| Objective:To investigate the intervention effect of compound Yindan decoction in rats with acute intrahepatic cholestasis induced by alpha-naphthyl isothiocyanate (ANIT),to observe the expression of Mrp2mRNA and protein, to explore the mechanism of compound Yindan decoction in the treatment of intrahepatic cholestasis.Methods:45 male wistar rats were randomly divided into three groups, normal control group (9), the model group (18),TCM group (18), the model of intrahepatic cholestasis can be induced by alpha-naphthyl isothiocyanate (ANIT) by gavage. The rats of TCM group were given compound Yindan decoction by gavage once a day.To collect the samples of serum and hepatic tissues after modeling 24 hours,48 hours,72 hours. To observe the changes of hepatic function at different time points; To observe the expression of Mrp2mRNA in hepatocytes at different time points by RT-PCR; To boserve protein expression of Mrp2 by Immunohistochemistry; To detect the protein quantitation of Mrp2 in the hepatic tissue by Immunoblotting (Wes tern-blot).To obtain data for statistical analysis by SARS software and compare of the differences in serum biochemical indicators, expression of Mrp2mRNA and Mrp2 protein at different time points between TCM group and model group.Results:1 Serum biochemical indexes:The rats of model group and TCM group appeared rapidly significant hepatic damage,manifested as the rapid rise of transaminases,biliary enzymes and serum bilirub in,while the hepatic function of normal control group had no significant change at three time points. ALT, AST, TBi1, DBi1, GGT, ALP and TBA of model group were significantly increased compared with normal control group (P<0.01).Hepatic function indexes were significantly decreased at 72 hours compared with model group (P<0.05, P<0.01).2 Hepatic tissue pathological changes:Comparing with normal control group, the rats of model group appeared damage to the hepatic lobules strueture,hepatocyte swelling,and scattering and exfoliation of biliary epithelial cells and infiltration of neutrophils in portal area at 24 hours after modeling; hepatic tissue lesions aggravated, hepatic lobules structure destructed severely,nucleus of different sizes, nuclear chromatin gathered,nucleolus enlarged and hyperstained. At 48 hours,we can see that significant hyperplasia of biliary epithelial cells,punctate and focal necrosis of hepatic cells around biliary and a lot of infiltration of neutrophils in portal area; At 72 hours, it can be still seen the hyperplasia of biliary epithelial cells and infiltration of neutrophils in portal area.The hepatic tissue pathological changes of TCM group were not very significant at 24 hours and 48 hours compared with model group,but neutrophils infiltration significantly decreased at 72 hours in portal area.3 RT-PCR quantitative results of Mrp2mRNA in hepatic tissue:The expression of Mrp2mRNA of rats of model group were significantly decreased compared with normal control group,at 24 hours and 72 hours, P<0.05,at 48 hours, P<0.01. The expression of Mrp2mRNA of rats of TCM group tended to increase, but there was no significant difference at 24 hours.The expression of Mrp2mRNA of rats of TCM group was ignificantly higher than model group at 48 hours and 72 hours (P<0.05)4 Expression results of Mrp2 transporter protein in hepatic tissue: Immunohistochemical results showed that Mrp2 expression were significantly decreased compared with the normal control group at all time points,P<0.01.There is no significant difference between TCM group and model group at 24 hours, the amount of expression of Mrp2 gradually to increase, significantly increased at 48 hours (P<0.05) and at 72 hours (P<0.01). Western-b lot gel image analysis showed that the expression ofMrp2 protein were significantly decreased at three time points compared with the normal control group(P<0.01);the expression of Mrp2 protein were significantly increased at 24 hours P<0.05,at 48 hours P<0.01, at 72 hours P<0.05 compared with the normal control group.Conclusions:1 Compound Yindan decoction can decrease the levels of serum bilirub in, reduce the injury of hepatic cells and bile duct, improve the hepatic function and reduce cholestasis and has a preventive effect to rat intrahepatic cholestasis caused by alpha-Naphthy1 isothiocyanate.2 The prescriptions and syndromes pathological basis of clearing heat and removing dampness, cooling blood and activating blood circulation is the intrahepatic cholestasis in rats indeced by alpha-Naphthy1 isothiocyanate and the down-regulation of the expression of Mrp2. 3 One of the key mechanism of control intrahepatic cholestasis in rats with Compound Yindan decoction is the up-regulated expression of Mrp2mRNA and protein.
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