Effect And Molecular Mechanism Of PDTC On Bsep And Ntcp Expression In Rats With Ethinyl Estradiol Induced Intrahepatic Cholestasis Of Pregnancy

Object:To establish a rat model of intrahepatic cholestasis of pregnancy (ICP) by injecting17-beta-ethinyl estradiol to pregnant rats, test the expression level of nuclear kappa B p65(NF-κB p6)in liver tissue of pregnant rats, and discuss the effect and possible molecular mechanism of NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC) on the expression level of Bsep and Ntcp protein of NF-κB signaling pathway.Method:Fifty female pregnancy SD rats as experimental subjects.(1) The50rats were randomly divided into five groups of pregnant rats after15days of pregnancy, including a normal pregnancy control group, a model group, a high dose treatment group, a low dose treatment group and a high dose control group, each group having10rats and being treated as follows:the normal pregnancy control group was given subcutaneous injection and intraperitoneal injection of propylene glycol (solvent)2.5ml/kg/day in; the ICP model group was given subcutaneous injection of2.5ml/kg/day ethinyl estradiol; the high dose treatment group was given subcutaneous injection of ethinyl estradiol2.5ml/kg/day and intraperitoneal injection of PDTC100mg/kg/day; the low dose treatment group was given subcutaneous injection of ethinyl estradiol2.5ml/kg/day and given intraperitoneal injection of PDTC50mg/kg/day; and the high dose control group was given intraperitoneal injection of PDTC100mg/kg/day; and all rats were killed on day20after continuous injection for5days in total;(2) the5groups of rats were sampled blood after administration for testing the total bile acid (TBA) level, alanine aminotransferase (ALT) level, and aspartate aminotransferase (AST) level in serum; After liver tissue was fixed by formalin, Hepatic pathological change was observed using HE staining, The expression level of NF-κB p65in liver tissue was detected by immunohistochemistry; the protein expression of bile salt export pump (Bsep) and Na+-taurocholatecotransporting polypeptide (Ntcp) was measured by Western Blot.Results:①It shows that the model of ICP was successfully established through the test of TBA, ALT, AST levels in serum and observation by HE staining for rats;②various serum indexes of rats in the ICP model group all increased, but increases in TBA, ALT, AST levels were all different (p<0.05,respectively) as compared to the normal pregnancy control group;③NF-κB was blocked by PDTC treatment, and the levels of serum TBA, ALT, AST in the high dose treatment group all lowered (p<0.05,respectively) as compared to the model group;④Compared with the normal pregnancy control group, the ICP model group showed obvious morphological changes, heavy inflammatory response and liver structural disorder, while the high dose treatment group and the low dose treatment group were better than that of the model group;⑤ the nuclear kappa B p65expression was tested in cell nucleus using immunohisochemistry, which increased in the model group as compared to the normal pregnancy control group and the high dose treatment group, and the differences were significant (p<0.05,respectively);⑥Bsep and Ntcp protein expression in liver tissue was tested using Western Blot, which increased in both the normal pregnancy control groups and the high dose treatment group as compared to the normal pregnancy control group, and the differences were significant (p<0.05,respectively).Conclusions:1.This study suggested that the NF-κB signaling pathway may be involved in SD rat model of intrahepatic cholestasis formed with estrogen and that cholestasis may be affected by downregulating Bsep/Ntcp protein expression.2. The NF-κB specific inhibitor PDTC can improve TBA and liver pathologic changes, suggesting that PDTC may have a therapeutic effect in cholestatic of pregnant rats.3. PDTC, as the NF-κB specific inhibitor, may block the inhibitory effect of estrogen on Bsep/Ntcp through inhibiting NF-κB activity.