细胞色素P450酶系对喹乙醇诱导的肾细胞凋亡的影响

Research on influence of CYP450 to renal cell apoptosis induced by OLA

目的采用体外细胞培养系统,研究肝脏微粒体细胞色素P450同工酶对喹乙醇(OLA)毒性的影响,筛选和确定影响喹乙醇毒性的主要细胞色素P450同工酶,探索CYP450酶系选择性介导OLA-ROS-细胞凋亡途径。方法(1)以体外培养的人类肾小管上皮细胞(HK-2)作为检测OLA致肾小管毒性的细胞模型,以脏微粒体混合酶系(S9)加入到HK-2细胞培养中模拟体内代谢环境,将CYP450同工酶(CYP2D6、NADPH:P450还原酶、CYP2A、CYP3A、CYP2C、CYP2E1和CYP1A1/CYP1A2)的化学抑制剂分别加入培养体系中,造成不同P450同工酶活性抑制状况,通过细胞增殖抑制率(MTT)试验来检测OLA单独染毒或OLA与抑制剂联合染毒情况下的细胞毒性。(2)通过流式细胞仪DCF法检测各CYP450同工酶抑制剂对OLA所致HK-2细胞ROS产生情况的影响,筛选HK-2细胞内影响OLA作用的主要CYP450同工酶,推测其代谢路径。结果 (1)MTT检测发现,OLA+S9+α-萘黄酮组与OLA+S9组之间以及OLA+4-甲基吡唑+S9组与OLA+S9组之间细胞活性差异有统计学意义(P<0.05),表明通过抑制CYP4501A酶以及CPY2E1活性可以使OLA的细胞毒性减轻。(2)OLA能够呈剂量依赖性的诱发细胞内ROS含量升高,且加入CYP1A抑制剂以及CPY2E1抑制剂后,可显著减少HK-2细胞ROS的产生量。结论 OLA通过CYP1A和CYP2E1的代谢诱导HK-2细胞生成ROS,进而可能诱发HK-2细胞的凋亡产生细胞毒性和肾毒性。

Objective Renal tubular epithelial cell was exposed to olaquindox and CYP450 inhibitors and was detected the cell activity and ROS,in order to screen and determine the major CYP450 isozymes and explore the olaquindox-ROS-apoptosis pathway mediated by CYP450.Methods（1） Olaquindox toxicity was tested by using human renal epithelial cells（HK-2） as model in vitro. The metabolism environment in vivo simulated by adding the liver microsomal mixed enzymes（S9） into HK-2 cell culture system. The inhibitors were added into HK-2 cell culture system respectively to form different activity inhibition situation for CYP450 isoenzymes. The cytotoxicities of olaquindox alone and the combination of olaquindox with inhibitors were determined by inhibition of cell proliferation（MTT） test.（2） The effect of CYP450 inhibitor on the ROS production of HK-2 cell induced by OLA was detected by flow cytometrx（DCF）. The main CYP450 isozymes in HK-2 cell was screened and the metabolic pathways were speculated. Results（1） There was a significant difference between OLA ＋ α-naphthoflavone/4-methylimidazole ＋ S9 group and OLA ＋ S9 group by MTT. Compared with OLA ＋ S9 group,OLA ＋ α-naphthoflavone/4-methylimidazole ＋ S9 could significantly enhanc the proliferation inhibition of OLA on HK-2 cell（P〈0. 05）.（2） OLA could induced the increase of ROS in a dose-dependent manner and significantly reduce the ROS of HK-2 cell after additions of inhibitors for CYP1 A/CYP2E1. The results showed that α-naphthoflavone/4-methylimidazole might be influence OLA cytotoxicity through inhibiting the activity of CYP1 A/CYP2E1. Conclusion CPY1 A and CYP2E1 were the major CYP450 isozymes that affected the metabolic activation and ROS production of OLA.