Therapeutic Effect And The Underlying Mechanism Of Yin-Chen-Hao Decoction And Its Formulae On Estrogen-induced Cholestasis In Rats

Objective:Based on the research about liver membrane transporter Ntcp and Mrp1-4, explore the therapeutic effect and the underlying mechanism of Yin-Chen-Hao decoction and its formulae on estrogen-induced cholestasis in rats.Methods:Male Wistar rats were subcutaneously administered estradiol benzoate (Estradio Benzoate, EB,5mg/kg/d) for consecutive5days to induce cholestasis, rats in normal group (N) were subcutaneously administered saline. After estradiol benzoate administration, bile flow rate, liver biochemical parameters and pathohistologic examination was measured, the expression of transporter Ntcp and Mrpl-4was also determined by Western blotting technique in order to evaluate whether the cholestasis models is successful or not. Estrogen-induced cholestasis rats were then randomly divided into six groups:Model group (M), control group (saline, CS), Yin Chen Hao extract (0.432g/kg/d, Y), Yin Chen Hao and Zhi Zi extract (1.08g/kg/d, YZ), Yin Chen Hao and Da Huang extract (1.09g/kg/d, YD), Yin Chen Hao, Zhi Zi and Da Huang extract (1.69g/kg/d, YCHT). The dose setting in each group was equivalent to the dose of crude herbs, namely Yin Chen Hao2.4g/kg/d, Zhi Zi1.8g/kg/d, Da Huang1.8g/kg/d, respectively. Drugs were orally administered to rats for consecutive14days. After drugs treatment, bile flow rate, liver biochemical parameters, pathohistologic examination, the expression of Ntcp and Mrpl-4were further measured.Results:1. Bile flow rate:Compared to the normal group, the bile flow rates cholestasis in model group and control group were significantly decreased (P<0.05), suggesting the success of model preparation. After the treatment of YCHT and its formulae, the bile flow rate was obviously increased and recovered to the normal level, suggesting the therapeutic effect of YCHT and its formulae in cholestasis rats.2. Liver biochemical parameters:Compared to the normal group, TBIL、DBIL and TBA level in model group and control group were all significantly increased (P<0.05). After the treatment of YCHT and its formulae, TBIL、IBIL and TBA in Y group and YD group were significantly decreased (P<0.05); while TBIL、IBIL and TBA in YZ group were all slightly decreased, only TBA showed significant difference(P<0.05). Compared to Y group, IBIL、TBIL、DBIL and TBA in YZ group were increased except IBIL; TBIL、IBIL、DBIL in YD group were decreased; while IBIL、TBIL、DBIL and TBA in YCHT group were increased, but did not show significant difference.3. Hepatic pathohistology and Knodell HAI scores:Tissue structure and the cell morphology in normal rats were clear without any pathological alterations. In cholestasis model group and control group, inflammation and necrosis could be seen around the liver portal area with obvious bile duct proliferation. After the treatment of YCHT and its formulae, lobular necrosis in YZ group was still obvious, but the pathological changes of cholestasis in other groups were all disappeared. Concerning the Knodell HAI score, they were significantly increased in model group and control group, but significantly reduced in Y group, YD group and YCHT group, but not YZ group (P>0.05)4. Western-blotting results:Compared to the normal group, the expression of transporter Mrpl and Mrp4were significantly increased, while Ntcp expression was significantly decreased in model group and control group (P<0.05); Mrp3expression was increased in model group but not in control group, and Mrp2expression was significantly decreased in model group, but significantly increased in control group (P<0.05). After the treatment of YCHT and its formulae, the expression of Ntcp, Mrp2and Mrp3was significantly increased, while Mrp1and Mrp4expression were significantly decreased in Y group (P<0.05); in YZ group, the expression Ntcp, Mrp2and Mrp3was significantly increased with reduced Mrp4expression (P<0.05); in YD group, Mrpl and Mrp4expression were significantly decreased with increased Ntcp and Mrp2expression (P<0.05); and in YCHT group, the alteration of transporter expression was same as that of YD group. Compared with Y group, Mrpl and Mrp4expression was increased, while Ntcp expression was decreased (P<0.05) with no alteration in Mrp3expression in YZ group; and the expression of Ntcp and Mrpl were increased with decreased expression of Mrp3and Mrp4in YD group and YCHT group (P<0.05). The Mrp2expression did not show any alteration in all of rats when compared to that of Y group.Conclusions:1. In estrogen-induced cholestasis rats, therapeutic effect can be seen in Y, YD and YCHT groups, the underlying mechanism may have correlation with their effect on regulating the expression of transporters which are responsible for the balance of bile acid salt and bilirubin. It is notable that YZ treatment did not show therapeutic effect on estrogen-induced cholestasis.2. Compared to the control group, the expression of Mrpl and Mrp4were decreased significantly in Y group, while the expression of Ntcp, Mrp2and Mrp3were significantly increased.3. Compared to Y group, Mrp1and Mrp4expression were significantly increased with reduced Ntcp expression in YZ group; Ntcp and Mrpl expression were significantly increased with reduced Mrp3and Mrp4expression in YD group and YCHT group; Mrp2expression was not altered in all of groups.4. Perspective research:The transporters that responsible for the balance of bile salt and bilirubin may also involve that distributed in sinusoidal membrane, such as Oatpl, Oatp2, Oatp4and canalicular membrane transporter Bsep. Therefore, it is necessary to further explore the expression of these transporter in order to have a comprehensive interpretation about the therapeutic effect and the molecular mechanism of Yin-Chen-Hao decoction and its formulae In estrogen-induced cholestasis rats.