| Objective:Perfroming bile duct ligation to induce cholestatic liver injury and exploring the expression of CTGF in BDL-induced cholestatic liver fibrosis.Method: Purchasing twelve male Sprague Dawley rats(body weight 200±10g)from the Experimental Animal Center of Tongji Medical College.We induced rats cholestatic liver injury through performing bile duct ligation(BDL).Performing H&E to evaluate the cholestasis model,Sirius Red,hydroxyproline assay,immunohistochemical,western blot,q RT-PCR to assess the level of fibrosis.The expression level of CTGF was checked by immunohistochemical,western blot,q RT-PCR.Results:The BDL model used to induce cholestatic liver injury was successfully constructed.Obvious liver fibrosis was observed 4 weeks post BDL,and BDL induced the enhanced expression of CTGF in livers and CTGF was mainly expressed in hepatocytes.Objective:To explore the mechanism by which conjugated bile acids regulate the expression of CTGF in hepatocyte.Method: Using mouse hepatocyte AML12 and rat hepatocyte BRL to perform experiments in vitro.AML12 and BRL were treated with conjugated bile acids.Performing western blot,q RT-PCR,luciferase gene reporter containing the fragment of CTGF promoter to check the effect of conjugated bile acid on CTGF.To explore which pathways could be activated by conjugated bile acids,we used western blot,then combining specific inhibitors and si RNA to check the pathways responsible for conjugated bile acids-induced CTGF.The activity of respective pathways was confirmed in the livers of BDL and sham rats using IHC,western blot and q RT-PCR.Results:Conjugated bile acids could enhance the synthesis of CTGF in AML12 and BRL hepatocytes.What’s more,we performed western blot and found that conjugated bile acids could activate ERK,Akt,p38 MAPK and YAP signaling pathways,but not JNK,TGFβ/Smad signaling pathways in AML12 and BRL hepatocytes.And ERK activity inhibitor U0126 and YAP function inhibitors Verteporfin(VP)could block the upregulation of conjugated bile acids on CTGF.Meanwhile,U0126 could partly inhibit the basal level of YAP and conjugated bile acids-induced YAP,but YAP function inhibitors VP had no effect on the activity of ERK.The activities of ERK,YAP were further confirmed in the livers of BDL rats,and the expression of ERK,YAP were partly co-expressed in hepatocytes.What’s more,Akt signaling pathway inhibitor MK2206 could not only enhance the upregulation of CTGF induced by conjugated bile acids but also the basal level of CTGF in AML12 and BRL hepatocytes,suggested that Akt signaling pathway could inhibit the expression of CTGF in normal physiological status.Furthermore,we found that Akt signaling pathway inhibitor MK2206 could enhance the activity of ERK signaling pathway and the level of YAP,on the contrary,U0126 and VP didn’t influence the Akt signaling pathway. |
