A Preliminary Study On The Role And Mechanism Of TGR5 In Intrahepatic Bile Duct Fibrosis Induced By Bile Salt

Objective:To determine the effect of TGR5 receptor on bile duct fibrosis in patients with hepatolithiasis and to preliminary discuss the molecular mechanism of signal transduction.Methods:1.The liver samples of 20 patients with hepatolithiasis and 20 patients with non-stone benign liver diseases were collected.HE staining and Masson staining were used to evaluate the difference of liver pathological changes and fibrosis between patients with hepatolithiasis and patients with non-stone benign liver disease.2.Immunofluorescence,Westernblot and RT-qPCR techniques were used to detect the expression of TGR5 receptor in liver of patients with hepatolithiasis and benign liver disease without hepatolithiasis.3.Culture of human intrahepatic bile duct epithelial cells(Human intrahepatic bile duct epithelial cell,HIBEC),The TGR5 overexpression vector(TGR5-PLVX-IRES-PURO)was constructed and the HIBEC stably expressing TGR5 was constructed by lentivirus transfection into HIBEC cells.A stable HIBEC cell line silencing TGR5 was constructed by interfering with TGR5 gene by sh TGR5 lentivirus.The overexpression and Silence effect of TGR5 were verified by RT-qPCR and Western blot techniques.4.After HIBEC cells were treated with taurodeoxycholic acid(TDCA)with concentration of 100μM and 200μM for 12 hours and 24 hours respectively,total cell proteins were extracted,and Western blot technique was used to detect the activation effect of TDCA on TGR5.5.HIBEC was divided into blank control group(untreated),positive control group(TDCA treated group),TDCA+TGR5 overexpression group(TDCA+HIBEC-TGR5)and TDCA+TGR5 silence group(TDCA+HIBEC-sh-TGR5).According to the experimental results of method 4,the TDCA processing time was selected to be treated with 100μM for 12 hours.RT-qPCR and Western blot techniques were used to verify the differences in the expression of fibrosis-related molecules such as transforming growth factor-β 1(TGF β1),fibronectin(FN)and type Ⅳ collagen(Col-Ⅳ),and the difference of phosphorylation of CREB.Results:1.HE staining showed that in patients with non-stone benign liver disease,the structure of hepatic lobules was intact,the hepatocytes were arranged neatly,and a small amount of inflammatory factors could be seen.Severe degeneration and necrosis of hepatocytes,dilatation of hepatic sinusoids with cholestasis,infiltration of inflammatory cells in portal area,including neutrophils and lymphocytes,and obvious proliferation of connective tissue around bile duct.2.Masson staining results: the proportion of blue-stained collagen in hepatolithiasis group was significantly higher than that in non-stone benign liver disease group(P <0.01).3.The results of immunofluorescence showed that the expression of TGR5 in liver of patients with hepatolithiasis was significantly higher than that of patients with benign liver disease without hepatolithiasis(P<0.001,P<0.01,P<0.01).4.The results of RT-qPCR showed that the expression of TGR5 mRNA in liver of patients with hepatolithiasis was significantly higher than that of patients with benign liver disease without hepatolithiasis.(P<0.01)5.The results of WB showed that the protein expression of TGR5 in liver of patients with hepatolithiasis was significantly higher than that of patients with benign liver disease without hepatolithiasis(P<0.01).6.The expression of TGR5 receptor in HIBEC treated by TDCA: the expression of TGR5 receptor in HIBEC treated with TDCA at the concentration of 100μM and200μM for 12 hours and 24 hours was significantly higher than that in the control group without TDCA treatment.When treated with TDCA at the concentration of 100μM for12 hours,the expression of TGR5 receptor was relatively high.7.The expression of fibrosis-related molecules(TGF β1,FN,Col-Ⅳ)was detected by RT-qPCR and WB.Results: after HIBEC was treated with TDCA,the expression of TGF β1,FN and Col-Ⅳ was increased(P < 0.05,P < 0.01).Compared with the positive control group,overexpression of TGR5 further increased the expression levels of TGF β1,FN and Col-Ⅳ(P < 0.05,P < 0.01,P < 0.001).After silencing TGR5,the expression levels of TGF β1,FN and Col-Ⅳ decreased significantly(P < 0.05,P < 0.01,P < 0.001).8.The phosphorylation level of CREB was detected by WB: TDCA increased the phosphorylation level of CREB(P < 0.01).Overexpression of TGR5 further increased the phosphorylation level of CREB.After silencing TGR5,the phosphorylation level of CREB was significantly decreased(P < 0.05,P < 0.01).Conclusion:TGR5 is highly expressed in the liver of patients with hepatolithiasis.To sum up,bile salt promotes intrahepatic bile duct fibrosis by activating TGR5,which may be related to CREB phosphorylation.