基于FXR信号通路研究藏族药二十五味松石丸对胆汁淤积型肝损伤大鼠的保护作用机制

Study on protective mechanism of Tibetan medicine Ershiwuwei Songshi Pills on cholestatic liver injury in rats based on FXR signaling pathway

基于法尼醇X受体(FXR)信号通路探讨藏族药(以下简称"藏药")二十五味松石丸对α-萘异硫氰酸酯(ANIT)致大鼠胆汁淤积型肝损伤的保护作用及机制。将SD大鼠随机分为空白组、模型组、熊去氧胆酸(UDCA)组以及藏药二十五味松石丸低、中、高剂量组(0.09,0.18,0.36 g·kg^-1),实验采用预防性给药方案。第1~4天UDCA组和藏药二十五味松石丸混悬液各组分别预防性灌胃给药;第5天空白组给予等体积橄榄油空白试剂,其余各组给予ANIT造模试剂,各给药组第5~6天继续给药2 d;第7天造模后48 h,大鼠股动脉取血。检测血清中碱性磷酸酶(ALP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、直接胆红素(DBIL)、总胆红素(TBIL)、总胆汁酸(TBA)水平,观察肝脏组织病理学变化。通过实时荧光定量法检测肝组织中相关基因FXR,SHP,CYP7A1,MRP2,MRP3,NTCP,BSEP mRNA相对表达量的变化,采用Western blot法检测肝组织中FXR,SHP,UGT2B4蛋白表达量的变化。结果显示,藏药二十五味松石丸能显著降低ANIT模型大鼠血清中ALT,ALP,DBIL,TBIL,TBA的含量水平(P<0.01,P<0.05);SHP和NTCP mRNA表达显著上调(P<0.01,P<0.05),CYP7A1和MRP3 mRNA表达显著下调(P<0.01,P<0.05);FXR,SHP的蛋白表达均显著上调(P<0.01,P<0.05)。藏药二十五味松石丸对ANIT致大鼠胆汁淤积型肝损伤具有明显的保护作用,其作用机制可能与FXR信号通路介导的胆汁酸代谢相关。

This paper aimed to investigate the protective effect and mechanism of the Tibetan medicine Ershiwuwei Songshi Pills onα-naphthalene isothiocyanate(ANIT)-induced cholestatic liver injury in rats based on the farnesol X receptor(FXR)signaling pathway.SD rats were randomly divided into blank group,model group,ursodeoxycholic acid(UDCA)group,Tibetan medicine Ershiwuwei Songshi Pills low,medium and high dose groups(0.09,0.18,0.36 g·kg^-1).A prophylactic dosing regimen was used in the experiment.From the 1 st to 4 th days,the UDCA group and the Tibetan medicine Ershiwuwei Songshi Pills suspension groups received prophylactic gavage administration;on the 5 th day,the blank control group was given an equal volume of olive oil blank reagent,and the remaining groups were given ANIT modeling reagent.Administration was continued on day 5 to 6 in each administration group.Forty-eight hours after modeling on the 7 th day,blood was collected from the femoral artery of rats.Serum alkaline phosphatase(ALP),alanine aminotransferase(ALT),aspartate aminotransferase(AST),direct bilirubin(DBIL),total bilirubin(TBIL),and total bile acid(TBA)levels were detected,and liver histopathological changes were observed.The relative expression changes of FXR,SHP,CYP7 A1,MRP2,MRP3,NTCP,BSEP mRNA in liver tissues were detected by Real-time fluorescence quantitative method,and the expression changes of FXR,SHP,UGT2 B4 protein in liver tissues were detected by Western blot.The results showed that the Tibetan medicine Ershiwuwei Songshi Pills significantly reduced the levels of ALT,ALP,DBIL,TBIL and TBA in the serum of the ANIT mo-del rats(P<0.01,P<0.05),significantly up-regulated the mRNA expressions of SHP and NTCP(P<0.01,P<0.05),significantly down-regulated the mRNA expression of CYP7 A1 and MRP3(P<0.01,P<0.05);and significantly up-regulated the protein expressions of FXR and SHP(P<0.01,P<0.05).The Tibetan medicine Ershiwuwei Songshi Pills have an obvious protective effect on ANIT-induced cholestatic liver injury in rats,and its mechanism may be related to the bile acid metabolism mediated by the FXR signaling pathway.