基于胆汁酸信号通路研究栀子苷治疗小鼠非酒精性脂肪性肝炎的效应机制

Study on Mechanism of Gardenoside in Treatment of Nonalcoholic Steatohepatitis Based on Bile Acid Signaling Pathway

目的观察栀子苷对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)小鼠胆汁酸代谢及其信号通路的干预作用,探讨栀子苷治疗NASH的作用机制。方法高脂饮食喂养14周诱导NASH小鼠模型,实验设正常组、模型组、栀子苷组和奥贝胆酸组,从第11周开始,栀子苷组和奥贝胆酸组小鼠分别按90 mg·kg^(-1)·d^(-1)和10 mg·kg^(-1)·d^(-1)的剂量灌胃给予栀子苷和奥贝胆酸,正常组和模型组小鼠灌胃给予等体积的饮用水。HE染色观察肝组织病理变化,生化试剂盒检测肝组织甘油三酯(triglyceride,TG)含量和血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆固醇(total cholesterol,TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、空腹胰岛素(fasting insulin,FINS)、空腹血糖(fasting blood glucose,FBG)水平,靶向代谢组学方法检测肠内胆汁酸谱,RT-PCR方法检测肝组织法尼醇X受体(Farnesoid X receptor,FXR)、小异二聚体伴侣(Small Heterodimer Partner,SHP)、固醇调节元件结合蛋白1(sterol-regulatory element binding protein 1,SREBP1)、脂肪酸合成酶(fatty acid synthase,FASN)、硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,SCD1)、肉毒碱棕榈酰基转移酶1A(carnitine Palmitoyltransferase 1A,CPT1A)、脂蛋白脂肪酶(lipoprotein lipase,LPL)、胆固醇7-羟化酶(cholesterol 7α-hydroxylase,CYP7A1)、胆汁酸盐输出泵(bile salt export pump,BSEP)、钠离子-牛磺胆酸共转运多肽(sodium taurocholate cotransporting polypeptide,NTCP)、成纤维细胞生长因子受体4(fibroblast growth factor receptor 4,FGFR4)和小肠FXR、成纤维细胞生长因子15(fibroblast growth factor 15,FGF15)的基因表达水平。结果栀子苷和奥贝胆酸均能显著改善NASH小鼠的肝组织病理变化,降低NASH小鼠肝组织TG含量及血清ALT、LDL-C、FINS、FBG水平(P<0.01)。栀子苷还能显著降低肠内容物TaMCA、TβMCA含量和肝组织SREBP1、FASN、SCD1、CYP7A1基因表达水平(P<0.01),显著升高肠内容物βCDCA的含量及小肠FXR、FGF15和肝组织FXR、SHP、BSEP、CPT1A、LPL的基因表达水平(P<0.05)。结论栀子苷能调节TaMCA、TβMCA、βCDCA等影响FXR活性的胆汁酸的表达,进而激活FXR信号通路,这可能是栀子苷治疗NASH的重要机制。

Objective To observe the intervention effect of geniposide on bile acid metabolism and bile acid signal pathway in nonalcoholic steatohepatitis(NASH)mice and explore the mechanism of geniposide in the treatment of NASH.Methods NASH models of mice were established by high-fat diet for 14 weeks,and the mice were divided into normal group,model group,geniposide group and obecholic acid group.From the beginning of the 11th week,the mice in geniposide group and obecholate group were given geniposide and obecholate by gavage at the dose of 90 mg·kg^(-1)·d^(-1)and 10 mg·kg^(-1)·d^(-1)respectively,while the mice in normal group and model group were given the same volume of drinking water by gavage for 4 weeks.HE staining was used to observe the pathological changes of liver tissue.The content of triglyceride(TG)in liver tissue and the levels of alanine aminotransferase(ALT),aspartate transaminase(AST),TG,total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),fasting insulin(FINS)and fasting blood glucose(FBG)in serum were detected by biochemical kit.Bile acid profile in intestinal content was detected by targeted metabonomics.The gene expression levels of farnesoid X receptor(FXR),small heterodimer partner(SHP),sterol regulatory element binding protein 1(SREBP1),fatty acid synthase(FASN),stearoyl COA desaturase 1(SCD1)and carnitine palmitoyltransferase 1A(CPT1A),lipoprotein lipase(LPL),cholesterol 7α-hydroxylase(CYP7A1),bile salt export pump(BSEP),sodium taurocholate cotransportering polypeptide(NTCP),fibroblast growth factor receptor 4(FGFR4),small intestine FXR and fibroblast growth factor 15(FGF15)were detected by RT-PCR method.Results Geniposide and obeticholic acid could significantly improve the pathological changes of liver tissue of NASH mice and reduce the content of TG in liver tissue and the levels of ALT,LDL-C,FINS and FBG in serum of NASH mice(P<0.01).Geniposide can also significantly reduce the contents of TaMCA and TβMCA in intestinal contents and the gene expressions of SREBP1,FASN,SCD1 and CYP7A1 in liver tissue(P<0.05),and significantly increased the content ofβCDCA in intestinal contents and the gene expressions of FXR and FGF15 in small intestine and the gene expressions of FXR,SHP,BSEP,CPT1A and LPL in liver tissue(P<0.05).Conclusion Geniposide can regulate TaMCA,TβMCA,βCDCA and other bile acids that affect FXR activity,and then activate FXR signaling pathway,which may be an important mechanism of geniposide in the treatment of NASH.