基于细胞色素氧化酶CYP2D6的何首乌肝细胞毒性及相关成分研究

Hepatotoxicity and Related Components of Polygoni Multiflori Radix Based on Cytochrome Oxidase CYP2D6

目的探究何首乌及其主要成分对人肝细胞L02中细胞色素氧化酶CYP2D6 mRNA表达的影响以及抑制CYP2D6活性或表达对何首乌肝细胞毒性的影响及相关成分辨识。方法利用实时荧光定量PCR(RT-qPCR)确定何首乌水提物(Polygoni Multiflori Radix,PMR)及其主要成分对L02细胞CYP2D6 mRNA表达的影响;使用CYP2D6抑制剂奎尼丁初步探究PMR对CYP2D6低活性L02细胞系的细胞毒作用;构建CYP2D6沉默的L02细胞系L02-shCYP2D6,进一步研究L02细胞中CYP2D6表达降低对PMR及其主要成分肝细胞毒性的影响;可能的肝细胞毒性成分与人肝微粒体进行体外孵育,通过考察加入不同CYP450酶特异性抑制剂对其代谢消除率的影响,探究影响其代谢的主要CYP450酶。结果在实验浓度下,PMR和芦荟大黄素(AE)对CYP2D6 mRNA表达有显著抑制作用(P<0.01),大黄素(EM)无明显影响,2,3,5,4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(TSG)仅在高浓度有一定激活作用,其余浓度无影响;PMR联合奎尼丁作用于L02细胞,发现PMR在高浓度下对L02细胞有一定毒性(P<0.01),各浓度抑制剂与之联用后均明显加重了其肝细胞毒性(P<0.01);PMR及其主要成分作用于CYP2D6敲低的L02细胞系,除TSG在高浓度下提升了肝细胞毒性外(P<0.01),PMR、EM、AE在实验浓度范围内肝细胞毒性均显著增加(P<0.01);人肝微粒体体外孵育实验显示,CYP2D6为EM、AE重要的代谢酶。结论PMR能够抑制肝细胞中CYP2D6的表达,而CYP2D6的低活性或低表达能够加重PMR肝细胞毒性,其中主要的毒性成分为EM和AE。

Objective To study the effects of Polygoni Multiflori Radix and its main components on the mRNA expression of CYP2D6 in L02 human liver cells and the effects of CYP2D6 inhibition on Polygoni Multiflori Radix and its main components induced hepatocyte toxicity.Methods The mRNA expression of CYP2D6 was determined by RT-XqPCR in L02 cells treated with Polygoni Multiflori Radix(PMR)or its main components.Quinidine(CYP2D6 inhibitor)was used to explore the cytotoxic effect of PMR on L02 cells with low CYP2D6 activity.Then,L02-shCYP2D6 cell line was constructed by RNA interference and used to investigate the main hepatotoxic components of PMR related to CYP2D6.Finally,the major CYP450 enzymes that affected the metabolism of possible hepatotoxic components were determined by the substrate metabolic clearance rate in human liver microsomes after different CYP450 enzyme specific inhibitors were added.Results PMR and aloeemodin(AE),rather than emodin(EM),inhibited the mRNA expression of CYP2D6 significantly(P<0.01),while 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(TSG)activated mRNA expression of CYP2D6 at a high concentration(P<0.01).PMR exerted more cytotoxic effect in L02 cells at a high concentration(P<0.01)when combined with quinidine than used alone.Hepatotoxicity was increased significantly when PMR was combined with quinidine at various concentrations(P<0.01).PMR,EM and AE increased hepatotoxicity significantly in L02-shCYP2D6 cells within the experimental concentration range(P<0.01),but TSG increased hepatotoxicity only at high concentrations(P<0.01).CYP2D6 was determined as an important metabolic enzyme for EM and AE in human liver microsomes.Conclusion PMR can inhibit the expression of CYP2D6 in hepatocytes.The low activity or low expression of CYP2D6 aggravates the hepatotoxicity of PMR and its main toxic components are EM and AE.