夫定(抗病毒药)等[18].药物代谢反应与众多酶系有关, 不仅包括CYP450, 还包括含黄素单氧化酶(flavin-containing monooxygenase, FMO), 环氧化物⽔解酶(epoxide hydrolase, EH), 其中CYP450起重要作⽤, 如CYP1A1、CYP1A2、CYP1B1、CYP2E1等. Gonzalez[19]研究发现在不同剂量的⼄酰氨基酚(acetaminophen, AP)产⽣肝毒性条件下, 缺失CYP2E1的⼩⿏存活率明显⾼于野⽣型⼩⿏, 缺失CYP2E1及CYP1A2的⼩⿏在最⾼剂量的AP(1200 mg/kg)条件下, 存活率仍约为90%.药物代谢可以通过多种途径引起的不同程度的肝损伤. 如AP经CYP450(主要为CYP2E1)代谢产⽣的活性中间体N-⼄酰-对苯醌亚胺(NAPQI), 与肝细胞⼤分⼦结合导致肝毒性及GSH的耗竭[20], 加剧LPO; 司他夫定, 通过抑制DNA pol-γ(多聚酶中唯⼀能复制mtDNA的酶类), 最终导致乳酸性酸中毒[21]; 曲格列酮能明显改变70种线粒体蛋⽩质, 并检测出Lon蛋⽩酶、CA T等含量增加[22].药物代谢过程中CYP2E1激活产⽣的ROS可能通过表⽪⽣长因⼦受体(epidermal growth factor receptor, EGRF)/c-Raf的信号增强细胞外信号调节激酶1/2(extracelluar signal-regulated kinase 1/2, ERK 1/2)的磷酸化导致细胞凋亡和坏死基因的激活或改变[23].图2 氧化应激在肝纤维化中的损伤机制.3 氧化应激与病毒性肝炎病毒性肝炎(viral hepatitis, VP)的发病机制⾄今尚未完全阐明, 越来越多的研究表明OS是重要的影响因素. VP患者的肝脏中促氧化/抗氧化系统处于严重失衡状态[24].⽬前, 研究肝炎病毒与OS的关系多集中在HBV(hepatitis B virus)、HCV(hepatitis C virus). Levent et al[25-26]发现HBV、HCV 患者⾎清中抗氧化酶CuZn-SOD、GSX-Px及GSH、β-胡萝⼘素(抑制单线氧活性)明显低于健康者. HBV、HCV蛋⽩的表达可以通过Ca2+信号产⽣OS[27-28]; OS 能导致GSH-Px(⼀种极少见的硒依赖酶)的耗竭使硒含量(主要以硒代半胱氨酸的型式存在)减少, 使免疫反应受到抑制, 并能促进病毒的复制[29]. HBV、HCV患者⾎清中MDA、共轭⼆烯(CD)含量也明显升⾼, 在丙肝患者表现得尤为突出, 这不仅与直接引起细胞溶解的病毒有关, 还与O2-引发的细胞膜损伤有关[30]. 另外, ROS还能激活NF-κB 及转录信号转导⼦与激活⼦-3(Signal Transducer and Activator of Transcription-3, STAT-3), ROS在NF-κB介导下可进⼀步激活环氧化酶-2(Cox-2), Cox-2与STAT-3在细胞增殖、分化、肿瘤形成中起重要作⽤[31-32].4 氧化应激与肝纤维化现代医学认为减少胶原纤维的⽣成和增强其降解, 肝纤维化是可以逆转的. 肝星状细胞(heptic stellate cells, HSCs)的激活转化为肌纤维母细胞, 并⼤量分泌细胞外基质[33](extracellular matrix, ECM)是肝纤维化发⽣和发展的核⼼病理环节, 发病机制复杂, OS引发的LPO是HSC活化、增殖、及胶原合成的重要原因.Wang et al[34]在研究褪⿊素对四氯化碳(CCl4)导致肝纤维化的保护作⽤时发现6个星期后, CCl4模型组⼤⿏的羟脯氨酸(胶原代谢的⽣化指标)、MDA、促炎症因⼦TNF-α及IL-1β显著性升⾼, ⽽抗氧化物酶GSH-Px及SOD的活性显著性降低. 另外, 平滑肌肌动蛋⽩(alpha-smooth muscle actin, α-SMA)、8-OhdG、转化⽣长因⼦β1(TGFβ1)mRNA、基质⾦属蛋⽩酶(matrix metalloproteinase, MMP-2)⽔平在肝纤维化模型中也明显升⾼[35-36].TGFβ1是⽬前研究发现的促纤维增⽣的最重要的细胞因⼦之⼀, ROS能促进HSC通过上调核转录因⼦KLF6(Kruppel-like factor 6, KLF6)分泌TGFβ1[37], 并激活HSC转化为肌纤维母细胞[38]. TGFβ1的激活与丝裂原活化蛋⽩激酶(mitogen activated protein kinase, MAPK)和Akt信号有关[35]. TGFβ1还能释放F2-异前列烷(F2-isoprostanes, F2-IsoPs), F2-IsoPs是肝细胞中LOP 反应⽣成的⼀类前列腺素F2样产物, 被认为是反应LPO⽔平最可靠的标志[39], 能介导HSC的分化和促进胶原的过度形成[40].对氧磷酶-1(paraoxonase-1, PON-1)对OS的调节和肝纤维化的形成起重要作⽤, PON-1是⼀类钙离⼦依赖性⾼密度脂蛋⽩(high-density lipoproteins, HDL)的酯酶, 在脂类代谢中具有重要的抗氧化活性. Ferre et al[41]发现在肝硬化患者的⾎清和肝脏中PON-1表达显著性增加, 但其活性显著性降低. 原因可能是PON-1在⽔解脂质过氧化物后活性降低或HDL的结构发⽣变化导致PON-1活性降低.另外, 过氧化物酶体增殖物启动受体-γ(peroxisome proliferator activated receptor gamma, PPARγ)活性[42]、ERK 1/2信号及Na+/H+交换[43]在激活HSC和胶原的合成中都起作⽤.5 氧化应激与肝癌OS在肝癌细胞增殖、凋亡机制中的作⽤也是不容忽视的. ROS在特定细胞内氧化还原环境下可通过激活蛋⽩激酶B(protein kinase B, PKB)途径传递信号, 促使激活蛋⽩-1(activator protein, AP-1)转录因⼦组成成员c-fos/c-jun基因的mRNA表达, 最终促进肝癌细胞⽣长[44]. 肝癌细胞凋亡与线粒体突变是密不可分的. 李国平et al[45]研究了OS诱导HepG2肝癌细胞凋亡及其机制时发现HepG2暴露于2 mmol/L的H2O2可以产⽣OS, OS作⽤后4 h, 细胞线粒体膜电位明显下降;作⽤8、12 h后细胞凋亡蛋⽩酶3、9(Caspase-3、Caspase-9)分别升⾼6.7和3.6倍; 作⽤12 h后细胞开始凋亡, 提⽰OS诱导HepG2肝癌细胞的凋亡与线粒体通路及Caspase启动有关. OS引起的mtDNA 突变也是肝癌细胞凋亡的⼀个重要原因. 张国强et al[46]⽤溴化⼄锭诱导建⽴mtDNA缺失肝癌细胞(ρ0SK-Hep1)模型, 探讨ROS及线粒体跨膜电位在ρ0SK-Hep1肝癌细胞中的改变时发现mDNA缺失后, 细胞内ROS荧光强度明显增强, 细胞膜电位下降, ROS的增加进⼀步加重线粒体膜的损伤.另外, 端粒酶的启动可能是细胞癌变的⼀个共同通路. 端粒酶的激活是原发性肝癌(hepatocellular carcinoma, HCC)的早期事件, Liu et al[47]发现端粒酶的活性与MDA的含量成正相关, OS对端粒还能引起端粒的损伤或缩短加速[48].6 结论作为细胞信号系统中第⼆信使⾓⾊的ROS, 具有独特的⽣理作⽤, 但过量的ROS通过多种途径在细胞因⼦等共同作⽤下引起不同程度的肝损伤.了解氧化应激在不同肝病中的损伤机制, 对今后肝病的预防和治疗⼯作、开发和利⽤⾼效低毒的抗氧化活性物质具有重要意义.背景资料⼈体内95%的⾃由基属于氧⾃由基, 他往往是其他⾃由基产⽣的起因. 过量的氧⾃由基容易引起细胞的损伤和死亡, 与多种疾病有密切的关系, 如衰⽼、肿瘤及冠⼼病等.同⾏评议者王蒙, 副教授, 中国⼈民解放军第⼆军医⼤学附属东⽅肝胆外科医院肝外综合治疗⼀科; 谭学瑞, 教授, 汕⼤医学院第⼀附属医院院长室研发前沿肝病的发病机制⼗分复杂, 氧⾃由基引起的氧化性损伤是其中⼀个⼗分重要的原因. ⽬前研究的热点是天然抗氧化性药物及其作⽤机制.应⽤要点本⽂综合阐述了氧化应激在多种肝病发病机制中的损伤机制, 为临床肝病的治疗中筛选⾼效的抗氧化活性药物提供了⼀定的理论依据.同⾏评价本⽂主题明确, 重点突出, 层次分明, 逻辑性强, 是⼀篇较⾼学术价值的综述.7参考⽂献1 张庆柱. 分⼦药理学. 第1版. 北京: ⾼等教育出版社,2007: 1902 光吉博则, ⾕仁烨. 氧化应激的病理⽣理作⽤. ⽇本医学介绍2007; 28: 150-1523 Day CP, James OF. Steatohepatitis: a tale of two "hits"? 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基于线粒体功能障碍和内质网应激探讨非酒精性脂肪性肝病发病机制

· 综述·基于线粒体功能障碍和内质网应激探讨非酒精性脂肪性肝病发病机制*薛春燕　饶晨怡　吴　玲　黄晓铨　陈世耀　李　锋#复旦大学附属中山医院消化科（200032）摘要　非酒精性脂肪性肝病（NAFLD ）是一种肝脏细胞脂肪异常堆积引起的慢性肝病，其患病率在全世界范围内呈上升趋势，已成为慢性肝病的最常见原因。

NAFLD 发病机制复杂多样，胰岛素抵抗、遗传和表观遗传因素、慢性全身性炎症、线粒体功能障碍、内质网应激、饮食和肠道菌群等均是NAFLD 发生、发展的重要因素。

本文主要讨论了线粒体功能障碍和内质网应激等参与NAFLD 形成的机制，旨在为NAFLD 防治提供新的认识和治疗思路。

关键词　非酒精性脂肪性肝病；　线粒体功能障碍；　内质网应激；　氧化性应激；　非折叠蛋白质应答Exploring Pathogenic Mechanisms of Non⁃alcoholic Fatty Liver Disease Based on Mitochondrial Dysfunction and Endoplasmic Reticulum Stress XUE Chunyan, RAO Chenyi, WU Ling, HUANG Xiaoquan, CHEN Shiyao, LI Feng. Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai (200032)Correspondence to: LI Feng, Email: li.feng2@zs⁃Abstract Non⁃alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by abnormal accumulation offat in the hepatocytes. Its prevalence is rising globally and has become the most common cause of chronic liver disease worldwide. The pathogenesis of NAFLD is multifaceted, involving insulin resistance, genetic and epigenetic factors, chronic systemic inflammation, mitochondrial dysfunction, endoplasmic reticulum stress, diet, gut microbiota, and other significantcontributors. This article primarily delves into the mechanisms of mitochondrial dysfunction and endoplasmic reticulum stress in the development of NAFLD, aiming to provide new insights and therapeutic strategies for NAFLD.Key words Non⁃Alcoholic Fatty Liver Disease;　Mitochondrial Dysfunction;　Endoplasmic Reticulum Stress;　Oxidative Stress;　Unfolded Protein ResponseDOI ： 10.3969/j.issn.1008⁃7125.2022.12.007*基金项目：复旦大学附属中山医院科研基金⁃308（2019ZSFZ09）#本文通信作者， Email: li.feng2@zs⁃非酒精性脂肪性肝病（non⁃alcoholic fatty liver disease, NAFLD ）指在无酒精作用下，以肝内细胞脂肪过度沉积为特征的慢性渐进性肝病。

Y. Geometric stability switch criteria in delay differential systems with delay dependent p

EDOARDO BERETTA† AND YANG KUANG‡ Abstract. In most applications of delay diﬀerential equations in population dynamics, the need of incorporation of time delays is often the result of the existence of some stage structure. Since the through-stage survival rate is often a function of time delays, it is easy to conceive that these models may involve some delay dependent parameters. The presence of such parameters often greatly complicates the task of an analytical study of such models. The main objective of this paper is to provide practical guidelines that combine graphical information with analytical work to eﬀectively study the local stability of some models involving delay dependent parameters. Speciﬁcally, we shall show that the stability of a given steady state is simply determined by the graphs of some functions of τ which can be expressed explicitly and thus can be easily depicted by Maple and other popular software. In fact, for most application problems, we need only look at one such function and locate its zeros. This function often has only two zeros, providing thresholds for stability switches. The common scenario is that as time delay increases, stability changes from stable to unstable to stable, implying that a large delay can be stabilizing. This scenario often contradicts the one provided by similar models with only delay independent parameters. Key words. delay diﬀerential equations, stability switch, characteristic equations, stage structure, population models AMS subject classiﬁcations. 34K18, 34K20, 92D25 PII. S0036141000376086

二乙烯三胺／一氧化氮聚合物诱导的奶牛乳腺上皮细胞损伤模型的建立

二乙烯三胺／一氧化氮聚合物诱导的奶牛乳腺上皮细胞损伤模型的建立郭咏梅;张博綦;石惠宇;闫素梅;史彬林;郭晓宇【摘要】Due to exuberant metabolism, dairy cows produce a great deal of nitric oxide ( NO) during lacta⁃tion, which leads to oxidative stress, and then results in cell toxicity. This experiment was conducted to investi⁃gate the suitable condition for oxidative damage model of bovine mammary epithelial cells ( BMEC) induced by diethylenetriamine/nitric oxide adduct ( DETA/NO) and establish an oxidative damage model. The oxida⁃tive damage model of BMEC was established using DETA/NO as the stimulation source. BMEC was exposed in DETA/NO with different medium concentrations (0, 10, 30, 100, 500 and 1 000μmol/L) and for differ⁃ent times (2, 4, 6, 8, 12 and 24 h). According to the analysis of cell survival rate and antioxidative parame⁃ters, inflammatory cytokines and nitric oxide ( NO) contents, as well as inducible nitric oxide synthase ( iN⁃OS) activity, the suitable treatment time and concentration of DETA/NO were screened. The results showed that the survival rate decreased to 74.27%, and the activitise of superoxide dismutase, catalase and glutathione peroxidase also significantly reduced ( P<0.05) after that BMEC was cultured with 1 000 μmol/L DETA/NO for 6 h. However, the activity of iNOS and the contents of NO, interleukin⁃1, interleukin⁃6, tumor necrosis factor⁃α and malondialdehyde showed opposite changes and increased significantly (P<0.05). In summary, the treatment of 1 000 μmol/L DETA/NO for 6 h issuitable for establishment of oxidative damage model.%泌乳期间的奶牛由于代谢旺盛，往往会产生大量一氧化氮（ NO），诱导发生氧化应激，进而对细胞产生毒害作用。

氧化应激心肌细胞prohibitin表达与分布变化及其生物学意义

3基金项目:国家自然科学基金资助课题(30570753)收稿日期:2006203231;修回日期:2006212228作者简介:任　哲(19792),女,内蒙古包头人,硕士在读,从事应激医学研究。

△通讯作者氧化应激心肌细胞prohibitin 表达与分布变化及其生物学意义3任　哲,钱令嘉△,杨志华(军事医学科学院卫生学环境医学研究所,天津300050)摘要　目的:探讨prohibitin 在氧化应激心肌细胞中的表达与分布变化的特点及其在心肌细胞损伤中的意义。

方法:H 2O 2干预体外培养乳鼠心肌细胞,建立氧化应激心肌细胞损伤模型;采用生物化学法检测细胞培养液中LDH 活性及M TT 实验观察心肌细胞损伤程度;以Western 印迹法检测氧化应激时prohibitin 蛋白表达变化与分布变化;线粒体H +2A TPase 合成活力实验检测线粒体氧化磷酸化功能;流式细胞术检测线粒体跨膜电位。

结果:氧化应激组LDH 活性显著高于对照组,而细胞存活率低于对照组34151%～6515%;线粒体H +2A TPase 合成活力降低60%;氧化应激组线粒体跨膜电位显著低于对照组;心肌细胞prohibitin 表达水平在H 2O 2处理3h 出现升高然后回落到正常水平,线粒体prohibitin 表达水平高于对照组。

结论:氧化应激心肌细胞prohibitin 表达水平代偿性增加并有向线粒体移位的趋势,氧化应激导致心肌细胞线粒体功能障碍。

关键词:　prohibitin ;　氧化应激;　线粒体;　心肌损伤中图分类号:R363文献标识码:A文章编号:100026834(2007)022******* 在一些损伤因素的作用下,细胞内的氧化代谢物增加,或细胞中抗氧化机制不足时,促使活性氧堆积,对细胞产生毒性,这种氧化和抗氧化的不平衡就是氧化应激[1]。

近年来的研究表明,氧化应激是多种不利因素,如运动、心理应激、缺血、缺氧等造成心肌细胞损伤的共同机制。

铁死亡调控机制及其在心血管疾病中的研究进展

㊃综述㊃铁死亡调控机制及其在心血管疾病中的研究进展马赛㊀左庆娟㊀和丽丽㊀张国瑞㊀郭艺芳050051石家庄,河北省人民医院疼痛科(马赛),老年心血管内一科(左庆娟㊁和丽丽㊁郭艺芳);050011石家庄市第三医院心血管内科(张国瑞)通信作者:郭艺芳,电子信箱:yifangguo@DOI:10.3969/j.issn.1007-5410.2023.06.013㊀㊀ʌ摘要ɔ㊀铁死亡是一种近期发现的可调节的程序性细胞坏死方式,涉及铁代谢㊁脂质代谢㊁氨基酸代谢等多种代谢过程,其主要特征为脂质过氧化物生成㊁活性氧超载和谷胱甘肽消耗等㊂研究证实铁死亡参与了多种心血管疾病病理生理过程㊂本文总结了铁死亡调控机制及国内外关于铁死亡在心血管疾病中的研究进展,旨在为心血管疾病的预防和治疗提供新思路㊂ʌ关键词ɔ㊀铁死亡;㊀心力衰竭;㊀心肌病;㊀心血管疾病基金项目:河北省创新能力提升计划项目(199776249D);河北省重点研发计划项目(19277787D)Ferroptosis homeostasis regulation and its research progress in cardiovascular diseases㊀Ma Sai,ZuoQingjuan,He Lili,Zhang Guorui,Guo YifangDepartment of Pain,Hebei General Hospital,Shijiazhuang050051,China(Ma S);Ward1,Department ofGeriatric Cardiology,Hebei General Hospital,Shijiazhuang050051,China(Zuo QJ,He LL,Guo YF); Department of Cardiology,The Third Hospital of Shijiazhuang,Shijiazhuang050011,China(Zhang GR)Corresponding author:Guo Yifang,Email:yifangguo@ʌAbstractɔ㊀Ferroptosis is a recently discovered regulated form of programmed cell death,involvingvarious metabolic processes such as iron metabolism,lipid metabolism,and amino acid metabolism.Its mainfeatures include lipid peroxide generation,oxidative stress,and glutathione depletion.Studies have confirmed the involvement of ferroptosis in various pathophysiological processes of cardiovascular diseases.This article summarizes the regulatory mechanisms of ferroptosis and the research progress on ferroptosis in cardiovascular diseases both domestically and internationally,aiming to provide new insights for the prevention and treatment of cardiovascular diseases.ʌKey wordsɔ㊀Ferroptosis;㊀Heart failure;㊀Cardiomyopathy;㊀Cardiovascular diseaseFund program:Hebei Province Innovation Capability Enhancement Plan Project(199776249D);Hebei Province Key Research and Development Plan Project(19277787D)㊀㊀铁是机体维持正常氧气运输㊁脂质代谢㊁氧化磷酸化等线粒体功能,DNA㊁蛋白质生物合成功能以及其他细胞生物学进程必不可少的微量元素㊂机体内铁稳态维持受多方因素调控,过量的游离铁可与过氧化氢发生芬顿反应,形成羟基自由基及活性氧(reactive oxygen species,ROS),从而造成核酸㊁蛋白质及细胞膜等损伤,水解酶转移,进而引发细胞死亡[1]㊂1981年Sullivan等[2]提出了铁源性心脏病假说,阐明铁超载在心血管疾病进程中发挥着重要作用㊂2012年Dixon等提出了铁死亡的概念,铁死亡是一种铁依赖的程序性细胞坏死方式,以脂质过氧化物生成㊁ROS超载和谷胱甘肽(glutathione,GSH)消耗等为标志性改变,参与了多种心血管疾病病理生理过程㊂但与其他细胞死亡方式不同,铁死亡既可由实验性小分子物质(如埃拉斯汀㊁原癌基因致死性小分子3和磺胺嘧啶等)以及某些药物(如柳氮磺吡啶㊁索拉非尼和青蒿琥酯等)所诱导,亦可被铁抑素1(ferrostatin-1,Fer-1)和脂血抑素1等物质所抑制[3]㊂即铁死亡机制本身具有可调控性,其中正向调节生物分子包括电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)2/3㊁原癌基因㊁还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶㊁p53等,负向调节生物分子包括谷胱甘肽过氧化物酶4 (glutathione peroxidase4,GPX4)㊁溶质载体家族7成员11 (solute carrier family7-member11,SLC7A11/xCT)㊁热休克蛋白B1㊁核因子E2相关因子2(nuclear factor E2related factor 2,Nrf2)等[4]㊂现将铁死亡调控机制及其在心血管疾病中的研究进展做一综述㊂1㊀铁死亡调控机制1.1㊀铁代谢转铁蛋白(transferrin,Tf)与细胞膜上转铁蛋白受体1 (transferrin receptor1,TFR1)结合后可将Fe3+由细胞外内吞至内含体中,经前列腺六跨膜表皮抗原3还原为Fe2+后,在二价金属离子转运体或锌铁转运蛋白的介导下释放到胞质内不稳定铁池中发挥其生理作用,多余的铁以Fe3+形式存储在铁蛋白中或经膜铁转运蛋白(ferroportin-1,FPN1)转至细胞外[5]㊂铁蛋白能够螯合4500个铁原子,从而保护细胞免受游离铁的干扰,维持铁稳态,铁反应元件结合蛋白2作为调控铁代谢的主要转录因子,可抑制其表达[6]㊂FPN1是目前唯一已知的铁输出蛋白[7]㊂在此过程中,TFR1是关键蛋白㊂Manz等[8]在对铁死亡敏感的原癌基因突变细胞的研究中发现,铁死亡细胞的TFR1表达升高,伴铁蛋白重链(ferritin heavy chain,FTH1)㊁铁蛋白轻链(ferritin light chain, FTL)表达降低,铁超载进而诱导铁死亡发生㊂铁调节蛋白与缺氧诱导因子1等均可增强TFR1的表达[9],热休克蛋白B1等可抑制TFR1的表达[10](图1)㊂1.2㊀脂质代谢细胞膜及细胞器膜上磷脂中多不饱和脂肪酸在酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member4,ACSL4)的作用下酰基化,在溶血磷脂酰胆碱酰基转移酶3的作用下酯化,生成含多不饱和脂肪酸磷脂㊂该物质结构不稳定,极易被脂氧合酶氧化为4-羟基壬烯醛(4-hydroxy-trans-2-nonenal,4-HNE)和丙二醛(malondialdehyde, MDA)[11]㊂其中ACSL4及脂氧合酶是限速酶,抑制其表达和生物活性可提高细胞对铁死亡的耐受性(图1)㊂1.3㊀氨基酸代谢在GPX4催化作用下GSH可将脂质过氧化产物转化为无毒的脂肪醇㊂GSH是由谷氨酸㊁半胱氨酸和甘氨酸组成的三肽,半胱氨酸由胱氨酸转化生成,胱氨酸经由xCT从胞外运输至胞内,此转运过程决定了GSH的合成效率㊂研究表明,铁死亡诱导剂埃拉斯汀靶向作用[12],p53抑制xCT转录[13],FTH1缺陷心肌细胞xCT表达下调[14]等,均可促进铁死亡发生㊂GSH还可通过NADPH氧化生成,NADPH作为清除脂质过氧化物所必需的还原剂,是铁死亡敏感性的生物标志物㊂GPX4是众所周知的铁死亡关键调节剂,各种内源性分子(如硒㊁多巴胺㊁维生素E㊁辅酶Q10等)和化学药品(如Fer-1㊁右雷佐生等)通过直接抑制或间接失活GPX4来激发铁死亡[15](图1)㊂1.4㊀甲羟戊酸途径甲羟戊酸途径是脂代谢中重要的生物合成途径,广泛存在于真核生物中,以乙酰辅酶A为原料,以类固醇等为主要产物㊂辅酶Q10作为该途径代谢产物之一,是铁死亡的内源性抑制剂,若消耗增多则可增加细胞对铁死亡的敏感性(图1)㊂1.5㊀铁自噬铁自噬的概念在2014年由Mancias等[16]提出,是指由核受体辅激活因子4介导的将铁蛋白靶向转运至自噬体中降解并释放游离铁的一种选择性自噬方式,是一种保守的细胞分解代谢过程㊂适当的铁自噬可以维持细胞内铁含量稳定,但是过度的铁自噬由于释放出大量游离铁而诱发铁死亡(图1)㊂1.6㊀电压依赖性阴离子通道VDAC是位于线粒体外膜的转运离子和代谢产物的跨膜通道蛋白,具有VDAC1㊁VDAC2和VDAC3三种亚型,可调AA/AdA:花生四烯酸/二十二碳四烯酸;CoA:辅酶A;GSS:谷胱甘肽合成酶;γ-GS:γ-谷胺酰半胱氨酸;GPX4:谷胱甘肽过氧化物酶4;ROS:活性氧;CoQ10:辅酶Q10;MVA:甲羟戊酸:HMG-CoA:3-羟基-3-甲基戊二酸单酰辅酶A;Acetyl-CoA:乙酰辅酶a羧化酶;ACSL4:酰基辅酶A 合成酶长链家族成员4;LPCAT3:溶血卵磷脂酰基转移酶3;ALOX15:15-脂氧合酶;VDAC:电压依赖性阴离子通道;NOX:NADPH氧化酶; DMT1:二价金属转运体1;ZIP8/14:锌-铁调节蛋白家族8/14;PCBP1/2:分子伴侣多聚结合蛋白1/2;LC3:微管结合蛋白1轻链3;NCOA4:核受体辅激活剂4图1㊀铁死亡调控机制模式图节线粒体代谢产能过程,参与细胞生存和死亡信号调控㊂有研究证明,铁死亡抑制剂脂血抑素1可通过下调VDAC1表达水平来抑制铁死亡[17]㊂铁死亡诱导剂埃拉斯汀可作用于VDAC2及VDAC3,使得线粒体通透性增加,进而诱导线粒体功能障碍和细胞铁死亡的发生[18]㊂敲低VDAC2和VDAC3基因可抑制铁死亡发生,但过表达VDAC2和VDAC3并没有显著诱发铁死亡,具体机制有待进一步研究[19](图1)㊂2　铁死亡在心血管疾病中的研究进展2.1㊀铁死亡与心力衰竭越来越多的证据表明,铁死亡是心力衰竭(heart failure, HF)病理生理机制中不可或缺的重要环节㊂Lapenna等[20]研究发现,老龄兔心脏组织中铁含量㊁氧化应激标记物等均明显高于正常成年兔,表明铁代谢可能与衰老㊁功能障碍等病理生理相关㊂Liu等[21]在体内通过主动脉缩窄术建立压力超负荷诱导大鼠HF模型,在体外培养经埃拉斯汀或异丙肾上腺素处理的H9c2心肌细胞,结果发现两者均可观察到以铁超载及脂质过氧化物生成增多为特征的铁死亡过程㊂在通过高盐饲料喂养盐敏感大鼠建立的射血分数保留HF模型中,铁死亡相关指标TFR1㊁ACSL4㊁4-HNE表达明显升高, FTH1㊁xCT表达明显降低,提示铁死亡是射血分数保留HF 发病机制之一[22]㊂为了探索铁死亡在HF中的调控机制, Zheng等[23]对GEO公共数据库进行了分析并发现,M2型巨噬细胞外泌体传递CircSnx12是参与铁代谢相关铁死亡的关键调节因子,可通过与miR-224-5p相互作用实现靶向调节与铁死亡相关的FTH1基因,进而调控诱导HF发生的铁死亡机制,所以环状RNA可能成为治疗HF的前瞻性靶标和新型药物研发的突破口㊂另外,阿尔茨海默病小鼠模型具有心脏结构和功能异常等特点,伴ACSL4㊁核受体辅激活因子4表达上调,xCT㊁GPX4表达下调,即存在脂质过氧化㊁氧化应激水平升高,GSH代谢异常以及铁死亡的激活㊂线粒体醛脱氢酶缺陷与阿尔茨海默病患者心功能不全相关,阿尔茨海默病小鼠铁死亡相关指标变化可通过线粒体醛脱氢酶转基因得以逆转,心脏结构和功能亦能得以改善[24]㊂这些发现表明铁死亡与HF㊁心功能不全密切相关㊂2.2㊀铁死亡与心肌缺血/再灌注损伤迄今为止,血运重建仍然是缺血性心肌病最有效的治疗方法,但心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤不可避免,并会造成多种类型细胞死亡,包括铁死亡㊂我们知道,多元醇途径参与了I/R损伤诱导的心肌梗死,Tang等[25]研究发现抑制多元醇途径关键酶可减弱I/R损伤介导的缺氧诱导因子1α㊁Tf㊁TFR1和细胞内铁含量的增加,减少心脏梗死区域面积,过表达多元醇途径关键酶可激活缺氧诱导因子1α,诱导TFR1的表达和铁的积累,加剧脂质过氧化和氧化损伤,故多元醇途径参与调节了I/R损伤介导的铁死亡㊂还有研究发现,泛素特异性蛋白酶7在心脏I/R损伤期间通过激活p53/TFR1通路参与调节铁死亡[26]㊂铁死亡可通过TLR4/Trif/Type1IFN信号通路促进中性粒细胞向冠状动脉内皮细胞黏附以及向受损心肌募集,造成坏死性炎症,加重心脏移植后心肌损伤[27]㊂铁死亡抑制剂可减轻心肌I/R损伤[28]㊂Gao等[29]研究证明细胞内谷氨酰胺分解代谢在铁死亡机制中发挥了关键作用,抑制谷氨酰胺代谢可抑制铁死亡,改善离体心脏模型I/R损伤,为I/R损伤治疗提供了新策略㊂亦有研究表明铁死亡不是发生在心肌缺血阶段,而是发生在心肌再灌注阶段[30],可能与这一阶段氧化的磷脂酰胆碱生成相关[31],为I/R损伤甚至心肌梗死患者建立精准医疗奠定了基础㊂同时,雷帕霉素可通过其靶标哺乳动物雷帕霉素靶蛋白基因过表达抑制铁死亡[32],进而改善心肌缺血,减少I/R损伤㊂在糖尿病心肌I/R损伤模型中,抑制铁死亡可减少内质网应激相关性心肌损伤[33]㊂故抑制铁死亡是I/R心肌损伤治疗的有效策略㊂2.3㊀铁死亡与蒽环类药物心脏毒性多柔比星(doxorubicin,DOX)是临床上常用的蒽环类化疗药物,具有心脏毒性,可造成DOX相关性心肌病,限制其临床应用并产生不良预后[34]㊂有研究表明,高铁基因可通过调节心肌细胞铁沉积来增加DOX诱导的心脏毒性的易感性[35]㊂Fang等[28]研究表明铁死亡机制在DOX诱导小鼠心肌病模型中发挥了关键作用,经Fer-1干预后可显著改善小鼠心肌病变及死亡率㊂通过全转录组测序发现DOX可通过Nrf2上调血红素加氧酶1表达,降解血红素铁,进而诱发铁死亡,且证实铁超载和脂质过氧化主要定位于心肌细胞线粒体,更加明确了线粒体损伤在DOX心肌损伤中的因果关系㊂Tadokoro等[36]同样证实线粒体依赖性铁死亡在DOX心肌损伤进展中的关键作用㊂Liu等[37]应用RNA测序方法发现,在DOX干预后小鼠心脏中,脂代谢途径中的Acot1基因明显下调,经Fer-1处理后部分逆转,且Acot1过表达可抑制铁死亡,进而实现心脏获益㊂因此,Acot1可能是通过抑制铁死亡来预防和治疗DIC的潜在靶点㊂2.4㊀铁死亡与糖尿病性心肌病糖尿病是心血管疾病常见的合并症,可增加心脏对I/R 损伤的易感性和糖尿病性心肌病(diabetic cardiomyopathy, DCM)的发生风险㊂氧化应激已被证实为DCM心脏结构和功能改变的重要因素㊂2022年发表的一项研究首次报道了铁死亡在DCM发病机制中起着至关重要的作用,萝卜硫素可通过AMPK激活NrF2,上调铁蛋白和xCT水平进而抑制铁死亡过程,改善DCM小鼠心脏病变[38]㊂2.5㊀铁死亡与败血症相关心脏损伤败血症致心脏损伤的发生率和死亡率均较高㊂盲肠结扎和穿刺是研究败血症最常用的造模方法,该模型可增加心脏铁含量和脂质过氧化水平,并降低GSH含量和GPX4表达水平,提示败血症引起的心脏损伤可能涉及铁死亡机制,而右美地托咪定可通过抑制铁死亡改善败血症引起的心脏损伤[39]㊂此外,铁死亡已被证明在脂多糖诱导的败血性心肌病模型中起重要作用[40]㊂2.6㊀铁死亡与心律失常目前,全球正面临新型冠状病毒(COVID-19)大流行,而COVID-19感染会导致小鼠心脏起搏细胞功能障碍并诱导铁死亡,酪氨酸激酶抑制剂伊马替尼和铁螯合剂去铁胺可阻断病毒感染和铁死亡相关过程,可能是改善病毒感染后心律失常的潜在机制[41]㊂另一项关于小鼠的研究亦表明铁死亡与心律失常相关,频繁过量饮酒会诱发铁死亡,并增加心房颤动发生率,而铁死亡抑制剂可部分逆转过量饮酒引起的不良反应[42]㊂2.7㊀铁死亡与心肌纤维化Wang等[43]发现在主动脉缩窄致压力超负荷HF模型中,HF晚期心肌纤维化主要由MLK3调节的JNK/p53信号通路介导的铁死亡引起,miR-351基因表达上调可抑制MLK3表达,进而改善心肌纤维化及心功能㊂xCT基因缺失亦可加剧血管紧张素Ⅱ介导的心肌纤维化和功能障碍,为铁死亡参与心肌纤维化提供了证据[44]㊂2.8㊀铁死亡与内皮功能障碍㊁动脉粥样硬化内皮功能障碍是糖尿病标志性病变,是糖尿病心血管并发症的起始和关键因素㊂有研究表明,在糖尿病db/db小鼠的主动脉内皮中观察到xCT表达下降㊁铁积累和脂质过氧化物生成增多以及去内皮化改变,且高糖和白细胞介素-1β可通过p53-xCT-GSH途径诱导静脉内皮细胞发生铁死亡[45]㊂高脂饮食可诱导ApoE-/-小鼠形成动脉粥样硬化,Bai等[46]发现在动脉粥样硬化血管中铁死亡相关蛋白明显上调,Fer-1干预后可部分抑制铁超载和脂质过氧化,并显著降低了xCT 和GPX4的表达水平,同时抑制铁死亡可改善主动脉内皮细胞的活力㊂另一项关于不同严重程度动脉粥样硬化的尸检报告数据表明,重度动脉粥样硬化患者的冠状动脉标本中前列腺素内过氧化物合成酶2㊁ACSL4表达上调,GPX4表达下调[47]㊂故铁死亡与内皮功能障碍和动脉粥样硬化病理学相关,并参与其发生及发展㊂2.9㊀铁死亡与其他镰状细胞病是一种以溶血㊁器官缺血和心血管并发症等为特征的遗传性疾病,该疾病小鼠血红素水平升高,导致心脏铁超载㊁脂质过氧化和铁死亡,抑制铁死亡减轻了与镰状细胞病相关的心肌病[48]㊂有研究发现,吸烟与腹主动脉瘤的发生㊁发展和破裂显著相关[49]㊂Sampilvanjil等[50]首次证实香烟提取物可引起血管平滑肌细胞发生铁死亡,并可能通过铁死亡机制诱导主动脉瘤或夹层㊂此外,Ma等[51]首次证实铁死亡是血管钙化发生的新机制㊂3㊀铁死亡抑制剂在心血管疾病中的应用由于铁死亡机制是治疗和预防心血管疾病的潜在靶点,铁死亡抑制剂在心血管疾病中的应用逐渐增多㊂UAMC-3203作为比Fer-1更稳定和有效的铁螯合剂,能更好地预防动物模型中铁死亡驱动的多器官功能障碍,可能更适合临床试验推广[52]㊂针对脂血抑素1的研究相对较少,但具有与Fer-1相似的保护作用,可显著减少棕榈酸诱导的心脏损伤[53]㊂抗氧化剂N-乙酰半胱氨酸可提高半胱氨酸的生物利用度,其抗铁死亡作用已得到证实[54],并可减少糖尿病大鼠心肌I/R损伤,为临床应用提供了理论基础[55]㊂去铁酮㊁化合物968在心脏I/R损伤中亦发挥了心脏保护作用[56,29]㊂右雷佐生是乙二胺四乙酸环状衍生物,是唯一一个被美国食品药品监督管理局批准的用来预防DOX相关性心肌病的铁螯合剂,可以直接进入心肌细胞线粒体并减少铁积累[57]㊂人脐带血中间充质干细胞的外泌体可通过抑制急性心肌梗死小鼠模型中的铁死亡来实现心脏保护作用[58]㊂卡格列净㊁葛根素㊁阿托伐他汀可抑制铁死亡改善心功能[21-22,59],为HF提供了潜在的治疗策略,而氧化锌纳米粒子可诱导铁死亡,促进内皮损伤发生㊂此外,常用的心脏药物可能具有未发现的抗铁死亡活性,如卡维地洛已被证明可以抑制铁死亡,而与其对β-肾上腺素能受体的作用无关㊂尽管抑制铁死亡已在多种动物模型中显示出心脏获益,但迄今为止尚未进行使用铁死亡抑制剂治疗心血管疾病的临床试验㊂4㊀小结铁死亡作为最近发现的程序性细胞死亡类型,是心血管疾病发生发展的关键机制之一㊂近年来日益引起人们的重视,相关研究不断增加㊂本文总结了铁死亡相关调控机制及其在心血管疾病中的研究进展和应用㊂但铁死亡研究领域的一些关键机制尚待研究和验证,需要我们进一步探索去揭示铁死亡的精细分子机制,从而为靶向铁死亡以减少主要不良心血管事件以及防治心血管疾病提供更加充分的理论依据,为预防和治疗心血管疾病提供新的生物标志物和前瞻性靶标㊂利益冲突:无参㊀考㊀文㊀献[1]Ward 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[58]Song Y,Wang B,Zhu X,et al.Human umbilical cord blood-derived MSCs exosome attenuate myocardial injury by inhibitingferroptosis in acute myocardial infarction mice[J].Cell BiolToxicol,2021,37(1):51-64.DOI:10.1007/s10565-020-09530-8.[59]Ning D,Yang X,Wang T,et al.Atorvastatin treatmentameliorates cardiac function and remodeling induced byisoproterenol attack through mitigation of ferroptosis[J].BiochemBiophys Res Commun,2021,574:39-47.DOI:10.1016/j.bbrc.2021.08.017.(收稿日期:2023-01-08)(本文编辑:李鹏)㊃消息㊃欢迎关注和投稿Aging Medicine㊀㊀Aging Medicine(ISSN:2475-0360)由北京医院国家老年医学中心主办,中华医学会老年医学分会学术指导,由国际著名出版集团Wiley出版,于2018年5月正式创刊发行㊂杂志是中华医学会老年医学分会的唯一一本面向国内外的官方英文国际刊物,2020年起先后被Pubmed Central(PMC)㊁DOAJ㊁EMBASE㊁EBSCOhost㊁Ovid㊁ProQuest㊁Reprints Desk㊁CCC和Scopus等数据库收录㊂2023年被ESCI数据库收录, 2024年入选‘科技期刊世界影响力指数(WJCI)报告“,影响因子为2.716,WAJCI为0.770,WI为0.181,WJCI为0.951, WJCI学科排名为56/90,位居医学综合类科技期刊Q3区㊂Aging Medicine办刊宗旨是贯彻党和国家的卫生方针政策,贯彻理论与实践㊁普及与提高相结合的方针㊂杂志现为双月刊,主要刊登老年人相关疾病的预防㊁临床诊断和治疗㊁康复㊁照护㊁心理及社会等方面的文章,旨在推动老年医学的发展,应对全球人口老龄化的挑战,积极打造融学术性㊁先进性㊁实用性于一体的国际交流平台,力争早日跻身国际化老年医学一流期刊行列,在国际舞台发出中国老年医学的最强音㊂Aging Medicine栏目包括指南与共识㊁专家论坛㊁临床研究㊁基础研究㊁流行病学调查㊁荟萃分析㊁疑难病例分析㊁病例报告㊁综述㊁社论㊁述评㊁来信等,设立衰老与长寿专栏㊁人工智能与老年医学专栏㊁老年医学公卫和流病专栏㊂期刊论文执行严格的同行评议,发表的全文免费开放获取(OpenAccess,OA),确保每篇文章可以第一时间被全世界的老年医学从业人员免费阅读㊁下载和分享㊂Aging Medicine正在进行2024年的专题约稿工作,约稿内容包括衰老与长寿㊁老年糖尿病㊁老年肌少症㊁老年脑血管疾病㊁老年心血管疾病㊁人工智能㊁老年营养㊁老年康复护理㊁老年心理健康等,热烈欢迎各位国内外医学同道积极投稿㊂欢迎访问Aging Medicine杂志官方网站(https:// /journal/24750360)进行投稿和获得电子版全文㊂联系方式通信地址:北京市东城区大佛寺东街6号院105室邮政编码:100010联系电话*************-8889电子信箱:agmeditor@投稿地址:https:///journal/24750360官方微信:Aging Medicine杂志。

parasitic oxidation学术意思

Parasitic oxidation refers to the unwanted oxidation of a substance or material, often resulting in degradation or deterioration of its properties. This phenomenon ismonly encountered in various fields, including materials science, chemistry, and industry. Understanding the mechanisms and implications of parasitic oxidation is crucial for the development of effective prevention and mitigation strategies.1. Definition and Mechanisms of Parasitic OxidationParasitic oxidation can be defined as the undesired reaction of a material with oxygen or other oxidizing agents, leading to the formation of oxides or other oxidized products. This process can occur through various mechanisms, including:1.1. Atmospheric ExposureExposure to ambient 本人r or oxygen can lead to the onset of parasitic oxidation in many materials, especially metals and alloys. The presence of moisture, pollutants, or other reactive species in the atmosphere can further accelerate this process.1.2. Catalytic EffectsCert本人n catalysts or impurities present in a material can promote the initiation and progression of parasitic oxidation.This ismonly observed in heterogeneous catalysis and industrial processes involving reactive substances.1.3. Thermal ActivationElevated temperatures can enhance the reactivity of materials with oxygen, leading to accelerated parasitic oxidation. This is particularly relevant in high-temperature applications such asbustion, metallurgy, and thermal processing.2. Impact and Consequences of Parasitic OxidationThe consequences of parasitic oxidation can be significant and detrimental to the performance and reliability of affected materials. Some of the key impacts include:2.1. Degradation of Mechanical PropertiesThe formation of oxides and other oxidized species canpromise the mechanical strength, ductility, and toughness of materials. This can lead to structural f本人lure, fractures, or reduced lifespan in engineeringponents.2.2. Loss of Functional PropertiesMany functional materials, such as semiconductors, catalysts, and coatings, can suffer a decline in their performance due toparasitic oxidation. This may result in diminished efficiency or effectiveness in their intended applications.2.3. Environmental and Health ConcernsIn some cases, the by-products of parasitic oxidation can pose environmental or health hazards. This is particularly relevant in the context of 本人r pollution, hazardous waste, and chemical safety.3. Prevention and Control Strategies for Parasitic Oxidation Given the widespread impact of parasitic oxidation, it is essential to develop effective strategies for its prevention and control. Some key approaches include:3.1. Material Selection and DesignChoosing materials with inherent resistance to oxidation, or employing protective coatings and surface treatments, can help mitigate the effects of parasitic oxidation. This is amon strategy in the development of corrosion-resistant alloys and high-temperature ceramics.3.2. Environmental ControlLimiting the exposure of materials to harsh or reactiveenvironments, such as controlling the humidity, temperature, orposition of the atmosphere, can reduce the likelihood of parasitic oxidation. This is relevant in storage, transportation, and industrial processing operations.3.3. Inhibitor AdditivesThe use of specific chemical additives or inhibitors can suppress the occurrence of parasitic oxidation in cert本人n materials. This approach ismonly used in the formulation of anti-corrosion agents and oxidation-resistantpounds.4. Research and Development in Parasitic Oxidation Continued research and development efforts are essential for advancing the understanding and management of parasitic oxidation. This includes:4.1. Fundamental StudiesInvestigating the mechanisms, kinetics, and thermodynamics of parasitic oxidation at the molecular and atomic levels is critical for developing predictive models and theoretical frameworks.4.2. Advanced Characterization TechniquesEmploying state-of-the-art analytical tools, such as electronmicroscopy, spectroscopy, and surface science techniques, can provide valuable insights into the behavior and evolution of materials undergoing parasitic oxidation.4.3. Multidisciplinary CollaborationCollaboration between researchers from diverse fields, including chemistry, physics, materials science, and engineering, can foster new perspectives and innovative approaches to addressing the challenges of parasitic oxidation.In conclusion, parasitic oxidation represents a pervasive and consequential phenomenon that warrants attention and action across various scientific and technological dom本人ns. By g本人ning a deeper understanding of its mechanisms, impacts, and control strategies, researchers and practitioners can work towards mitigating the adverse effects of parasitic oxidation and enhancing the performance and longevity of materials and products.。

Oxidative stress, antioxidants and stress tolerance

1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(02)02312-9Ron MittlerDept of Botany, Plant Sciences Institute,353Bessey Hall, Iowa State University, Ames,IA 50011, USA.e-mail: rmittler@Reactive oxygen intermediates (ROIs) are partially reduced forms of atmospheric oxygen (O 2). Theytypically result from the excitation of O 2to form singlet oxygen (O 21) or from the transfer of one, two or three electrons to O 2to form, respectively , a superoxide radical (O 2−), hydrogen peroxide (H 2O 2) or a hydroxyl radical (HO −). In contrast to atmospheric oxygen, ROIs are capable of unrestricted oxidation of various cellular components and can lead to the oxidative destruction of the cell [1–4].Production of ROIs in cellsThere are many potential sources of ROIs in plants (Table 1). Some are reactions involved in normalmetabolism, such as photosynthesis and respiration.These are in line with the traditional concept,considering ROIs as unavoidable byproducts ofaerobic metabolism [1]. Other sources of ROIs belong to pathways enhanced during abiotic stresses, such as glycolate oxidase in peroxisomes during photorespiration. However, in recent years, new sources of ROIs have been identified in plants,including NADPH oxidases, amine oxidases and cell-wall-bound peroxidases. These are tightlyregulated and participate in the production of ROIs during processes such as programmed cell death (PCD) and pathogen defense [2,4,5].Whereas, under normal growth conditions, the production of ROIs in cells is low (240 µM s −1O 2−and a steady-state level of 0.5 µM H 2O 2in chloroplasts) [6],many stresses that disrupt the cellular homeostasis of cells enhance the production of ROIs (240–720 µM s −1O 2−and a steady-state level of 5–15 µM H 2O 2) [6].These include drought stress and desiccation, salt stress, chilling, heat shock, heavy metals, ultravioletradiation, air pollutants such as ozone and SO 2,mechanical stress, nutrient deprivation, pathogen attack and high light stress [2,7–10]. The production of ROIs during these stresses results from pathways such as photorespiration, from the photosynthetic apparatus and from mitochondrial respiration. In addition, pathogens and wounding or environmental stresses (e.g. drought or osmotic stress) have been shown to trigger the active production of ROIs byNADPH oxidases [4,11–13]. The enhanced production of ROIs during stress can pose a threat to cells but it is also thought that ROIs act as signals for the activation of stress-response and defense pathways [9,14]. Thus, ROIs can be viewed as cellular indicators of stress and as secondary messengers involved in the stress-response signal transduction pathway .Although the steady-state level of ROIs can be used by plants to monitor their intracellular level of stress, this level has to be kept under tight control because over-accumulation of ROIs can result in cell death [1–4]. ROI-induced cell death can result from oxidative processes such as membrane lipidperoxidation, protein oxidation, enzyme inhibition and DNA and RNA damage (the traditional concept).Alternatively , enhanced levels of ROIs can activate a PCD pathway , as was recently demonstrated by the inhibition of oxidative stress (paraquat)-induced cell death in tobacco by anti-apoptotic genes [15].Because ROIs are toxic but also participate in signaling events, plant cells require at least two different mechanisms to regulate their intracellular ROI concentrations by scavenging of ROIs: one that will enable the fine modulation of low levels of ROIs for signaling purposes, and one that will enable the detoxification of excess ROIs, especially during stress.In addition, the types of ROIs produced and the balance between the steady-state levels of different ROIs can also be important. These are determined by theinterplay between different ROI-producing and ROI-scavenging mechanisms, and can change drastically depending upon the physiological condition of the plant and the integration of different environmental,developmental and biochemical stimuli.Scavenging of ROIs in cellsMajor ROI-scavenging mechanisms of plants include superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) [1,7,16] (Table 1). The balance between SOD and APX or CAT activities in cells is crucial for determining the steady-state level ofT raditionally,reactive oxygen intermediates (ROIs) were considered to be toxic by-products of aerobic metabolism,which were disposed of using antioxidants.However,in recent years,it has become apparent that plants actively produce ROIs as signaling molecules to control processes such as programmed cell death,abiotic stress responses,pathogen defense and systemic signaling.Recent advances including microarray studies and the development of mutants with altered ROI-scavenging mechanisms provide new insights into how the steady-state level of ROIs are controlled in cells.In addition,key steps of the signal transduction pathway that senses ROIs in plants have been identified.These raise several intriguing questions about the relationships between ROI signaling,ROI stress and the production and scavenging of ROIs in the different cellular compartments.Published online: 12 August 2002Oxidative stress,antioxidants and stress toleranceRon Mittlersuperoxide radicals and hydrogen peroxide [17]. This balance, together with sequestering of metal ions, is thought to be important to prevent the formation of the highly toxic hydroxyl radical via the metal-dependent Haber–Weiss or the Fenton reactions [1]. The different affinities of APX (µM range) and CAT (m M range) for H 2O 2suggest that they belong to two differentclasses of H 2O 2responsible for the fine modulation of ROIs forsignaling, whereas CAT might be responsible for or the removal of excess ROIs during stress.The major ROI-scavenging pathways of plants (Fig. 1) include SOD, found in almost all cellular compartments, the water–water cycle inchloroplasts (Fig. 1a), the ascorbate–glutathione cycle in chloroplasts, cytosol, mitochondria, apoplast and peroxisomes (Fig. 1b), glutathione peroxidase (GPX; Fig. 1c), and CAT in peroxisomes (Fig. 1d). The finding of the ascorbate–glutathione cycle in almost all cellular compartments tested to date, as well as the high affinity of APX for H 2O 2, suggests that this cycle plays a crucial role in controlling the level ofROIs in these compartments. By contrast, CAT is only present in peroxisomes, but it is indispensable for ROI detoxification during stress, when high levels of ROIs are produced [16]. In addition, oxidative stress causes the proliferation of peroxisomes [18]. Drawingupon the model for bacteria [19], a dense population of peroxisomes might be highly efficient in scavenging of ROIs, especially H 2O 2, which diffuses into peroxisomes from the cytosol.The water–water cycle (Fig. 1a) draws its reducing energy directly from the photosynthetic apparatus [3]. Thus, this cycle appears to be autonomous with respect to its energy supply . However, the source of reducing energy for ROI scavenging by theascorbate–glutathione cycle (Fig. 1b) during normal metabolism and particularly during stress, when the photosynthetic apparatus might be suppressed or damaged, is not entirely clear. In animals and yeast,the pentose-phosphate pathway is the main source of NADPH for ROI removal [20,21]. Because CAT does not require a supply of reducing equivalents for its function, it might be insensitive to the redox status of cells and its function might not be affected during stress, unlike the other mechanisms (Fig. 1).Antioxidants such as ascorbic acid and glutathione,which are found at high concentrations in chloroplasts and other cellular compartments (5–20 m M ascorbic acid and 1–5 m M glutathione) are crucial for plant defense against oxidative stress [8]. Consequently ,both mutants with suppressed ascorbic acid levels [22] and transgenic plants with altered content of glutathione [23] are hypersensitive to stress conditions. It is generally believed that maintaining a high reduced per oxidized ratio of ascorbic acid and glutathione is essential for the proper scavenging of ROIs in cells. This ratio is maintained by glutathione reductase (GR),monodehydroascorbate reductase (MDAR) anddehydroascorbate reductase (DHAR) using NADPH as reducing power (Fig. 1) [3,8]. In addition, the overall balance between different antioxidants has to betightly controlled. Enhanced glutathione biosynthesis in chloroplasts can result in oxidative damage to cells rather than their protection, possibly by altering the overall redox state of chloroplasts [23]. It has also been suggested that the oxidized:reduced ratio of the different antioxidants can serve as a signal for the modulation of ROI-scavenging mechanisms [24].Avoiding ROI productionAvoiding ROI production might be as important as active scavenging of ROIs. Because many abiotic stress conditions are accompanied by an enhanced rate of ROI production, avoiding or alleviating the effects of stresses such as drought or high light on plant metabolism will reduce the risk of ROI production. Mechanisms that might reduce ROI production during stress (Table 1) include:(1)anatomical adaptations such as leaf movement and curling, development of a refracting epidermis and hiding of stomata in specialized structures;(2)physiological adaptations such as C 4and CAM metabolism; and (3) molecular mechanisms that rearrange the photosynthetic apparatus and its antennae in accordance with light quality andintensity or completely suppress photosynthesis. Bybalancing the amount of light energy absorbed by the plant with the availability of CO 2, these mechanisms might represent an attempt to avoid the over-reduction of the photosynthetic apparatus and the transfer of electrons to O 2rather than for CO 2fixation.ROI production can also be decreased by the alternative channeling of electrons in the electron-transport chains of the chloroplasts and mitochondria by a group of enzymes called alternative oxidases (AOXs). AOXs can divert electrons flowing through electron-transport chains and use them to reduce O 2to water (Fig. 2). Thus, they decrease ROI production by two mechanisms: they prevent electrons fromreducing O 2into O 2−and they reduce the overall level of O 2, the substrate for ROI production, in theorganelle. Decreasing the amount of mitochondrial AOX increases the sensitivity of plants to oxidative stress [25]. In addition, chloroplast AOX is induced in transgenic plants that lack APX and/or CAT, and in normal plants in response to high light [50].Production and scavenging of ROIs in different cellular compartmentsRecent manipulations of ROI-scavenging pathways in different cellular compartments suggest someintriguing possibilities. For years, the chloroplast was considered to be the main source of ROI production in cells and consequently one of the main targets for ROI damage during stress. However, it has recently been suggested that the chloroplast is not as sensitive to ROI damage as previously thought [26]. The mitochondrion is another cellular site of ROI production. However,recent studies suggest that the mitochondrion is also a key regulator of PCD in plants and that enhanced ROIs levels at the mitochondria can trigger PCD [27].Both the mitochondrion and the chloroplast contain ROI-scavenging mechanisms. By contrast, little is known about the ROI-scavenging properties of the nucleus, which might contain redox-sensitivetranscription factors [28]. Because H 2O 2can diffuse through aquaporins [29], ROIs produced at a specific cellular site (e.g. the chloroplast during stress or the apoplast during pathogen attack) can affect other cellular compartments, overwhelm theirROI-scavenging capabilities and alter the pattern of gene expression during stress, pathogen infection or PCD. In support of this assumption, stresses that result in the enhanced production of ROIs at thechloroplast induce cytosolic and not chloroplastic ROI-scavenging mechanisms [24,30], and ROI production at the apoplast induces the production of pathogenesis-response proteins [4]. Because the plant mitochondria and nuclei are involved in the activation of PCD [27],the level of ROIs that reaches these compartments during stress or pathogen challenge needs to be tightly controlled to prevent abnormal PCD activation. The cytosol, with its ascorbate–glutathione cycle, and the peroxisomes, with CAT, might therefore act as a buffer zone to control the overall level of ROIs that reaches different cellular compartments during stress and normal metabolism.The importance of peroxisomes in ROI metabolism is beginning to gain recognition [31]. Peroxisomes are not only the site of ROI detoxification by CAT but also the site of ROI production by glycolate oxidase and fatty acid β-oxidation. In addition, peroxisomes might be one of the cellular sites for nitric oxide (NO)biosynthesis [31]. In animal cells, NO activates fatty acid β-oxidation and enhances the production of ROIs in cells. However, although NO has been shown to be involved in ROI-induced cell death in plants [32] and NO is known to be a key regulator of pathogen responses [5], little is known about how NO isinvolved in the response of plants to abiotic stresses.Redundancy in ROI-scavenging mechanismsSome of the complex relationships between the different ROI-scavenging and ROI-producingmechanisms have been revealed in transgenic plants with suppressed production of ROI-detoxifying mechanisms. Thus, plants with suppressed APXFig. 1.Pathways for reactive oxygen intermediate (ROI)scavenging in plants.(a)The water –water cycle.(b) The ascorbate –glutathione cycle. (c) The glutathione peroxidase (GPX) cycle. (d) Catalase (CAT). Superoxidedismutase (SOD) acts as the first line of defense converting O 2−into H 2O 2.Ascorbate peroxidases (APX), GPX and CAT then detoxify H 2O 2. In contrast to CAT (d), APX and GPX require an ascorbate (AsA)and/or a glutathione (GSH)regenerating cycle (a –c).This cycle uses electrons directly from thephotosynthetic apparatus (a) or NAD(P)H (b,c) as reducing power. ROIs are indicated in red,antioxidants in blue and ROI-scavenging enzymes in green. Abbreviations:DHA, dehydroascorbate;DHAR, DHA reductase;Fd,ferredoxin; GR,glutathione reductase;GSSG, oxidized glutathione; MDA,monodehydroascorbate;MDAR, MDA reductase;PSI,photosystem I; tAPX,thylakoid-bound APX.production induce SOD, CAT and GR to compensate for the loss of APX, whereas plants with suppressed CAT production induce APX, GPX and mitochondrial AOX [16,50]. CAT and APX are not completely redundant because they do not compensate for the lack of each other, as shown by the sensitivity of plantswith reduced APX or CAT levels to environmental stresses and pathogen attack [33]. Interestingly ,plants with suppressed APX and CAT appeared, at least under a defined set of environmental conditions,to be less sensitive to oxidative stress than plants with lowered APX or CAT levels. These plants had reduced photosynthetic activity , enhanced chloroplastic AOX production and enhanced expression of genes of the oxidative and reductive pentose-phosphate pathway and MDAR, possibly to avoid ROI production as well as to enhance the non-enzymatic detoxification of H 2O 2by ascorbic acid [50].ROIs at the interface between biotic and abiotic stressesROIs play a central role in the defense of plants against pathogen attack. During this response, ROIs are produced by plant cells via the enhanced enzymatic activity of plasma-membrane-bound NADPH oxidases,cell-wall-bound peroxidases and amine oxidases in the apoplast [4,5]. H 2O 2produced during this response (up to 15 µM ; directly or as a result of superoxide dismutation) is thought to diffuse into cells and,together with salicylic acid (SA) and NO [34], toactivate many of the plant defenses, including PCD [35].The activity of APX and CAT is suppressed during this response by the plant hormones SA and NO [34], the production of APX is post-transcriptionally suppressed [36] and the production of CAT is downregulated at the level of steady-state mRNA [37]. Thus, the plantsimultaneously produces more ROIs and at the same time diminishes its own capacity to scavenge H 2O 2,resulting in the over-accumulation of ROIs and the activation of PCD. The suppression of ROI-scavenging mechanisms together with the synthesis of NO appears to be crucial for the activation of PCD because, in their absence, increased ROI production at the apoplast does not result in the induction of PCD [32,33].The role ROIs play during PCD appears, therefore,to be opposite to the role they play during abiotic stresses, during which ROIs induce ROI-scavenging mechanisms such as APX and CAT that decrease the steady-state level of ROIs in cells (Fig. 3). The differences in the function of ROIs between biotic and abiotic stresses might result from the action of hormones such as SA and NO, from cross-talkbetween different signaling pathways (Fig. 4) or from differences in the steady-state level of ROIs produced during the different stresses. The apparent conflict in ROI metabolism between biotic and abioticstresses (Fig. 3) raises the question of how the plant manipulates its rate of ROI production and ROIscavenging when it comes under biotic attack during an abiotic stress. In support of the possible existence of such a conflict, tobacco plants that were previously subjected to oxidative stress (and consequently had a higher level of antioxidative enzymes) had a reduced rate of PCD compared with unstressed control plants [33]. In addition, plants that overproduce CAT have a decreased resistance to pathogen infection [38].Fig. 2.Involvement of alternative oxidase (AOX) in reactive oxygen intermediate (ROI) avoidance.In both the mitochondrial electron-transport chain (a) and the chloroplast electron-transport chain (b),AOX diverts electrons that can be used to reduce O 2into O 2−and uses these electrons to reduce O 2to H 2O. In addition, AOX reduces the overall level of O 2, the substrate for ROI production, in theorganelle. AOX is indicated in yellow and the different components of the electron-transport chain are indicated in red, green or gray. Abbreviations: Cyt-b 6f , cytochrome b 6f ; Cyt-c, cytochrome c ;Fd,ferredoxin; PC, plastocyanin; PSI, PSII, photosystems I and II.Fig. 3.Differences in the steady-state levels of reactive oxygen intermediates (ROI) during biotic stress and abiotic stress. Biotic stress (a) results in the activation of NADPH oxidase and the suppression of ascorbate peroxidase (APX) and catalase (CAT). This leads to the over-accumulation of ROI and the activation of defense mechanisms. Abiotic stress (b) enhances ROI production by chloroplasts and mitochondria. However, by inducing ROI-scavenging enzymes such as APX and CAT , it reduces ROI levels. The question mark indicates that little is known about the regulation of ROI metabolism during a combination of biotic and abiotic stresses. Chloroplasts are indicated in green, mitochondria in gray and the steady-state levels of ROI in yellow.ROI signal transduction pathwayRecent studies have identified several components involved in the signal transduction pathway of plants that senses ROIs. These include the mitogen-activated protein (MAP) kinase kinase kinases AtANP1 and NtNPK1, and the MAP kinases AtMPK3/6 and Ntp46MAPK [39,40]. In addition, calmodulin has been implicated in ROI signaling [9,41]. A hypothetical model depicting some of the players involved in this pathway is shown in Fig. 4. H 2O 2is sensed by a sensor that might be a two-component histidine kinase, as in yeast [9]. Calmodulin and a MAP-kinasecascade are then activated, resulting in the activation or suppression of several transcription factors. These regulate the response of plants to oxidative stress [9,42]. Cross-talk with the pathogen-response signal transduction pathway also occurs and might involve interactions between different MAP-kinase pathways,feedback loops and the action of NO and SA as key hormonal regulators. This model (Fig. 4) is simplified and is likely to change as research advances our understanding of this pathway .ROIs act as signals that mediate the systemic activation of gene expression in response to pathogenpathway by ROIs? It is possible that the level of H 2O 2that is currently thought to kill cells by direct cellular damage actually induces PCD [15,27], and it might require a higher level of ROIs to kill cells by direct oxidation. Perhaps future studies applying oxidative stress to mutants deficient in different PCD pathways will answer this question.Many questions related to ROI metabolism remain unanswered (Box 1). We are currently at an exciting time, when most of the technologies required to answer these questions are in place. Thus, acomprehensive analysis of gene expression using microarrays and chips, coupled with proteomics andAcknowledgementsI apologize to all colleagues whose work could not be reviewed here because of space limitation. I thank Barbara A.Zilinskas and Eve Syrkin Wurtele for critical reading of the manuscript. Research at my laboratory is supported by funding from the Israeli Academy of Sciences and the Biotechnology Council of Iowa State University.Fig. 4.A suggested model for the activation of signal transduction events during oxidative stress.H 2O 2is detected by a cellular receptor or sensor. Its detection results in the activation of a mitogen-activated-protein kinase (MAPK) cascade and a group of transcription factors that control different cellular pathways. H 2O 2sensing is also linked to changes in the levels of Ca 2+and calmodulin, and to the activation or induction of a Ca 2+–calmodulin kinase that can also activate or suppress the activity of transcription factors. The regulation of gene expression by the different transcription factors results in the induction of various defense pathways, such as reactive oxygen intermediate (ROI) scavenging and heat-shock proteins (HSPs), and in the suppression of some ROI-producing mechanisms and photosynthesis. There is also cross-talk with the plant –pathogen signal transduction pathway, which might depend on pathogen recognition by the gene-for-gene mechanism and can result in an inverse effect on the regulation of ROI-production and ROI-scavenging mechanisms, as well as on theactivation of programmed cell death (PCD). The plant hormones nitric oxide (NO) and salicylic acid (SA) are key regulators of this response.metabolomics to follow different antioxidants and related compounds during oxidative stress, should answer many of these questions. 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中药复方治疗急性胰腺炎作用机制的研究进展

中药复方治疗急性胰腺炎作用机制的研究进展牛小龙1，2，姚广涛1，31 上海中医药大学研究生院，上海201203；2 上海中医健康服务协同创新中心；3 上海中医药大学创新中药研究院摘要：急性胰腺炎（AP）是临床常见的一种急腹症。

大多数AP患者为轻症，病程具有自限性，通常1～2周即可恢复。

但约20% AP患者会发展为重症急性胰腺炎（SAP），病死率为20%～40%。

西医治疗AP易引起继发性感染、腹膜炎、休克等并发症，整体治疗效果并不理想。

中医认为，AP起因于诸多病邪，包括热、湿、水、气、瘀等壅阻于胰、肝、胆、胃、脾、肠等脏腑，在治疗上应以“攻下通腑”“疏肝退热”“清热解毒”为突破点。

常用的中药复方包括大承气汤、大柴胡汤、大黄牡丹汤、柴芩承气汤、清胰汤等，其作用机制包括改善胃肠功能，修复肠黏膜屏障；抑制炎症反应，提高免疫功能；促进胰腺微循环；诱导胰腺腺泡细胞凋亡等。

这些中药复方以其多组分、多途径、多靶点相互作用，协同发挥治疗作用。

关键词：急性胰腺炎；中药复方；作用机制doi：10.3969/j.issn.1002-266X.2024.01.023中图分类号：R657.5+1 文献标志码：A 文章编号：1002-266X（2024）01-0093-05急性胰腺炎（AP）是临床常见的消化系统急症之一。

大多数AP患者为轻症，病程具有自限性，通常1～2周即可恢复。

但仍有约20% AP患者会发展为重症急性胰腺炎（SAP），病死率为20%～40%［1］。

西医治疗AP的主要方法包括立即禁食水、持续胃肠减压、静脉输液支持、抑制胃酸和胰液分泌等［2］。

但西医治疗易引起继发性感染、腹膜炎、休克等并发症，整体治疗效果并不理想。

中医药以其多组分、多途径、多靶点相互作用，协同发挥治疗作用，在治疗AP方面具有独特优势。

经典中药复方大承气汤、清胰汤能够减轻胰腺炎症，抑制病情加重［3］。

此外，大柴胡汤、大黄牡丹汤、柴芩承气汤等中药复方亦能通过改善胃肠功能、修复肠黏膜屏障、诱导细胞凋亡等基金项目：上海市科技计划项目资助（22S21901300）。

鱼肉蛋白肽在模拟胃肠消化吸收过程中的抗氧化活性和生物利用度_霍艳姣

Science and Technology of Food Industry研究与探讨鱼肉蛋白肽在模拟胃肠消化吸收过程中的抗氧化活性和生物利用度霍艳姣1，王波1，郭珊珊2，李博1，*，罗永康1（1.中国农业大学食品科学与营养工程学院，北京100083；2.滨州万嘉生物科技有限公司，山东滨州256600）摘要：以鳕鱼鱼肉蛋白肽为研究对象，构建体外胃肠消化模型和Caco-2细胞吸收模型，模拟连续的胃肠消化、吸收过程，测定抗氧化活性和生物利用度。

结果显示：在模拟胃肠消化过程中，鱼肉蛋白肽的水溶性维生素E 抗氧化能力（TEAC ）变化不显著（p ＞0.05），DPPH 自由基清除率在胃消化阶段下降而在肠消化阶段升高（p ＜0.05），但低于消化前的水平（p ＜0.05）。

在模拟转运2h 过程中，鱼肉蛋白肽吸收产物的肽氮含量逐渐增加，TEAC 和氧自由基抗氧化能力（ORAC ）显著升高（p ＜0.05）。

鱼肉蛋白肽的生物利用度显著高于鱼肉（9.1%），且分子量最小的蛋白肽的生物利用度最高（46.2%）。

鱼肉蛋白肽经胃肠消化吸收后，具有一定的抗氧化活性且生物利用度较高，为鳕鱼蛋白肽的开发利用提供了理论依据。

关键词：胃肠消化，Caco-2细胞模型，抗氧化活性，生物利用度Antioxidant activity and bioavailability of the Pacific cod meat peptidesduring simulated gastrointestinal digestion and absorptionHUO Yan-jiao 1，WANG Bo 1，GUO Shan-shan 2，LI Bo 1，*，LUO Yong-kang 1（1.College of Food Science and Nutritional Engineering ，China Agricultural University ，Beijing 100083，China ；2.Binzhou Wan Jia Biological Science and Technology Co.，Ltd.，Binzhou 256600，China ）Abstract ：Antioxidant activity and bioavailability of the Pacific cod （Gadus macrocephalus ）protein peptide weredetermined during in vitro gastrointestinal digestion model and Caco -2cell monolayer model simulated the process of gastrointestinal digestion and absorption.The results showed that TEAC activity of fish protein peptide had no obvious variation during gastrointestinal digestion ，while DPPH scavenging capacity significantly （p ＜0.05）reduced after pepsin digestion and obviously picked up but below （p ＜0.05）the level of before digestion after intestinal digestion.The peptide nitrogen content and antioxidant activity of the absorption components increased （p ＜0.05）during absorption.The bioavailability of protein peptides was higher than fish meat （9.1%），especially ，low-molecular-weight fraction of protein peptides had the highest bioavailability （46.2%）.The cod protein peptide had antioxidant activity and pretty bioavailability after gastrointestinal digestion and absorption ，which provided theoretical evidence for the cod protein peptide development.Key words ：gastrointestinal digestion ；Caco-2cell model ；antioxidant activity ；bioavailability 中图分类号：TS201.1文献标识码：A 文章编号：1002-0306（2016）06-0174-06doi ：10.13386/j.issn1002-0306.2016.06.027收稿日期：2015－07－29作者简介：霍艳姣（1990-），女，硕士研究生，研究方向：功能食品，E-mail ：huoyjiao@ 。

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。

硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响

硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响许晖;李亚男【摘要】The effects of selenium salt stress on the activities of antioxidant enzymes in selenium -tolerant mutant 58(5) and wild type WT of Arabidopsis thaliana were researched .It was found that:under the stress of high concentration of selenium salt , the activities of GSH-Px, SOD and POD in the plants of Arabidopsis thaliana all increased , and the activities of these antioxidant en-zymes in 58(5) were obviously higher than those in WT .The above results indicated that selenium -tolerant mutant 58(5) of Ara-bidopsis thaliana had stronger tolerance to selenium .%研究了硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响，发现在高浓度硒胁迫下，植物体内的谷胱甘肽过氧化物酶（GSH－Px）、超氧化物歧化酶（SOD）、过氧化物酶（POD）的活性均升高，而且拟南芥耐硒突变体58（5）中这几种酶的活性均明显高于野生型拟南芥WT中的。

说明突变体58（5）具有较强的耐硒特性。

【期刊名称】《江西农业学报》【年(卷),期】2014(000)012【总页数】3页(P63-65)【关键词】硒盐胁迫;拟南芥;耐硒突变体;抗氧化酶;活性【作者】许晖;李亚男【作者单位】湖北省荆州农业科学院，湖北荆州 434010;长江大学农学院，湖北荆州434023【正文语种】中文【中图分类】Q945.78当植物受辐射、干旱、低温、病虫害等逆境伤害时,植物体内会产生大量的自由基,这些自由基可被谷胱甘肽过氧化物酶(GSH-Px)、过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等抗氧化酶系统所清除,因而,增强植物的抗氧化能力能够提高植物对环境胁迫的抗性[1]。

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。

症状性许莫氏结节治疗的研究进展

- 168 -Neurobiology，2002，53（4）：479-500.[16] SU J，YANG L，YU L，et al. Long-term effectiveness ofrivastigmine patch or capsule for mild-to-severe Alzheimer's disease: a meta-analysis[J]. Expert Review of Neurotherapeutics，2015，15（9）：1093-1103.[17]王荫华，陈清棠，张振馨，等.卡巴拉汀治疗阿尔茨海默病患者的临床研究[J].中华神经科杂志，2001，34（4）：210-213.[18]裘卫东，杨建英，申屠燕琴，等.石杉碱甲对老年患者腹部手术后认知功能障碍的双盲研究[J].解放军药学学报，2009，25（3）：222-224.[19]李琳，王晓良，彭英.抗阿尔茨海默病天然产物及其药理学研究进展[J].中国药理学通报，2016，32（2）：149-155.[20] MCGEER P L，MCGEER E G. The inflammatory responsesystem of brain: implications for therapy of Alzheimer and other neurodegenerative diseases[J]. Brain Res Brain Res Rev，1995，21（2）：195-218.[21] O'BRYANT S E，FAN Z，JOHNSON L A，et al. A precisionmedicine model for targeted NSAID therapy in Alzheimer's disease[J]. Journal of Alzheimer's disease: JAD，2018，66（1）：1-8.[22]马晓玮，李金泽，张天泰，等.非甾体抗炎药抗阿尔茨海默病神经炎症的研究进展[J].药学学报，2014，49（9）：1211-1217.[23] MAYER E A. Gut feelings: the emerging biology of gut-braincommunication[J]. Nature Reviews Neuroscience，2011，12（8）：453-466.[24]聂志玲，张腾.阿尔茨海默病从肾论治的理论依据[J].湖北中医杂志，2014，36（10）：27.[25]周友龙，韩红艳.针灸治疗阿尔兹海默病的临床研究进展[J].针灸临床杂志，2006，（11）：50-53.[26]陈振兴，王鹤淞，张沥丹，等.针灸治疗阿尔茨海默病机制的研究进展[J].国际老年医学杂志，2022，43（6）：738-741.[27] COTMAN C W，BERCHTOLD N C. Exercise: a behavioralintervention to enhance brain health and plasticity[J]. Trends in Neurosciences，2002，25（6），295–301.[28] ROSENBERG A，SOLOMON A，NGANDU T，et al.Multidomain lifestyle intervention benefits a large elderly population at risk for cognitive decline: subgroup analyses of the finnish geriatric intervention study to prevent cognitive impairment and disability (finger)[J]. Alzheimer's & Dementia，2017，13（7）：P265.[29] THAPA A，CARROLL N J. Dietary modulation of oxidativestress in Alzheimer's disease[J]. International Journal of Molecular Sciences，2017，18（7）：1583.（收稿日期：2023-08-05）*基金项目：济宁市重点研发计划项目（2022YXNS150；2020JKNS008）；山东省医药卫生科技发展计划项目（20210407383）①济宁医学院临床医学院　山东　济宁　272067②济宁市第一人民医院通信作者：倪勇症状性许莫氏结节治疗的研究进展*张云鑫①　张存鑫②　吕超亮②　倪勇② 【摘要】　目前症状性许莫氏结节（Schmorl's nodes，SNs）的治疗首选服用消炎镇痛药、卧床休息及腰围保护等保守治疗，但对于经正规保守治疗无效的患者，通常可采取手术治疗的方式缓解症状。

炎症与细胞应激

(BULK)
Control Compounds
Neutral-ODN (Control for iODNs) (endotoxin-free) (synthetic)
5‘-tcctgcaggttaagt-3‘ (BULK) ALX-746-256-C100 ALX-746-356-M001 100 µg 1 mg
iodns - Potent inhibitors of tlr9 signAling
Class III: Class IV:
LIT: Immunotherapeutic utility of stimulatory and suppressive oligodeoxynucleotides: K.J. Ishii, et al.; Curr. Opin. Mol. Ther. 6, 166 (2004) Inhibitory oligodeoxynucleotides - therapeutic promise for systemic autoimmune diseases?: P. Lenert; Clin. Exp. Immunol. 140, 1 (2005) DNA motifs suppressing TLR9 responses: A. Trieu; Crit. Rev. Immunol. 26, 527 (2006)
Inhibitory ODNs (iODNs)
Product/Sequence Prod. No. (BULK) ALX-746-250-T050 ALX-746-350-M001 ALX-746-251-T050 ALX-746-351-M001 ALX-746-252-C100 ALX-746-352-M001 ALX-746-253-C100 ALX-746-353-M001 ALX-746-254-C100 ALX-746-354-M001 ALX-746-255-C100 ALX-746-355-M001 Size 50 tests 1 mg 50 tests 1 mg 100 µg 1 mg 100 µg 1 mg 100 µg 1 mg 100 µg 1 mg