FLLL32_JAK2-STAT3抑制剂_CAS号1226895-15-3_M9142说明书_AbMole中国
当归多糖抑制铁调素中JAK2和SMAD158通道作用
Study to establish the role of JAK2and SMAD1/5/8pathways in the inhibitionof hepcidin by polysaccharides from Angelica sinensisYu Zhang a,Ming-Ming Li a,Fang Zeng a,Cheng Yao a,Kai-Ping Wang b,na Union Hospital of Huazhong University of Science and Technology,Department of Pharmacy,No.1227,Jiefang Road,430030Wuhan,Chinab Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,Tongji Medical College of Huazhong University of Science and Technology,No.13,Hangkong Road, 430030Wuhan,Chinaa r t i c l e i n f oArticle history:Received22May2012Received in revised form20September2012Accepted25September2012Available online2October2012Keywords:Angelica sinensisPolysaccharidesErythropoietinHepcidinPhospho-SMAD1/5/8JAK2a b s t r a c tEthnopharmacological relevance:Angelica sinensis polysaccharide(ASP)is one of the major activeingredients in Angelica sinensis(Oliv.)Diels.This traditional Chinese medicine has been used forthousands of years for treating gynecological diseases.Aim of the study:Previous studies have suggested that ASP from the roots of Angelica sinensis(Oliv.)Diels suppresses hepcidin expression,but the underlying molecular mechanisms are not known.Thepresent study was designed to establish the role of the janus-kinases2(JAK2)and son of mothersagainst decapentaplegic1/5/8(SMAD1/5/8)pathways in the inhibition of hepcidin by polysaccharidesfrom Angelica sinensis in normal rats.Materials and methods:ASP was administered orally(0.3,0.6and 1.2g/kg body weight)to maleSprague–Dawley rats every day for20days.Intraperitoneal injections of recombinant humanerythropoietin(rhEPO;800and2000U/kg body weight)were given to the positive control groupevery day for3days.After administration,hepcidin levels,blood parameters,serum iron status andnon-heme iron concentrations in the liver were examined.Western blot analyses were used toinvestigate the expression offive relevant signaling proteins in the liver.Results:RhEPO injection significantly stimulated erythropoiesis and expression of the serum transferrinreceptor(sTfR),and decreased serum iron status and non-heme iron concentrations in the liver.However,blood parameters barely changed in the ASP groups.sTfR,serum iron,and liver iron levelsaltered only in the ASP high-dose group(1.2g/kg body weight).rhEPO and ASP significantly reducedhepcidin expression by inhibiting the expression of phospho-SMAD1/5/8and JAK2in the liver,but notthrough transmembrane protease serine6(TMPRSS6)and extracellular signal-regulated kinase1/2(ERK1/2).Conclusions:These data suggested that ASP can interrupt the JAK2and SMAD1/5/8pathways,whicheventually results in lower expression of hepcidin.Crown Copyright&2012Published by Elsevier Ireland Ltd.All rights reserved.1.IntroductionThe roots of Angelica sinensis(Oliv.)Diels,a well-knownChinese herbal medicine,have been used for thousands of yearsas a hematopoietic,tonic and anti-inflammatory agent for thetreatment of menstrual disorders,dysmenorrheal and amenor-rhea(Jin et al.,2012).As one of the active components,Angelicasinensis polysaccharide(ASP)has various bioactivities,such ashematopoiesis(Lee et al.,2012),immunomodulation(Liu et al.,2010a),radioprotection(Jin et al.,2012),and anti-tumor activity(Cao et al.,2010).Liu et al.(2010b)found that ASP is the majorcomponent responsible for the hematopoietic effect of Angelicasinensis(AS).This hematopoietic activity is improved mainlythrough the stimulation of secretion of interleukin(IL)-6andgranulocyte macrophage colony-stimulating factor.Previously,we reported that ASP can participate in the regulation of ironhomeostasis.In addition,we found that intragastric administration of ASPreduces hepcidin expression,which has become a target for thetreatment of iron-deficiency diseases(Ganz and Nemeth,2006).Hepcidin(a25amino-acid antimicrobial peptide produced byhepatocytes)is thought to be a principal iron-regulatory hor-mone.Hepcidin inhibits the entry of iron into the plasmacompartment.Iron can be from three main sources:dietaryabsorption in the duodenum,the release of recycled iron frommacrophages and release of stored iron from hepatocytes(Ganzand Nemeth,2012).Chronic increases in hepcidin levels causeContents lists available at SciVerse ScienceDirectjournal homepage:/locate/jepJournal of Ethnopharmacology0378-8741/$-see front matter Crown Copyright&2012Published by Elsevier Ireland Ltd.All rights reserved./10.1016/j.jep.2012.09.040n Corresponding author.Postal address:Tongji Medical College of HuazhongUniversity of Science and Technology,430030Wuhan,China.Tel./fax:þ862783692762.E-mail address:wkpzcq@(K.-P.Wang).Journal of Ethnopharmacology144(2012)433–440systemic iron deficiency(Andrews,2008).Our previous study reported that ASP significantly reduces hepcidin expression by inhibiting the expression of signal transducer and activator of transcription3/5(STAT3/5)and sons of mothers against deca-pentaplegic protein4(SMAD4),and stimulates the secretion of erythropoietin,which further down-regulates hepcidin expres-sion by repressing CCAAT/enhancer-binding protein a(C/EBP a), SMAD4,and the phosphorylation of STAT3/5(Wang et al.,2011). Hence,how ASP influences the circulation of iron and the path-ways of hepcidin regulation in vivo is worthy of investigation.Therefore,the present study had two objectives.Thefirst objective was to evaluate the influence of ASP in iron homeostasis involving hepcidin.We tested hematological indices and serum measurements including iron,transferrin(TF),total iron-binding capacity(TIBC)and the soluble transferrin receptor(sTfR)(Kong et al.,2008,Sun Zhou and Zhao,2006).The other objective was to clarify the underlying mechanism in hepcidin suppression caused by ASP.We monitored the liver expressions offive proteins demonstrated to be important for hepcidin regulation,i.e.,JAK2/ phospho-JAK2;phospho-SMAD1/5/8;TMPRSS6and ERK1/2 (Maliken et al.,2011;Gardenghi et al.,2010;Theurl et al.,2011; An et al.,2012;Ramey et al.,2009).2.Materials and methods2.1.Plant collectionThe dry roots of Angelica sinensis(Oliv.)Diels were collected from Minxian(Gansu Province,China)in October2010.Plant identification was undertaken by Professor Jin-Lan Ruan(Faculty of Pharmaceutical Sciences,Tongji Medical College of Huazhong University of Science and Technology,Wuhan,China)in accor-dance with the identification standard of the Pharmacopoeia of the People’s Republic of China.2.2.Preparation of raw polysaccharide(ASP)The sliced roots of AS(200g)were extracted in1L of boiling water for30min.Acidic and basic proteins were separated by adjusting the pH to5and adding4L of95%ethanol and maintaining the extract at41C for12h.The precipitate was lyophilized after suspension in distilled water.The yield of polysaccharide was19.6%, with a carbohydrate content of76.8%.Phytochemical screening demonstrated two subtypes of ASP:ASP I and II.The mean molecular weight was8000and76,000Da,respectively.ASP I and II(7.5:1) comprised glucose,galactose,and arabinose.2.3.Animals and experimental designAll the methods used for plant collection,preparation of raw polysaccharide(ASP)and drug administration were identical to those employed in a previous study(Wang et al.,2007).Male Sprague–Dawley(SD)rats(Animal Center of Tongji Medical College, Wuhan,China)were kept on a standard rodent diet(45mg Fe/kg) until they reached the age of6weeks and weighed$200g.Rats had free access to food and water.They were housed in a room with a 12-h light–dark cycle and a mean temperature of20711C.In addition to the experimental groups,48male SD rats were randomly divided into six groups.Group A(negative control group) received normal(0.9%)saline.Groups B and C were the recombinant human erythropoietin(rhEPO)treatment groups(800and2000U/kg body weight,respectively).Groups L,M and H were given ASP(0.3, 0.6and 1.2g/kg body weight,respectively).The negative control group and experimental groups were administered orally once daily for20consecutive days.The positive groups were given rhEPO (Sunshine Pharmaceutical Co.,Shenyang,China)by intraperitoneal injection once daily for3consecutive days.2.4.Hematological parametersBlood(0.5mL)was taken from the retro-orbital sinus before thefirst administration and24h after the last administration.The times for taking blood were in accordance with our previous study(Wang et al.,2011).Blood in ethylenediamine tetra-acetic acid(EDTA)-containing vacuum tubes was analyzed by a MEK-6318K Multispecies Hematology Analyzer(Nihon Kohden,Tokyo, Japan)for the determination of complete blood count,hemoglo-bin concentration(Hb),red blood cell count(RBC),hematocrit (HCT),and mean cell volume(MCV).Blood without anticoagulant was collected into eppendorf tubes.After4h at room tempera-ture,serum was obtained after centrifugation at5000rpm/min for10min,and then stored atÀ201C until analysis.Hepcidin levels in serum were determined by enzyme-linked immunosorbent assay(ELISA)according to manufacturer instruc-tions(USCN Life Co.,Houston,TX,USA).Serum TF and sTfR levels were determined with a commercially available ELISA kit(R&D Systems,Minneapolis,MN,USA).Serum TIBC was determined using commercially available kits(Nanjing Jiancheng Bioengi-neering Institute,Nanjing,China).Measurements for hematological parameters were all carried out in the Hubei Key Laboratory at the China Medical Center (Wuhan,China).2.5.Determination of iron statusRats were anesthetized with urethane(0.9g/kg body weight) after thefinal blood-taking.Normal saline was perfused to remove blood in the liver.After perfusion,liver tissues from each rat were stored atÀ801C.For quantitative determination of iron content,standards were prepared by serial dilution of Fe standards purchased from vendors that could provide traceability to National Institute of Metrology(NIM)standards.Two-hundred microliters of each serum sample was diluted directly with1800m L of deionized water.The wet livers were placed in a constant-temperature drying stove until they reached a constant weight.Dry liver tissue samples were digested using the conventional wet acid digestion method.Briefly,liver samples were digested in a(10:1)nitric acid (HNO3)and perchloric acid(HClO4)solution(China National Medicines Corporation Ltd.,Beijing,China).Dry tissues were weighed accurately into150-mL Erlenmeyerflasks.Then,5mL HNO3(65%)and0.2mL HClO4(70%)were added to eachflask.The contents of theflasks were heated on an electric hotplate at 2801C for15–30min until clear-white crystalline digested con-tent was obtained.Digested samples were diluted by10mL deionized water and kept at41C until further analyses.The contents of the trace element Fe in serum and liver were measured using a SpectrAA-240FS Atomic Absorption Spectro-meter(Varian Medical Systems,Palo Alto,CA,USA).2.6.Western blotting in liver tissueLiver tissue was homogenized supersonically in TBS buffer[1mL TBS(pH7.5)buffer containing1%NP-40,1m g/mL pepstatin,1m g/mL leupeptin,1m g/mL aprotinin,100m g/mL phenylmethylsulfonyl fluoride(PMSF)].After centrifugation at12,000Âg for30min at 41C,the supernatant was collected.Protein concentrations were determined by a protein assay(Bio-Rad,Hercules,CA,USA).Total cellular proteins(100m g/lane)were run on10%sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE)under redu-cing conditions,and electroblotted onto nitrocellulose membranesY.Zhang et al./Journal of Ethnopharmacology144(2012)433–440 434(Amersham Pharmacia Biotech,Buckinghamshire,UK).Membranes were blocked with5%non-fat milk in TBS and incubated with primary antibody(Table1).They were washed and incubated with horseradish peroxidase(HRP)-conjugated secondary antibody (1:4000dilution)for2h at room temperature,washed,then incu-bated with chemiluminescence substrate(ECL Plus)for1min and immediately exposed to X-rayfilm.Band density was quantified using Labworks(GelPro4.0;Media Cybernetics,Bethesda,MD,USA) by calculating the average optical density in eachfield.Relative and normalized protein expressions were calculated using the ratio of density of each protein to the density of b-actin.2.7.Statistical analysesAll parameters were recorded for individuals within all groups. Data are the means7S.D.Data were analyzed using SPSS ver13.0 (SPSS,Chicago,IL,USA).P o0.05was considered significant.3.Results3.1.Suppression of hepcidin expressionRhEPO groups(800and2000U/kg)reduced hepcidin expres-sion by24.3%and29.5%,respectively.After20-day treatment of ASP at three doses(0.3,0.6and1.2g/kg),hepcidin levels were reduced significantly by19.4%,27.1%and31.2%,respectively. Hepcidin inhibition was enhanced with increasing ASP dose.The inhibitory effect of rhEPO at high dose was stronger than that seen at lower doses(Fig.1).3.2.Hematological parameters and indices of iron statusIt has been demonstrated that erythropoietin(EPO)can promote the differentiation and development of RBCs as well as initiate the production of Hb(Kong et al.,2008).The hematolo-gical parameters of all the treatment groups were determined. The HCT and blood concentrations of RBCs and Hb increased significantly only in the rhEPO groups(Table2,P o0.05),but barely changed in the ASP groups.The mean corpuscular volume (MCV)of ASP and rhEPO groups was not significantly different from that of the control group(Table2).The effects of rhEPO and three oral doses of ASP on iron as well as the indices of iron status are shown in Table3.The data suggested that rats treated with rhEPO had increased expression of sTfR.In addition,0.6and1.2g/kg of ASP dramatically increased sTfR expression(P o0.05).However,TF level and TIBC in the drug groups did not show significant differences compared with the negative control group.Both rhEPO injection groups had lower levels of iron in the serum and liver than those in the controlTable1Antibodies used in the western blot assay.Protein Classification Molecular weight(kDa)Antibody source Antibody dilution Antibody manufacturer Catalog number b-actin Total protein42Mouse1:4000Sigma-Aldrich A5441Erk1/2Total protein4244Rabbit1:1000CST4695P-SMAD1/5/8Total protein60Rabbit1:800CST9511JAK2Total protein125Rabbit1:1000CST3230P-JAK2Total protein125Rabbit1:500CST3776TMPRSS6Total protein89Rabbit1:800AbcamAb28286Fig.1.Changes of hepcidin levels in rats of different treatment groups.Rats administrated normal saline and ASP for20days,respectively.Rats administrated rhEPO for 3days.Results are expressed as mean7SEM,n¼8.*P o0.05or**P o0.01as compared with those before administration.Y.Zhang et al./Journal of Ethnopharmacology144(2012)433–440435group (P o 0.05),whereas the amount of iron showed no change in the ASP groups.ASP (0.3g/kg)-treated rats demonstrated significant increases in iron concentrations in the liver (P o 0.05)compared with the negative control group (Table 3).This finding may reveal another mechanism by which ASP regulates iron metabolism compared with that seen with rhEPO.3.3.Variation of signal proteins involved in the systemic regulation of hepcidinThe dose dependency of ASP between low (0.3g/kg)and medium (0.6g/kg)doses seemed to be apparent (Fig.1).However,the reduction in hepcidin expression at 1.2g/kg of ASP was not significantly different to that seen with 0.6g/kg of ASP.In our preliminary western blot tests,no significant difference was found in regulation of the levels of signaling proteins between 0.6g/kg of ASP and 1.2g/kg of ASP (data not shown).It was probable that the high and medium doses of ASP were close to the maximum effective dose,which was in between the two doses.Hence,we chose group L (ASP 0.3g/kg)and group H (ASP 1.2g/kg)to study the dose dependency of ASP on the regulation of signaling proteins.3.3.1.Phospho-SMAD1/5/8expressionAfter phosphorylation,SMAD1/5/8complexes with SMAD4,which translocates to the nucleus to activate hepcidin transcrip-tion.The expression of phospho-SMAD1/5/8in liver tissues from ASP groups (0.3and 1.2g/kg)was decreased by about 27.9%and 63.4%,respectively (P o 0.05)(Fig.2).rhEPO injection of both doses reduced expression of phospho-SMAD1/5/8in the liver by 26%and 53.6%,respectively (P o 0.05).3.3.2.Expression of JAK2and phospho-JAK2JAK2transmits signals for various cytokine receptors,includ-ing the erythropoietin receptor (EpoR),which is essential for RBC production.Upon stimulation by Epo,JAK2activates downstream signaling such as STAT5and phosphatidylinositol 3-kinase/AKTpathways (Constantinescu et al.,1999).Mice deficient in JAK2,Epo,or EpoR die as embryos because of the absence of definitive erythropoiesis.In the present study,ASP administration and rhEPO injection significantly decreased the expression of JAK2(P o 0.05,Fig.3A).Moreover,JAK2inhibition was enhanced with increasing doses of ASP.The effects of low and high doses of ASP on the inhibition of JAK2were 49.5%and 63.2%,respectively (P o 0.05).Both rhEPO treatment groups (800and 2000U/kg)reduced the expression ofTable 2Effect on blood parameters of ASP (i.g.)and rhEPO (i.p.)in of the group Hb (g/l)RBC (106/mm 3)HCT (%)MCV (fL)Control group 159.678.97.570.638.672.252.572.5ASP (0.3g/kg)152.8715.37.670.737.573.548.771.6ASP (0.6g/kg)153.6714.67.9670.539.371.949.172.2ASP (1.2g/kg)159.3711.87.9370.440.072.052.772.1RhEPO (800U/kg)172.3712.9n 8.570.7nn 45.272.1n 54.371.3RhEPO (2000U/kg)174.5710.9nn8.370.5nn45.772.3n55.470.7Results are expressed as mean 7SEM,n ¼8.n P o 0.05versus control group.nnP o 0.01versus control group.Table 3Effect on iron status of ASP (i.g.)and rhEPO (i.p.)in of the group Serum iron (mg/L)Liver iron (ug/g)TIBC (mg/L)sTfR (nmol/L)Serum TF (nmol/L)Control group 2.7170.53265.68717.6470.33724.9152.54711.485.04710.69ASP (0.3g/kg) 3.1470.36314.97711.2n 485.62713.8177.9175.5583.1777.14ASP (0.6g/kg) 2.4370.24243.78727.9478.54720.7219.63711.82n 79.3679.64ASP (1.2g/kg) 1.6870.27nn 166.69727.1nn 453.17721.3312.5078.58nn 76.4978.48RhEPO (800U/kg) 2.0870.39n 210.89710.7n 465.27727.82169.2977.53n 83.7478.52RhEPO (2000U/kg)1.7070.18nn157.33712.4nn435.34723.91331.0973.17nn81.1377.71Results are expressed as mean 7SEM,n ¼8.n P o 0.05versus control group.nnP o 0.01versus controlgroup.Fig. 2.Suppression of phospho-smad1/5/8by ASP and rhEPO.Bars represent mean (7S.D.)of arbitrary densitometric units (ADUs)following Western blot analysis.Values are expressed as percentages relative to negative control group.Control,rats (n ¼6)administrated normal saline (1mL)for 20days;ASP,rats (n ¼6)administrated with ASP (0.3and 1.2g/kg,respectively)for 20days;EPO,rats (n ¼6)administrated with rhEPO (800and 2000U/kg,respectively)for 3days.*P o 0.05;**P o 0.01versus control group.Y.Zhang et al./Journal of Ethnopharmacology 144(2012)433–440436JAK2by E 55%,and there was no significant difference between these two groups (Fig.3A).Similarly,western blot analyses of phospho-JAK2revealed a reduction in expression of 70.4%(P o 0.01)in rats from the ASP (1.2g/kg)treatment group.Low-dose ASP could barely suppress phospho-JAK2expression (P 40.05).rhEPO treatment groups (800and 2000U/kg)reduced the expression of phospho-JAK2by about 56.2%and 75.6%,respectively (P o 0.05,Fig.3B).There were no apparent effects on the ratio of JAK2and phospho-JAK2at 1.2g/kg of ASP,suggesting that phosphorylation was not affected by ASP.However,the ratio of JAK2and phospho-JAK2in rhEPO groups (especially if treated with 2000U/kg)was significantly increased 80.6%(P o 0.05)compared with the nega-tive control (Fig.3C).This finding suggested that EPO inhibited the phosphorylation of JAK2rather than the proteins themselves.3.3.3.Expression of ERK1/2and TMPRSS6Reports have shown that hepcidin can express through the ERK1/2kinase pathway,and that the ERK-specific inhibitor U0-126blunts holotransferrin-mediated induction of hepcidin (Ramey et al.,2009).The western blot results suggested that a significant decrease in Erk1/2levels was not induced by treat-ment with ASP and rhEPO (Fig.4).The cell-surface protein TMPRSS6is a potent suppressor of hepcidin expression,having a key role in iron homeostasis.To understand the mechanism of change in iron absorption upon treatment with rhEPO and ASP,levels of TMPRSS6were analyzed using western blotting.Treatment with rhEPO and ASP did not increase TMPRSS6expression as compared with the control group (Fig.5,P 40.05).4.DiscussionIn our previous studies (Wang et al.,2011),ASP from the roots of Angelica sinensis (Oliv.)Diels demonstrated hepcidin-suppressing effects in normal rats.To investigate how rhEPO and ASP decrease hepcidin in vivo,we focused on the changes in the C/EBP a ,SMAD4,STAT3/5signaling pathways as well as STAT3/5phosphorylation after treatment with rhEPO and ASP.Western blotting analyses revealed that expression of STAT3/phospho-STAT3,STAT5/phospho-STAT5,C/EBP a and SMAD4in the treatment groups was significantly reduced compared with those of the normal controls.We designed different dose treatment groups to investigate the correlation between inhibition and treatment dose.Moreover,the iron indices in the blood and livers of rats were detectedafterFig.3.Suppression of JAK/STAT pathway by ASP and rhEPO.(A)Total JAK2expression,(B)Phospho-JAK2expression,(C)The ratio of JAK2and phospho-JAK2.For (A)and (B),bars represent mean (7S.D.)of arbitrary densitometric units (ADUs)following western blot analysis.Values are expressed as percentages relative to negative control group.For (C)stripes represent the ratio of ADUs of JAK2to ADUs of phospho-JAK2.Rats administrated normal saline and ASP for 20days,respectively.Rats administrated rhEPO for 3days.Results are expressed as mean 7SEM,n ¼6.*P o 0.05or **P o 0.01as compared with the negative control.Y.Zhang et al./Journal of Ethnopharmacology 144(2012)433–440437administration of rhEPO and ASP.Research such as that under-taken on the effect of ASP on JAK/STAT signal transduction in response to inflammatory mediators (Stoian et al.,2007)and thebone morphogenetic protein (BMP)–SMAD pathway (Kautz et al.,2008;Finberg et al.,2010),which mediates hepcidin upregulation by iron and hypoxia,is needed.Interestingly,the effect of ASP and rhEPO on the suppression of hepcidin expression increased with increasing administration dose.RBC production is triggered by the action of EPO through its binding to surface receptors (EPO-R)on erythroid precursors in the bone marrow (Fibach,2011).Our data suggested that,in rats treated with rhEPO,erythropoiesis was promoted.However,the HCT as well as the levels of RBCs and Hb in three ASP treatment groups barely changed.These findings suggested that ASP could not directly regulate hematological parameters in rats except for increasing rhEPO levels.Hence,the activation of erythropoiesis may not be a key factor to lessen hepcidin expression.Iron availability is regulated at cellular and systemic levels by iron regulatory proteins (IRPs)and the liver hormone hepcidin,respectively (Camaschella and Poggiali,2011).The present study showed that treatment with rhEPO and ASP (1.2g/kg)led to a reduction in iron storage in the liver and to a significant decrease in the serum levels of iron.Iron is redistributed to meet the need of erythropoiesis (Collard,2009).However,iron contents in the liver increased significantly after ASP treatment (0.3g/kg).This was probably due to the different ways in which doses of ASP regulate iron levels.ASP (0.3g/kg)improved iron contents in the liver mainly by suppressing hepcidin expression,whereas 1.2g/kg of ASP (which can stimulate appreciable secretion of EPO)affected iron homeostasis in vivo in a similar way to that seen with rhEPO.We demonstrated that sTfR expression increased dramatically after administration with rhEPO and ASP (0.6,1.2g/kg),whereas serum levels of TIBC and TF did not change.rhEPO treatment can increase TfR1expression in the bone marrow and increase iron uptake into cells,resulting in reduced levels of diferric TF (Weiss et al.,1997).Fig.4.Suppression of ERK1/2by ASP and rhEPO.Bars represent mean (7S.D.)of arbitrary densitometric units (ADUs)following Western blot analysis.Values are expressed as percentages relative to negative control group.Control,rats (n ¼6)administrated normal saline (1mL)for 20days;ASP,rats (n ¼6)administrated with ASP (0.3and 1.2g/kg,respectively)for 20days;EPO,rats (n ¼6)administrated with rhEPO (800and 2000U/kg,respectively)for 3days.Fig.5.Suppression of TMPRSS6by ASP and rhEPO.Bars represent mean (7S.D.)of arbitrary densitometric units (ADUs)following western blot analysis.Values are expressed as percentages relative to negative control group.Control,rats (n ¼6)administrated normal saline (1mL)for 20days;ASP,rats (n ¼6)administrated with ASP (0.3and 1.2g/kg,respectively)for 20days;EPO,rats (n ¼6)admini-strated with rhEPO (800and 2000U/kg,respectively)for 3days.Y.Zhang et al./Journal of Ethnopharmacology 144(2012)433–440438Western blot analyses showed that ASP could significantly down-regulate JAK2expression with the same degree of phospho-JAK2suppression,whereas the inhibitory effect of EPO on phos-phorylation was more intense than that on JAK2.Therefore,one could speculate that the slight inhibitory activity of ASP on phosphorylation could be attributed to the effect of EPO stimula-tion by ASP (Fig.6).In hepatocytes,hepcidin transcription is iron-dependent through BMP6,which activates its receptors in the presence of the co-receptor hemojuvelin (HJV)and signal through SMAD1/5/8(Lenoir et al.,2011).Increasing evidence suggests that the extent of phosphorylation of SMAD1/5/8is related directly to iron-dependent expression of hepcidin in vivo.In hepcidin regulation,BMP signaling can enhance SMAD1/5/8phosphorylation.Then,phospho-SMAD1/5/8makes a complex with SMAD4,which translocates to the nucleus to activate hepcidin (HAMP)transcrip-tion (Kautz et al.,2008).Injection of iron dextran and adminis-tration of an iron-rich diet have been shown to increase phospho-SMAD1/5/8expression in the mouse liver.Conversely,sHJV injected into mice has been shown to decrease serum levels of iron,and decreased phospho-SMAD1/5/8expression in the liver eventually lessens hepcidin expression (Ramey et al.,2009).The results of the present study suggested that rhEPO and ASP could suppress hepatic phospho-SMAD1/5/8expression,and could interrupt the entire BMP–SMAD pathway by further inhibition of BMP6and hemojuvelin.The membrane protein TMPRSS6also influences hepcidin synthesis by modulation of the hemojuvelin concentration on the cell membrane (Silvestri et al.,2008).Nevertheless,EPO and ASP could not activate TMPRSS6expres-sion in hepatocytes.It was recently shown that binding of holotransferrin to TfR2can activate the ERK signaling pathway in K562cells,and that ERK activation is necessary for holotransferrin-induced hepcidin gene expression (Calzolari et al.,2006;Ramey et al.,2009).Ramey et al.(2009)found that ERK inhibition leads to a strong decrease in the amount of phospho-SMAD1/5/8,which could be respon-sible for the decrease in hepcidin gene expression observed in the presence of the ERK inhibitor.However,our western blot results suggested that treatment with rhEPO and ASP did not signifi-cantly alter Erk1/2expression as compared with that seen in the control group.We therefore conclude that rhEPO and ASP reduced phospho-SMAD1/5/8levels not through Erk1/2,and that the expression of phospho-SMAD1/5/8was not directly associated with ERK.The sTfR level reflects the total body TfR concentration.It has been hypothesized that ASP and rhEPO lead to an increased transfer of receptors on the cell surface,resulting in increased uptake of extracellular iron (Fig.6).ASP and rhEPO down-regulate the transcription regulators phospho-SMAD1/5/8and JAK2(which phosphorylates STAT3/5)and stimulate endogenous secretion of EPO (which further decreases the expression of signaling proteins in the liver).As a result,hepcidin expression is suppressed.EPO also inhibits JAK2phosphorylation,which causes a reduction in the expression of activation factors such as phospho-STAT5and phospho-STAT3,and the eventual inhibi-tion of hepcidin levels.In conclusion,it was demonstrated that ASP and rhEPO can interrupt the BMP–SMAD and JAK–STAT pathways of hepcidin regulation.This resulted in decreased expression of hepcidin in a dose-related manner in vivo (Fig.6).In addition,ASP could stimulate EPO secretion and regulate the erythropoietic pathway (Fig.6),which decreased hepcidin mRNA levels after an increase in the rate of erythropoiesis (Viatte and Vaulont,2009).This finding supported the speculation that ASP could be useful fortheFig. 6.Current understanding of the molecular mechanisms involved in hepcidin (HAMP)suppression by ASP (schematic).The iron-regulated pathway and the inflammatory pathway are the two well-studied regulation pathways of hepcidin.In the iron-regulated pathway,the circulating amount of transferrin-bound iron binds to its two receptors (TfR1and TfR2),which communicate with each other via iron-specific adaptor HFE and sensitize the BMP receptor to its ligands (e.g.,BMP6).Membrane-linked co-receptor hemojuvelin (HJV)also potentiates activation of the BMP receptor,which then controls hepcidin transcription via SMAD1/5/8.Hepcidin transcription is increased appreciably by inflammation,predominantly through the activity of IL-6,its receptor,and its canonical JAK2–STAT3pathway.Y.Zhang et al./Journal of Ethnopharmacology 144(2012)433–440439。
美国药店上市2型糖尿病药-达格列净-Farxiga
根据这项声明，对该药物的核准是基于在9,400多例2型糖尿病患者中进行的16项临床试验的结果。这些研究显示，该药物治疗可改善糖化血红蛋白A1c。达格列净是FDA批准的第二种钠-葡萄糖协调转运蛋白2 (SGLT2)抑制剂，首个SGLT2为2013年3月获准上市的canagliflozin (Invokana)。这两种口服药物的作用机制为，通过抑制表达于肾脏的SGLT2，减少肾脏的葡萄糖重吸收，增加尿液中葡萄糖的排泄，从而降低血浆葡萄糖水平。
达格列净将由Haoeyou Pharmacy（好医友美国药房）销售，商品名为Farxiga。
因达格列净此前已在欧洲获准上市，故在欧洲已经开始为一项心血管结局研究招募患者，该研究最终将招募17,000例以上的2型糖尿病伴明确心血管疾病或至少有2项心血管风险因素的患者
FDA要求的上市后试验包括一项评估基线时心血管疾病高危患者使用达格列净治疗后的心血管风险的心血管结局试验和一项评估招募患者的膀胱癌风险的研究。另有一项研究将评估该药物对啮齿类动物的膀胱肿瘤促进效应。两项研究将在儿科患者中评估达格列净的药代动力学、疗效和安全性；一项加强的药物警戒计划将在接受达格列净治疗的患者中监测肝脏异常和妊娠结局报告。
jaktinib结构
jaktinib结构
Jaktinib（也称为INCB039110或INCB39110）是一种新型的针对Janus激酶（JAK）的口服小分子抑制剂，是一种治疗类风湿性关节炎等自免疫病的有前景、突破性新药。
Jaktinib的分子式为C22H27N5O3，分子量为413.49。
其分子结构式如下：
Jaktinib的化学名称是（R）
-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanen itrile，其中（R）-为左旋构型的指示符。
Jaktinib的化学结构含有大的杂环骨架，具有极强的生物活性。
它的选择性抑制剂作用针对的“JAKs”即JAK1和JAK2等是Janus激酶家族中最广泛的成员之一。
JAK1和JAK2是JAK家族中较为常见的两个亚型，作用于细胞信号转导的多种路径中。
Jaktinib的研究发现，它特异性的抑制JAK1/2可以有效抑制体内细胞因子介导的炎症反应，减轻部分自免疫疾病炎性紊乱的程度，能够有效抑制IL-6以及因IL-6降低引起的C反应蛋白的升高。
临床试验的研究表明，Jaktinib在治疗自炎性疾病方面可产生良好的临床疗效和耐受性，并且其分子结构中含有环肽结构，具有较好的口服吸收和组织分布特性，使其成为该领域的突破性新药，备受关注和研究。
维格列汀
结语
结语
综上所述，维格列汀具有降低空腹及餐后血糖水平、餐后胰高血糖素分泌及提高B细胞功能的作用，为2型糖 尿病患者的治疗提供了新选择。该药与其他药物联合使用的安全性和有效性，禁忌证、不良反应等还有待于更多 的临床研究观察。
谢谢观看
药动学
药动学
健康人体的药动学研究表明，本品口服给药后迅速吸收，吸收率为85%，蛋白结合率为4%～17%，Tmax为1～ 2 h。当给药剂量在25～200mg时，血药浓度与给药剂量呈线性关系，t1/2为1．5～4．5 h。按25～200 mg单剂 量给药时，在30～60 min内，血浆中DPP-IV的抑制率能达到90%以上，但抑制的持续时间与剂量有关，当剂量为 50和100 mg时，DPP-IV的12 h抑制率分别为70%和90%，而100 mg组DPP-I V的24 h抑制率为40%。本品主要 (55%)通过水解氰基进行代谢，此外，22%的药物以原形药通过肾排泄，极少部分药物通过细胞色素P450酶代谢， 但本品既不是该酶的抑制剂，也不是诱导剂。多剂量给药时未出现药物蓄积现象，肝肾功能不全患者亦无需调整 剂量，且药动学结果也不受食性质
合成方法
基本性质
中文名称：维达列汀 中文别名：(-)-(2S)-1-[[(3-羟基三环[3.3.1.1[3,7]]硅烷-1-基)氨基]乙酰基]吡咯烷-2-甲腈；维他列 汀；(-)-(2S)-1-[[(3-羟基三环[3.3.1.1[3,7]]癸烷-1-基)氨基]乙酰基]吡咯烷-2-甲腈；维格列汀;维达利汀； 维达列汀VILDAGLIPTIN(研发中)；维他列汀S型； 英文名称：Vildagliptin 英文别名：Vildagliptin (NVP-LAF 237)；Galvus；Laf 237 ；Unii-I6B4B2U96p ；(-)-(2S)-1[[(3-Hydroxytricyclo[3.3.1.1[3,7]]dec-1-yl)amino]acetyl]pyrrolidine-2-carbonitrile； (-)(2S)-1-[[(3-Hydroxytricyclo[3.3.1.1[3,7]]dec-1-yl)amino]acetyl]pyrrolidine-2-carbonitrile；2Pyrrolidinecarbonitrile,1-(((3-hydroxytricyclo(3.3.1.13,7)dec-1-yl)amino)acetyl)-(2S)-；； CAS号：-16-5 分子式：C17H25N3O2 维格列汀结构式： 分子量：303.
巴瑞替尼的合成研究
·药物研发·巴瑞替尼的合成研究丁若洋 唐春雷（江南大学生命科学与健康工程学院 无锡 214122）摘要目的：改进Janus激酶抑制剂（JAKi）巴瑞替尼（baricitinib,1）的合成工艺。
方法：3-(氰基亚甲基)氮杂环丁烷-1-甲酸叔丁酯（2）脱Boc得到中间体2-(3-氮杂环丁基亚基)乙腈盐酸盐（3），再经磺酰化反应得到2-[1-(乙基磺酰基)-3-氮杂环丁亚基]乙腈（4），再发生迈克尔加成反应得到1-(乙基磺酰基)-3-[4-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)-1H-吡唑-1-基]-3-氮杂环丁烷乙腈（5），最后与4-氯-7H-吡咯并[2,3-d]嘧啶发生偶联反应得到1。
结果与结论：总收率为63.6%，纯度为99.9%（HPLC面积归一化法），目标终产物及关键中间体的结构经MS和1H-NMR确证正确。
该方法所使用的起始原料价廉易得。
反应后处理简单，总收率较高，适合大量制备，可为巴瑞替尼的生产及其衍生物的合成研究提供参考。
关键词巴瑞替尼 Janus激酶抑制剂 工艺改进中图分类号：O626.21 文献标志码：A 文章编号：1006-1533(2024)05-0073-04引用本文丁若洋, 唐春雷. 巴瑞替尼的合成研究[J]. 上海医药, 2024, 45(5): 73-76.Synthetic studies of baricitinibDING Ruoyang, TANG Chunlei(College of Life Science and Health Engineering, Jiangnan University, Wuxi 214122, China) ABSTRACT Objective: To improve the synthetic route of Janus kinase inhibitor baricinib (1). Methods:3- (cyanomethylene) azocyclobutane-1-carboxylic acid tert-butyl ester (2) was deprotected to obtain the intermediate2-(3-azocyclobutanylidene) acetonitrile hydrochloride (3), which was then subjected to sulfonation reaction to obtain2-[1-(ethylsulfonyl)-3-azocyclobutanylidene] acetonitrile (4), followed by Michael addition reaction to obtain 1-(ethylsulfonyl)-3-[4-(4,5,5-tetramethyl-1,3,2-dioxocyclopentane-2-yl)-1H-pyrazole-1-yl]-3-azocyclobutane acetonitrile (5). Finally, a coupling reaction was carried out with 4-chloro-7H-pyrrolo[2,3-d] pyrimidine to obtain 1. Results & Conclusion: The total yield was 63.6% with the purity 99.9% (HPLC area normalization method). The structures of the target end product and key intermediates were confirmed to be correct by MS and 1H-NMR. The starting materials used in this method are inexpensive and readily available and the post-treatment of the reaction is simple. The new route has a high overall yield and is suitable for large-scale preparation, and can provide reference for the production of baricitinib and the synthesis research of its derivatives.KEY WORDS baricitinib; Janus kinase inhibitor; process improvementJAK是一个由4种酪氨酸受体激酶组成的家族，通过与信号转导子和转录蛋白激活子的相互作用，在细胞因子受体信号通路中发挥关键作用。
抗体公司
赛信通（上海）生物试剂有限公司
上海市浦东南路1101号远东大厦514室，200120 info@cst www.cst 2158356288 公司总部: 美国
Established in Beverly, MA in 1999, Cell Signaling Technology (CST) is a privatelyowned company with over 400 employees worldwide. We are dedicated to providing innovative research tools that are used to help define mechanisms underlying cell function and disease. Since its inception, CST has become the world leader in the production of the highest quality activationstate and total protein antibodies utilized to expand knowledge of cell signaling pathways. Our mission is to deliver the world's highest quality research tools that accelerate progress in biological research and personalized medicine. 总引用数为4670，来自于1966篇文章。最常引用的试剂包括： Akt, ERK2, ERK1, p38, Akt1。
AbD Serotec (BioRad)
Siglec-15靶向激活STAT3通路促进甲状腺癌细胞增殖及转移
Siglec-15靶向激活STAT3通路促进甲状腺癌细胞增殖及转移Siglec-15靶向激活STAT3通路促进甲状腺癌细胞增殖及转移甲状腺癌是甲状腺最常见的恶性肿瘤，其发病率在全球范围内呈不断增长的趋势。
虽然目前通过手术切除、放射治疗和化疗等方法可以有效控制部分病例，但仍有相当一部分患者无法完全治愈或出现复发转移，严重影响患者的生活质量和预后。
因此，寻找新的治疗靶点和策略具有重要的临床意义。
Siglec-15（sialic acid binding Ig-like lectin 15）是一种糖蛋白，在免疫反应和炎症过程中发挥重要的调控作用。
近年来研究发现，Siglec-15在多种恶性肿瘤中高表达，包括甲状腺癌。
研究显示，Siglec-15与甲状腺癌的恶性程度和预后相关，提示其在甲状腺癌发生发展中可能具有重要的功能。
Siglec-15的一个主要作用是通过激活信号转导和转录激活子3（STAT3）通路来促进肿瘤细胞的增殖和转移。
STAT3是一种关键的调节因子，在多种肿瘤中被发现过度激活。
Siglec-15结合细胞表面的糖基化修饰物，进而通过下游蛋白激酶激活STAT3通路，增强细胞的增殖和生存能力。
此外，激活STAT3通路还可以通过影响细胞外基质蛋白的合成与分解、上皮间质转化等途径，促进甲状腺癌细胞的转移。
研究表明，通过干扰Siglec-15与细胞表面糖基化修饰物的结合以及干扰STAT3通路的激活，可以有效抑制甲状腺癌细胞的增殖和转移。
近年来，一些研究已经开始探索针对Siglec-15和STAT3的新治疗策略，以期提供新的治疗选择。
其中，抗体药物是最具有潜力的治疗方法之一。
预期的结果显示，通过抑制Siglec-15的表达或使用针对Siglec-15的单克隆抗体，可以抑制甲状腺癌细胞的生长和转移，并且提高化疗和放疗等传统治疗的疗效。
此外，一些研究还发现，使用小分子化合物干扰STAT3通路的激活，也能够有效抑制甲状腺癌细胞的增殖和转移。
卡格列净分子量
卡格列净分子量1. 介绍卡格列净是一种降糖药物，属于二肽酶-4抑制剂（DPP-4抑制剂）类药物，用于治疗2型糖尿病。
卡格列净分子量是指卡格列净这种化合物的分子质量，它是评估药物性质和药物效果的重要指标。
2. 卡格列净的结构和化学性质卡格列净（英文名：Sitagliptin）的化学名称是7-[[(3R)-3-氨基-1-氧代-4-(2,4,5-三氟苯基)丁基]氨基]-3,4-二氢-[1,2,4]噻二嗪-4-羧酸钠盐。
卡格列净的分子式为C16H15F6N5O4S，并且其结构式如下所示：卡格列净是一种白色结晶性粉末，可溶于水和甲醇，不溶于乙醇和氯仿。
它具有稳定的化学性质，在常规药物保存条件下不易发生分解。
3. 卡格列净的生物活性和作用机制卡格列净是一种高选择性的DPP-4抑制剂，通过抑制DPP-4的活性来提高胰岛素的分泌和降低胰岛素升高激素（GLP-1）的降解。
这种药物具有以下作用机制：•抑制糖尿病患者肝脏中葡萄糖生成酶的活性，从而减少血糖的生成；•促进胰岛素释放，增加胰岛素的分泌量；•阻止胰岛素升高激素的降解，延长其半衰期，从而增加GLP-1的浓度；•增加肠道中GLP-1的分泌，提高胰岛素的释放。
通过这些作用机制，卡格列净可以显著降低血糖水平，改善2型糖尿病患者的胰岛素分泌功能。
4. 卡格列净分子量的意义和测定方法卡格列净分子量是指卡格列净分子的质量，通常以Dalton（Da）为单位表示。
卡格列净分子量的测定对药物的生产和质量控制非常重要。
目前，常用的测定卡格列净分子量的方法有质谱法、核磁共振法和高效液相色谱法等。
其中，质谱法是最常用的测定方法。
质谱法通过测量药物分子的质荷比来确定分子量，并可以进一步研究药物的碎裂和结构。
5. 卡格列净分子量的应用卡格列净分子量的确定对于药物研发、药代动力学及药物效果评价等方面具有重要意义。
在药物研发方面，药物的分子量可以作为药物设计和合成的参考。
通过调整药物分子量，可以改变药物的药代动力学性质，进而提高药物的疗效和安全性。
西格列汀结构式
西格列汀结构式西格列汀（Sildenafil）是一种用于治疗男性勃起功能障碍的药物，常见的商品名为“伟哥”。
它属于磷酸二酯酶-5（PDE-5）抑制剂类药物，通过抑制PDE-5酶的活性，增加一氧化氮（NO）的生物效应，从而促进阴茎海绵体的血管舒张，增加血液流量，帮助男性实现和维持足够的勃起。
西格列汀的结构式如下：西格列汀的化学名称为1-[[3-(6,7-二甲基-1,3-二氮杂苯并[2,3-d]哌啶-4-基)基]-4-甲基苯基]甲基]-4-哌啶醇。
它的分子式为C22H30N6O4S，相对分子质量为474.58。
西格列汀为白色结晶性粉末，在水中溶解度较高。
西格列汀的作用机制是通过抑制PDE-5酶的活性，增加一氧化氮（NO）的生物效应。
在性刺激下，一氧化氮释放增加，促使环磷酸鸟苷（cGMP）水平升高。
cGMP是一种介导海绵体血管舒张的重要物质，它通过激活蛋白激酶G（PKG）来促进血管平滑肌的松弛。
而PDE-5酶主要负责降解cGMP，因此西格列汀的作用是通过抑制PDE-5酶，使cGMP得以积累，进而增加海绵体血管的舒张，增加血液流量，从而促进勃起。
西格列汀的用法是口服，一般建议在需要性活动前30分钟至4小时内服用。
剂量一般为50毫克，根据个体反应可适当调整剂量。
不建议每天超过一次服用。
虽然西格列汀在治疗男性勃起功能障碍方面取得了显著的成效，但它并非适用于所有人群。
对于存在心血管疾病、严重肝肾功能损害、视网膜病变等的患者，应谨慎使用或避免使用西格列汀。
此外，西格列汀也存在一些常见的副作用，如头痛、面部潮红、消化不良等。
总的来说，西格列汀是一种有效治疗男性勃起功能障碍的药物，通过抑制PDE-5酶的活性，增加一氧化氮的生物效应，从而促进阴茎海绵体的血管舒张，增加血液流量，帮助男性实现和维持足够的勃起。
但在使用时需谨慎，遵循医生的指导，并注意可能的副作用和禁忌症。
阿法替尼中文说明书
【药物名】Afatinib（阿法替尼）【商品名】Gilotrif【美国上市时间】非小细胞肺癌，2013年【类别】激酶抑制剂【分子式】C32H33ClFN5O11【靶点】EGFR【生产公司】Boehringer Ingelheim Pharmaceuticals 勃林格殷格翰公司【购买地】美国【剂型和规格】口服片剂，规格有：40mg/片、30mg/片、20mg/片。
40毫克药片：浅蓝色，薄膜包衣，圆形双凸面，斜角边。
一面有“T40”字样，另一面标有勃林格殷格翰的标志，国家药品验证号NDC: 0597-0138-30。
30毫克药片：深蓝色，薄膜包衣，圆形双凸面，斜角边。
一面有“T30”字样，另一面标有勃林格殷格翰的标志，国家药品验证号NDC: 0597-0137-30。
20毫克药片：白色到浅黄色，薄膜包衣，圆形双凸面，斜角边。
一面有“T20”字样，另一面标有勃林格殷格翰的标志，国家药品验证号NDC: 0597-0141-30。
【适应症和用法】EGFR突变阳性，转移性非小细胞肺癌。
使用限制：目前没有数据支持阿法替尼能够用于治疗肿瘤含有其他EGFR突变的病人; 铂化疗后的转移性非小细胞肺鳞癌。
【用法用量】病人的选择：根据病人肿瘤切片中EGFR19号外显子缺失或21号外显子替换突变的样式。
推荐剂量：口服40毫克/次/天，直到出现耐受性或者疾病的进展。
患者有严重的肾损伤（肾小球滤过率为15到29 毫升/分钟/1.73 m2）：推荐剂量为：口服30毫克/次/天，一天口服一次。
用药时间：饭前1小时或餐后2小时。
在错过一剂量用药的十二个小时内不要进行下次用药。
出现副反应时的剂量调整：出现任何如下副反应，请立即停止用药：•3级或者更高级别的副作用•2级或更高级别腹泻；也可以在服用抑制腹泻药物的同时，持续坚持2天或两天以上•持续超过7天或难以忍受的皮肤反应•2级或者更高级的肾损伤当副作用降为1级或者回到基准线水平或者患者恢复正常状态时，恢复给药；但是剂量需要减少，如比原剂量减少10毫克/次/天。
伊立替康分子量
伊立替康分子量
伊立替康分子量是多少？这是很多人关心的问题。
作为一款常被用于治疗糖尿病的口服药物，伊立替康的分子量对药物的疗效和副作用产生重要影响。
1. 什么是伊立替康？
伊立替康（Empagliflozin）是一种双吡嗪类抑制剂，用于治疗2型糖尿病。
它起到促进肾脏排泄过多的糖分，从而降低血糖水平的作用。
2. 伊立替康的分子结构
伊立替康的化学名为（编号：761423-87-4）-（S）-1，5-二羟甲基-4-（4-甲基苯基）-2- [4-（三氟甲基）苯氧]吡咯烷-3-羧酸二乙酯。
其分子式为C23H27F3O7，分子量为僅僅450.46。
3. 伊立替康分子量和药效的关系
伊立替康分子量较小，因此可以更容易地进入肾脏和其他器官。
这种小分子量也是许多糖尿病患者更喜欢选择口服药物的原因之一。
由于其分子量越小，越容易口服，被吸收和运输到靶细胞位置。
4. 小结
伊立替康分子量为僅僅450.46，这使得它能够更容易地进入肾脏和其他器官，发挥其治疗糖尿病的作用。
因此，掌握伊立替康分子量是非常重要的，关于伊立替康的更多知识应该通过各种途径，包括药品说明书和医疗专业人员来获取。
达格列净原研标准
达格列净原研标准一、活性成分达格列净（Dapagliflozin）是一种钠-葡萄糖协同转运蛋白2（SGLT2）抑制剂，化学结构式为：N-[(1S)-1-（4-chloro-3-(trifluoromethyl)phenyl)-2-propynyl]tetrahydro-2H-pyran-4-carboxamide。
二、性状达格列净为白色至类白色薄膜衣片，除去包衣后显白色。
三、鉴别1. 取本品细粉适量（约相当于达格列净50mg），加甲醇10ml，超声处理5分钟，滤过，取滤液作为供试品溶液；另取达格列净对照品适量，加甲醇溶解并制成每1ml中含0.2mg的溶液，作为对照品溶液。
照薄层色谱法试验，吸取上述两种溶液各10μl，分别点于同一硅胶G薄层板上，以乙酸乙酯-甲醇-水（60:35:5）为展开剂，展开后，晾干，置五氧化二磷干燥器中减压干燥，在105℃干燥至恒重，供试品溶液所显主斑点的位置和颜色应与对照品溶液的主斑点一致。
2. 取含量测定项下的供试品溶液，照紫外-可见分光光度法测定，在287nm 的波长处有最大吸收。
四、纯度按高效液相色谱法测定，本品按干燥品计算，含C20H25ClF3NO3应为99.0%～101.0%。
五、有关物质按高效液相色谱法测定，本品中的有关物质主要包括杂质A（对氯苯乙酰胺）、杂质B（4-氯-3-(三氟甲基)苯乙酮）、杂质C（4-氯-3-(三氟甲基)苯丙酮酸）、杂质D（N-[(1R)-1-（4-氯-3-(三氟甲基)苯基)-2-丙ynyl]tetrahydro-2H-pyran-4-carboxamide）、杂质E（N-[(1S)-1-（4-氯-3-(三氟甲基)苯基)-2-丙ynyl]tetrahydro-2H-pyran-4-carboxamide）。
其中杂质A 的含量不得超过0.5%，杂质B、C、D、E的含量均不得超过0.1%。
六、含量按高效液相色谱法测定，本品每片中含达格列净的含量应为标示量的95.0%～105.0%。
FLLL-32产品说明书
FLLL-32；FLLL32产品编号：MB4106 质量标准：>98%,BR 包装规格：5MG;25MG 产品形式：solid 基本信息 简介：FLLL32是一种有效的 JAK2/STAT3 抑制剂。
别名：(2E,2'E)-1,1'-cyclohexylidenebis[3-(3,4-dimethoxyphenyl)-2-propen-1-one 物理性状及指标：外观：………………白色至类白色粉末溶解性：...............DMSO ：92 mg/mL (198.04 mM)；Water ：Insoluble ；Ethanol ：25 mg/mL warmed (53.81 mM) 含量： (98)储存条件：-20℃，避光防潮密闭干燥 生物活性储液配置●我司产品为非无菌包装，若用于细胞培养，请提前做预处理，除去热原细菌，否则会导致染菌。
●部分产品我司仅能提供部分信息，我司不保证所提供信息的权威性，以上数据仅供参考交流研究之用。
活性化合物操作注意事项1产品分装：您收到货物后最好不要自己进行分包，因为分包环境、包装材料等因素可能导致分包后的产品变质；如您有特殊包装要求，请在订购时候与我们客服代表阐明，当然价格会做适当调整。
对于开盖后，长期未使用的，请务必重新密封好，建议Parafilm封口膜，并按照相应储存条件使用。
如果放置时间过长，超过产品有效期，建议您重新购买，以免影响实验质量。
2储备液制备：大部分试剂的溶液形式稳定性较差，请优先采用现用现配的方式。
如需制备储存液，请选用合适溶剂，细胞培养类多选择DMSO，储备液制备完成后请于零下80摄氏度储存，一般可以稳定存在3-6个月以上。
在使用前，再对储备液进行稀释。
避免储备液反复冻融。
3细胞培养工作液制备：请根据个人需要正确计算浓度，稀释储备液或者直接用粉末配置工作液。
由于大部分化合物是脂溶性的，所以使用水性溶剂（如PBS）稀释时，可能会析出沉淀，可通过超声使固体重新溶解，不会对实验产生影响。
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分子量464.55
溶解性（25°C）
DMSO ≥ 60 mg/mL
分子式C28H32O6Water Insoluble
CAS号1226895-15-3Ethanol
储存条件3年 -20°C 粉末状
生物活性
FLLL32是一种有效的JAK2/STAT3抑制剂，IC50 <5 μM。
FLLL32在MDA-MB-231乳腺癌和PANC-1胰腺癌细胞中，下调STAT3磷酸化作用和DNA结合活性。
在MDA-MB-231异种移植的小鼠体内，FLLL32 (50 mg/kg, i.p.)显著减少肿瘤负担。
在OS-33骨肉瘤细胞异种移植的小鼠体内，FLLL32 (50 mg/kg, i.p.)通过靶向作用于STAT3，也会抑制肿瘤生长。
不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）
小鼠大鼠兔豚鼠仓鼠狗
重量 (kg)0.020.15 1.80.40.0810
体表面积 (m)0.0070.0250.150.050.020.5
K系数36128520
动物 A (mg/kg) = 动物 B (mg/kg) ×
动物 B的K系数
动物 A的K系数
例如，依据体表面积折算法，将白藜芦醇用于小鼠的剂量22.4 mg/kg 换算成大鼠的剂量，需要将22.4 mg/kg 乘以小鼠的K系数（3），再除以大鼠的K系数（6），得到白藜芦醇用于大鼠的等效剂量为11.2 mg/kg。
参考文献
A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells.
Lin L, et al. Mol Cancer. 2010 Aug 16;9:217. PMID: 20712901.
Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells.
Lin L, et al. Cancer Res. 2010 Mar 15;70(6):2445-54. PMID: 20215512.
FLLL32 目录号M9142
化学数据
2
m
m
m
m m。