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2019, v.44(04) 787-795

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基于Sirt1-FXR通路探究京尼平苷酸对胆汁淤积大鼠胆汁酸肝肠循环的影响
Effect of geniposidic acid on hepato-enteric circulation in cholestasis rats through Sirt1-FXR signaling pathway

陈浩;李甲;胡蕾;赵威;俞浩;刘汉珍;马世堂;
CHEN Hao;LI Jia;HU Lei;ZHAO Wei;YU Hao;LIU Han-zhen;MA Shi-tang;Anhui Science and Technology University;Institute of Clinical Pharmacology,Guangzhou University of Chinese Medicine;

摘要(Abstract):

通过体内外结合的方式探究京尼平苷酸(GPA)对胆汁淤积大鼠胆汁酸肝肠循环作用影响及其基于去乙酰化酶1(Sirt1)-法尼醇X受体(FXR)通路的作用机制探究。将60只SD大鼠随机分为6组,分别为空白对照组、α-萘异硫氰酸酯(ANIT)模型组、阳性对照组(100 mg·kg~(-1)·d-1UDCA),以及100,50,25 mg·kg~(-1)·d-1GPA高、中、低剂量组,每组10只,给药10d。在第8天药后,除空白大鼠其余均一次性灌胃65 mg·kg~(-1)ANIT,末次药后,测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、γ-谷氨酰转移酶(γ-GGT)和碱性磷酸酶(ALP)活性以及总胆红素(TB)和总胆汁酸(TBA)的含量; RT-PCR法检测肝组织中胆汁酸肝肠循坏关键基因Sirt1,FXR,多药耐药相关蛋白2(MRP2),胆盐输出泵(BSEP),钠离子-牛磺胆酸协同转运多肽(NT-CP),回肠中顶端钠-胆汁酸转运体(ASBT),回肠胆汁酸结合蛋白(IBABP) mRNA转录水平的影响;免疫荧光三染法检测MRP2,BSEP和NTCP在肝脏中表达,ASBT,IBABP在回肠中表达; Western blot法检测肝组织中Sirt1,FXR表达;体外培养原代肝细胞观察GPA对EX 527(Sirt1抑制剂)抑制与否,RT-PCR和Western blot法检测细胞中Sirt1和FXR的mRNA和蛋白表达。在体实验表明,GPA能显著降低或改善ANIT诱导的胆汁淤积大鼠血清ALT,AST,γ-GGT,ALP活性和TB,TBA的含量(P<0. 01)以及肝组织病理损伤; GPA能显著升高肝组织Sirt1,FXR,MRP2,BSEP,NTCP和回肠中ASBT,IBABP mRNA转录水平和蛋白表达(P<0. 01);体外原代肝细胞实验表明,EX-527能通过抑制原代肝细胞中Sirt1基因和蛋白功能来抑制FXR基因和蛋白表达(P<0. 01),GPA可显著提高改善EX-527抑制的原代肝细胞中Sirt1和FXR基因和蛋白的表达(P<0. 01)。以上结果发现GPA的改善作用呈剂量正相关性。GPA可改善ANIT诱导的胆汁淤积大鼠的胆汁酸肝肠循环发挥保肝利胆作用,其可能机制是GPA通过激活改善氧化应激损伤关键调控基因Sirt1来激活核受体FXR,激活的FXR再调控胆汁酸肝肠循环的相关蛋白来实现。
To investigate the effects of geniposidic acid( GPA) on hepato-enteric circulation in cholestasis rats,and to explore the mechanism based on the sirtuin 1( Sirt1)-farnesol X receptor( FXR) pathway,sixty SD rats were randomly divided into 6 groups:blank control group,ANIT model group,ursodeoxycholic acid group( 100 mg·kg~(-1)·d-1 UDCA),and GPA high,medium and low( 100,50 and 25 mg·kg~(-1)·d-1) dosage groups,10 rats in each group. Corresponding drugs were intragastrically( ig) administered for10 days. After administration on day 8,all rats except blank rats were administered with 65 mg·kg~(-1)α-naphthalene isothiocyanate( ANIT) once. After the last administration,the serum levels of alanine aminotransferase( ALT),glutamine oxalacetate aminotransferase( AST),gamma-glutamyltransferase( γ-GGT),alkaline phosphatase( ALP),total bilirubin( TB) and total bile acid( TBA)were measured,and the mRNA transcription levels of Sirt1,FXR,multidrug resistant associated protein 2( MRP2),bile salt export pump( BSEP),sodium taurocholate contractible polypeptide( NTCP) in liver and apical sodium bile acid transporter( ASBT),ileum bile acid binding protein( IBABP) in ileum were detected by reverse transcription-polymerase chain reaction( RT-PCR). The protein expression levels of Sirt1,FXR and NTCP were detected by Western blot; the expression of MRP2,BSEP in liver and ASBT,IBABP in ileum were determined by immunofluorescence three staining. Primary rat hepatocytes were cultured in vitro to investigate the inhibitory effect of GPA on a potent and selective Sirt1 inhibitor( EX 527),and the mRNA and protein expression levels of Sirt1 and FXR were detected by RT-PCR and Western blot. GPA significantly decreased the levels of ALT,AST,γ-GGT,ALP,TB,TBA in serum( P<0.01) and improved the pathological damage of liver tissues in ANIT-induced cholestasis rats; significantly increased the mRNA and protein expression levels of Sirt1,FXR,MRP2,BSEP,NTCP in liver and ASBT,IBABP in ileum( P< 0.01). In vitro primary hepatocytes experiment indicated that the gene and protein expression levels of FXR and Sirt1 were noticeably improved by GPA in primary hepatocytes inhibited by EX-527( P<0.01). It was found that the improvement of GPA was in a dose-dependent manner. GPA could improve bile acid hepatointestinal circulation and play a liver protection and cholagogu role in cholestasis rats induced by ANIT.The mechanism may be that GPA activated FXR by regulating Sirt1,a key regulator of oxidative stress injury,and then the activated FXR could regulate protein of bile acid hepato-enteric circulation.

关键词(KeyWords): 京尼平苷酸;法尼醇X受体;去乙酰化酶1;胆汁淤积;肝肠循环
geniposidic acid;farnesoid X receptor;sirtuin 1;cholestasis;hepato-enteric circulation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81403268);; 安徽省教育厅高校自然科学研究重点项目(KJ2018A0528);; 安徽科技学院稳定高层次人才项目(SPYJ201701);安徽科技学院校级大学生创新训练项目(2017X059)

作者(Author): 陈浩;李甲;胡蕾;赵威;俞浩;刘汉珍;马世堂;
CHEN Hao;LI Jia;HU Lei;ZHAO Wei;YU Hao;LIU Han-zhen;MA Shi-tang;Anhui Science and Technology University;Institute of Clinical Pharmacology,Guangzhou University of Chinese Medicine;

Email:

DOI: 10.19540/j.cnki.cjcmm.20181204.013

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