Effects Of Huoxue Qingjie Decoction On The TGR5-NLRP3 Signaling Pathway And Bile Acid Metabolism In Cholestatic Liver Injury

ObjectiveThe purpose of this study is to explore the therapeutic effect of Huoxue Qingjie decoction on cholestatic liver injury induced by ANIT and the role of TGR5 in cholestatic liver injury by TGR5-NLRP3-Caspase-1 pathway.We also investigate the mechanism of bile acid metabolism in rats with cholestasis and the effect of Huoxue Qingjie Decoction on bile acid metabolism.MethodWistar male rats were randomly divided into 6 groups,namely the control group,the model group,the compound Glycyrrhizin group,and the low,medium,high dose of Huoxue Qingjie decoction group(4.1 g·kg^-1,8.1 g·kg^-1,16.2 g·kg^-1).The cholestatic model was prepared by ANIT once.Compound Glycyrrhizin and Huoxue Qingjie decoction were administered after modeling for 4 days.On the fifth day,the rats were anesthetized,and bile,serum,liver tissue were collected after bile duct intubation.TBIL,TBA,DBIL,IBIL and ALT in serum were detected by automatic biochemical analyzer.Hepatic histopathological changes were observed by HE staining.The expression of TGR5,NLRP3,Caspase-1,α-SMA and NTCP in liver tissues were analyzed by Western blot and Immunohistochemical.The flow rate of bile was calculated by observing the volume and retention time of bile.The contents of 18 bile acids in bile were determined by HPLC-MS/MS.Results（1）Compared with the control group,the bile flow rate in the model group decreased significantly（P<0.01）.Compared with the model group,the bile flow rate of Huoxue Qingjie decoction high dose group increased significantly（P<0.05）.（2）Compared with the control group,the serum levels of TBIL,DBIL,IBIL,ALT,and TBA in the model group were significantly increased（P<0.01）.Compared with the model group,the serum indexes of the Huoxue Qingjie decoction high dose group were decreased significantly（P<0.05）.（3）The structure of liver tissue was complete and the cell morphology was normal in the control group.In the model group,the bile duct proliferated and expanded in the portal area,a large number of inflammatory cells were infiltrated,and both the bile duct and bile duct epithelial cells showed some morphological changes.The compound Glycyrrhizin group and Huoxue Qingjie decoction group had different degrees of improvement in the above lesions,and the Huoxue Qingjie decoction high dose group had significant improvement in the medium and high dose groups.（4）Compared with the control group,the expressions ofα-SMA were significantly increased（P<0.01）.Compared with the model group,the expression ofα-SMA in the high-dose group of Huoxue Qingjie decoction was significantly decreased（P<0.01）.（5）Compared with the control group,the expressions of NLRP3 in the model group was significantly decreased（P<0.01）,while the expressions of TGR5 and Caspase-1were significantly increased（P<0.01）.Compared with the model group,the expression of NLRP3 in the high-dose groups of Huoxue Qingjie decoction was significantly increased（P<0.01）,while the expression of TGR5 and Caspase-1 in the high-dose group of Huoxue Qingjie decoction was significantly decreased（P<0.05）.（6）Compared with the control group,the expressions of NTCP in the model group was significantly decreased（P<0.01）.Compared with the model group,NTCP in the high-dose groups of Huoxue Qingjie decoction was significantly increased（P<0.05）.（7）Compared with the control group,all the bile acids concentrations decreased except for the increase TCDCA content in the model group（P<0.05）.Compared with the model group,the DCA,TUDCA,GUDCA,THDCA,GCA concentrations were increased,while the concentration of TCDCA was significantly reduced in Huoxue Qingjie decoction high dose group（P<0.05）.Conclusion（1）UDCA can effectively treat cholestatic liver injury by inhibiting liver inflammation,reducing liver cell injury,anti-fibrosis,promoting bile secretion,affecting bile acid spectrum composition,and actively regulating intrahepatic bile acid metabolism,providing experimental basis for the cholagogic effect of this Chinese medicine compound.（2）In the ANIT-induced cholestasis liver injury model,the high expression of TGR5in liver tissue significantly inhibited the expression of NLRP3,further demonstrating the association.（3）Different bile acids have different effects on cholestatic liver injury,which may be caused by specific changes in TCDCA concentration of bile acid.