The Antioxidative Effects Of Baicalin, Matrine Or Berberine On L-FABP In Vivo And Vitro

Nonalcoholic steatohepatitis (NASH) is the chronic liver disease associated with genetic, environmental, metabolic factors and stress. NASH can progress to more severe liver diseases including fibrosis, hepatocellular necrosis and hepatocellular carcinoma. Although the exact mechanisms of NASH remain unknown, a large amount of information on the key mechanisms is oxidative stress and lipid peroxidation, which induce liver inflammation, necrosis and fibrosis. Thus, antioxidants should be as a target in NASH treatment. However, there are no optimal drugs for NASH treatment so far. Recent reports show that liver fatty acid binding protein (L-FABP) possesses all the characteristics necessary to be an effective endogenous protectant. Clinical research reported that clofibrate, a fibrate role in reducing blood cholesterol, have improved the antioxidativc capability of L-FABP. Are there any other drugs with similar effects as clofibrate? Some monomers extracted from Chinese herbs have the property of antioxidative stress, such as baicalin, matrine and berberine. Its mechanisms of antioxidative stress have not been elucidated clearly.In this study, we established a vivo rat model of NASH induced by high fatty diet. The antioxidative effects of baicalin, matrine and berberine on NASH were observed in the rat model. Meanwhile, we established a vitro model of oxidative stress induced by hydrogen peroxide (H2O2) to investigate whether the drugs improve the antioxidative effect of L-FABP, and the mechanism of L-FABP regulation as well. The main results and conclusions are as follows:1. In vivo modelAfter the establishment of the rat model of NASH, the NASH rats were intragastric administrated by baicaiin at the dose of30mg/kg/d, matrine at the dose of36mg/kg/d, berberine at the dose of18mg/kg/d, respectively for3weeks. Initially, we found that the NASH rats lost weight in the treatment of all the three drugs, but little or no changes on liver weight. Histopathology showed that all the three drugs ameliorate the hepatocytes steatosis, inflammation, ballooning degeneration and spotty necrosis. Furthermore, after exposure to baicalin, matrine and berberine respectively, the serum levels of ALT, AST and MDA in hepatic tissue were decreased, but the levels of GSH and SOD in liver were increased. Finally, Realtime-PCR indicated that all the three drugs exerted no effects on L-FABP mRNA expression of hepatic tissue. However, immunoblotting showed that the amount of L-FABP protein increased in liver treated by baicalin and matrine respectively, but no changes by berberine treatment2. In vitro modelCellular oxidative stress in vitro was induced by incubating cells with400μmol/L hydrogen peroxide (H2O2) for20minutes at37℃in the dark. Firstly, MTT assay showed that baicalin treatment at a concentration below120μ mol, matrine treatment at a concentration below67.8μ mol, and berberine treatment at a concentration below78.2μmol for48hours were feasible and safe. Next, ROS assay showed baicalin-treated at50μmol, matrine-treated at32μmol, berberine-treated at50μmol, respectively for48hours were the optimal condition against ROS generation. Furthermore, activity of intracellular SOD and GSH was increased significantly after exposure to baicalin at50μmol, matrine at32μmol, berberine at50μmol, respectively for48hours. However, they exerted no effect on activity of intracellular CAT. In addition, Realtime PCR analysis indicated50μmol baicalin treatment,32μmol matrine treatment,50μmol berberine treatment, respectively for48hours showed no change of L-FABP mRNA expression under the oxidative stress condition. However, western blotting analysis indicated50μmol baicalin treatment,32μ mol matrine treatment increased the amount of L-FABP protein under the same stress surrounding, while50μ mol berberine treatment have no effects on the amount of L-FABP protein in consistent with Real-time PCR results. Subsequently, in combination with10μ g/ml CHX and50μ mol MG132treatment respectively, we demonstrated that the L-FABP amount was down-regulated by CHX treatment, but up-regulated by MG132treatment.In the present study, we indicated that baicalin, matrine and berberine treatment were used to treat NASH of rats effectively. Baicalin and matrine function as antioxidation, which involves in the amount of L-FABP protein and the activities of intracellular SOD and GSH. Moreover, baicalin and matrinc treatment increased the level of L-FABP not via transcriptional activation but through enhancing the stability and decreasing protein degradation. Berberine played the role of antioxidation by enhancing the activities of intracellular SOD and GSH, possibily unrelated to L-FABP protein.