Inhibitory Effect Of The Ginkgo Biloba Extract On Cytochrome P450 2E1 Expression

Background and Objective The number of obesity crowd is rising increasingly with improvement of people's living standard. As a result, patients suffering Nonalcoholic fatty liver disease (NAFLD) are also largely increasing. At present, it is thought that the "Second Strike" theory can be a common pathogenesy of alcoholic liver disease (ALD) and NAFLD. The first strike, such as alcohol, obesity and diabetes mellitus result in the store of fat leading to simple fatty liver by unbalance between synthesis and excretion of triglyceride in hepatocytes. The second strike is that lipid peroxidation and inflammatory cytokine relating to oxidative stress induces inflammation, necrosis and fibrosis of steatosis hepatocytes. Some researches find that the pathogenesy of the liver in ALD is remarkably similar to that of NAFLD, suggesting one common link, namely the induction of cytochrome P4502E1 (CYP2E1). Ethanol, fatty acids, isoniazid and other chemical materials induce the activity of the enzyme which thereby contributes to the release of oxygen free radical and liver injury, especially mitochondrial damage. Mitochondrial damage in turn exacerbates the oxidative stress. GBE, a traditional Chinese medicine, has many pharmacological activities, such as anti-inflammation, anti-allergy and lowering blood viscosity. It has been reported that GBE can inhibit the activity of CYP2E1, however, it has no study on molecular level.This study aimed to investigate the effect of GBE on CYP2E1 expression and lipid peroxidation in induced E47 cells by ethanol and fish oil.Methods E47 cells (HepG₂ transfected CYP2E1 gene can express stably CYP2E1 gene) were divided into five groups, the control group, ethanol group, fish oil group, GBE+ethanol group, and GBE+fish oil group, respectively. GBE was given to E47 cells while E47 cells were induced by ethanol and fish oil. The expression of CYP2E1 protein was observed by immunocytochemical staining method and Western blotting. The CYP2E1 activity in intact cells was measured by p-nitrophenol (PNP) as the probe. Malondialdehyde (MDA) and superoxide dismutase (SOD) contents were also determined and compared with those in the control group.The data was analyzed by SPSS13.0. One-way ANOVA and bivariate correlation were used to analyze the difference between groups. P<0.05 was considered as significant difference.Results 1. Immunocytochemical staining revealed that the expression of CYP2E1 in control group E47 cells was in endochylema. The expression intensity of ethanol and fish oil groups were markedly higher than that of controls (P<0.05) and nuclear expression could be observed in some cells. GBE+ethanol .group and GBE+fish oil group were significantly lower than ethanol group and fish oil, respectively (P<0.05), which were similar to the controls. The color of negative control had no stain.2. Western blotting displayed that the IOD (integrated optical density) ratio of CYP2E1 to GADPH in controls, ethanol, fish oil, GBE+ethanol and GBE+fish oil was 1.195±0.287, 2.911±0.642, 2.691±0.448, 1.073±0.370, and 1.345±0.168, respectively. The CYP2E1 protein expression of Ethanol group and fish oil group is markedly higher than that of the controls (P<0.05) . GBE treatment decreased the CYP2E1 protein expression. Compared with ethanol group and fish oil group, GBE+ethanol and GBE+fish oil group is significantly lower (P<0.05). Otherwise, However, there is not notably different between two groups and the controls (P>0.05)3. Compared with the controls, The CYP2E1 activity of E47 cells in ethanol-induced group and fish oil-induced group elevated (P<0.05). The CYP2E1 activity of GBE+ethanol group was markely lower than that of ethanol group(P<0.05). GBE+fish oil group is also significantly lower than fish oil group (P<0.05).4. GBE could evidently decrease MDA and increase SOD in cells. In comparison with the controls, MDA notably elevated after ethanol and fish oil treatment (P<0.05). Meanwhile, SOD level is decline (P<0.05). MDA and SOD level recovered to the controls in GBE+ethanol group and GBE+fish oil group (P<0.05).5. Gray level of CYP2E1 expression was significantly positively correlated with the concentration of MDA(r =0.850, P< 0.01), but it was negative correlated with the concentration of SOD (r=- 0.826 , P<0.01) . MDA was negatively correlated with SOD (r=-0.759, P<0.01) .Conclusions GBE can inhibit the expression and activity of CYP2E1 and lessen lipid peroxidation markedly.