抑制CYP2E1增强何首乌水提物肝毒性作用及其毒性成分筛选研究

目的研究肝细胞色素氧化酶P450(CYP450)亚型CYP2E1与何首乌肝毒性的关系并筛选有关毒性成分。方法MTT法检测CYP2E1特异性抑制剂二乙基二硫代氨基甲酸钠(DDTC)作用后何首乌水提物(PMR)对人肝细胞L02的毒性作用,及PMR和6种成分2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(2,3,5,4-tetrahydroxy stilbene glycoside,TSG)、大黄素-8-O-β-D-葡萄糖苷(emodin-8-O-β-D-glucoside,EG)、大黄素(emodin,EM)、没食子酸(gallic acid,GA)、大黄素甲醚(physcione,PH)和大黄酸(rhein,RH)对稳定转染shCYP2E1的HepaRG细胞的毒性作用;利用CYP2E1特异性抑制剂4-甲基吡唑(4-methylpyrazole,4-MP)作用大鼠,进一步验证何首乌对大鼠的肝毒性作用与CYP2E1抑制的关系。结果DDTC处理L02细胞24 h后,CYP2E1活性显著下降(P<0.05),PMR联用DDTC后细胞活力明显下降(与PMR、DDTC组相比,P<0.01);成功构建了HepaRG-shCYP2E1细胞系,与空质粒对照组相比,其CYP2E1 mRNA表达下降70%以上;与对照组相比,何首乌提取物PMR、EM、PH及RH对HepaRG-shCYP2E1的细胞毒作用显著增加(P<0.01),而TSG和EG在一定浓度下细胞毒作用降低(P<0.01);大鼠实验表明,何首乌与CYP2E1抑制剂联合作用于大鼠后,与空白组、抑制剂组相比,ALT及AST的水平均明显升高,与何首乌组相比,AST水平显著升高,说明大鼠体内CYP2E1被抑制后,何首乌的肝毒性作用增强。结论CYP2E1活性或表达下降可导致何首乌水提物肝细胞毒性及大鼠肝毒性增加,其毒性成分可能为游离蒽醌类成分EM,PH及RH。

Enhanced Hepatotoxicity of Extract of Polygoni Multiflori Radix after Inhibition of CYP2E1 and Screening of Toxic Constituents

Objective To study the relationship between cytochrome oxidase P450(CYP450)subtype CYP2E1 and the hepatotoxicity of Polygoni Multiflori Radix(PMR),and screen the toxic components.Methods MTT assay was used to detect the toxic effects of aqueous extract of PMRE on L02 cells treated with CYP2E1 specific inhibitor sodium diethyldithiocarbamate(DDTC),and the toxic effects of PMR and 6 major ingredients 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside(TSG),emodin-8-O-β-D-glucoside(EG),emodin(EM),gallic acid(GA),physcione(PH)and rhein(RH)on stably transfected HepaRG-shCYP2E1 cells.Then,the effects of PMR combined with CYP2E1 specific inhibitor 4-methylpyrazole(4-MP)on hepatotoxicity were studied in rats.Results CYP2E1 activity was decreased significantly when treated with DDTC,so was cell viability after being treated with PMR combined with DDTC compared with PMR alone(P<0.01).A stably transfected cell line,HepaRG-shCYP2E1,was successfully constructed,and the mRNA expression of CYP2E1 in HepaRG-shCYP2E1 cells decreased by more than 70%.Compared with the control group,the cytotoxic effect of PMR,EM,PH and RH on HepaRG-shCYP2E1 was significantly increased(P<0.01),while TSG and EG showed a decreasing toxicity at certain concentrations(P<0.01).The rat experiment showed that the levels of ALT and AST in PMR combined with CYP2E1 specific inhibitor 4-MP group were significantly increased compared with the control group and the 4-MP group,and the levels of AST in PMR combined with 4-MP group were significantly increased compared with PMR group,indicating that the hepatotoxicity of PMR increased after the inhibition of CYP2E1 in rats.Conclusion The decrease of CYP2E1 activity or expression in rats could increase the hepatotoxicity of PMR.The main cytotoxic components are free anthraquinones including EM,PH and RH.