基于FXR信号通路研究藏药十五味赛尔斗丸对胆汁淤积型肝损伤大鼠的作用机制

Study on Mechanism of Tibetan Medicine Shiwuwei Sai’erdou Pills on Liver Injury with Cholestatic in Rats Based on FXR Signaling Pathway

目的 评价藏药十五味赛尔斗丸对 α-萘异硫氰酸酯(ANIT)诱导的胆汁淤积型肝损伤大鼠的影响,并探讨其作用机制。方法 将雄性 Wistar 大鼠随机分为空白对照组、模型对照组、熊去氧胆酸(60 mg·kg^(-1))组、藏药十五味赛尔斗丸低、中、高(0.262 5、0.525、1.05 g·kg^(-1))剂量组,各组每天灌胃相应药物 1 次,连续 9 d。至第 7 天除空白对照组外,其余各组灌胃 60 mg·kg的 ANIT 试剂进行模型复制。观察大鼠的生活状态并计算肝脏指数;HE 染色分析大鼠肝脏组织病理变化;检测血清中碱性磷酸酶(ALP)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBIL)、直接胆红素(DBIL)与总胆汁酸(TBA)含量;Elisa 法检测大鼠肝组织中超氧化物歧化酶(SOD)、还原性谷胱甘肽(GSH)与丙二醛(MDA)的含量;采用 qRT-PCR 法和 Western-Blot 法测定大鼠肝组织中法尼醇 X 受体(FXR)、Tgr5、CYP7A1、Mrp2、BSEP 与 NTCP mRNA 及蛋白表达情况。结果与空白对照组比较,模型对照组大鼠肝脏指数明显升高(P<0.01),肝索结构处肝细胞核严重皱缩且坏死细胞较多,血清中 ALT、AST、ALP、TBIL、DBIL 和 TBA 含量明显升高(P<0.01),肝组织中 SOD、GSH 含量均降低(P<0.05,P<0.01),MDA 含量明显升高(P<0.01),肝组织中 FXR、Tgr5、NTCP、BSEP 与 Mrp2 的 mRNA相对表达明显下调(P<0.01),CYP7A1 的 mRNA 相对表达明显上调(P<0.01),肝组织中 FXR、Tgr5、NTCP、BSEP 与 Mrp2 蛋白表达量均降低(P<0.05,P<0.01),CYP7A1 蛋白表达量明显上升(P<0.01)。与模型对照组比较,十五味赛尔斗丸各剂量组大鼠肝脏指数明显降低(P<0.01),肝索结构清晰,肝细胞排列紧密,未见明显坏死,血清中肝功能指标、肝脏中氧化应激指标的水平均有不同程度改善(P<0.05,P<0.01),十五味赛尔斗丸各剂量组均可下调肝组织中 CYP7A1 的 mRNA 表达(P<0.05,P<0.01),上调 BSEP mRNA 表达(P<0.05,P<0.01),中、高剂量组可明显上调 FXR mRNA 表达(P<0.01),且高剂量组可上调 Tgr5、BSEP与 Mrp2 mRNA 表达(P<0.05),高剂量组可上调肝组织中 FXR、Tgr5、NTCP、BSEP 与 Mrp2 蛋白表达量(P<0.05),下调 CYP7A1 蛋白表达量(P<0.05)。结论 藏药十五味赛尔斗丸对胆汁淤积型肝损伤大鼠具有明显的保护作用,其机制可能是通过激活 FXR 及其下游的转运体与代谢酶,达到维持胆汁酸稳态的目的。

Objective To evaluate the effect of Shiwuwei Sai’erdou Pills on α-naphthalene isothiocyanate(ANIT)-induced liver injury with cholestatic in rats and explore its mechanism. Method Wistar rats were randomly divided into blank control group,model control group,ursodeoxycholic acid(60 mg·kg^(-1))group,low-,medium- and highdose(0.262 5,0.525,1.05 g·kg^(-1))Shiwuwei Sai’erdou Pills groups. The corresponding drugs were intragastrically administrated once a day for consecutive 9 days. On the seventh day,except the blank control group,the other groups were given 60 mg·kgANIT to copy rat models. The living conditions of the rats were observed and the liver index was calculated. HE staining was used to analyze the pathological changes in rat liver tissue. The serum levels of alkaline phosphatase(ALP),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),direct bilirubin(DBIL)and total bile acids(TBA)were detected. The contents of SOD,GSH and MDA in rat liver tissue were detected by Elisa. The mRNA and protein expressions of FXR,TGR5,CYP7A1,Mrp2,BSEP and NTCP in rat liver tissue were measured by qRT- PCR and Western- Blot. Results Compared with the blank group,the liver index of rats in the model group was significantly increased(P<0.01),the hepatocyte nuclei at the hepatic- cord structure were severely shrunken and more necrotic cells were found, the contents of ALT, AST,ALP,TBIL,DBIL and TBA in serum were significantly increased(P<0.01),the contents of SOD and GSH in liver tissue were decreased(P<0.05,P<0.01)and the content of MDA was significantly increased(P<0.01). The relative mRNA expressions of FXR,Tgr5,NTCP,BSEP and Mrp2 in liver tissue were significantly down-regulated(P<0.01),and the relative mRNA expression of CYP7A1 was significantly up-regulated(P<0.01). The protein expressions of FXR,Tgr5,NTCP,BSEP and Mrp2 in liver tissue were all decreased(P<0.05,P<0.01),and the protein expression of CYP7A1 was significantly increased(P<0.01). Compared with the model group,the liver index of rats in the Shiwuwei Sai’erdou Pills administration groups was significantly reduced(P<0.01). The hepaticcord structures were clear, and the hepatocytes were closely arranged. There was no significant necrosis was observed. The serum test index of liver function and oxidative stress index of liver were improved to different degrees(P<0.05,P<0.01). The mRNA expression of CYP7A1 in liver tissue was down-regulated(P<0.05,P<0.01)and the m RNA expression level of BSEP was up- regulated(P<0.05, P<0.01) by Shiwuwei Sai’erdou Pills at different concentrations. The FXR mRNA expression levels were significantly increased in the medium- and highdose Shiwuwei Sai’erdou Pills groups(P<0.01). Besides,high-dose Shiwuwei Sai’erdou Pills could up-regulate the mRNA expressions of Tgr5,BSEP and Mrp2(P<0.05). It could also up-regulate the protein expressions of FXR,Tgr5,NTCP,BSEP and Mrp2 in liver tissue(P<0.05)and down-regulate the protein expression of CYP7A1(P<0.05). Conclusion Shiwuwei Sai’erdou Pills has obvious protective effect on cholestasis type liver injury in rats,the mechanism may be related to the activation of FXR and its downstream transporters and metabolic enzymes,resulting in maintaining bile acid homeostasis.