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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 To investigate the effects of liver microsomal cytochrome P450 isoenzymes on olaquindox (OLA) cytotoxicity in vitro and to screen and identify the major cytochrome P450 isoenzymes that affect olaquindox toxicity and to explore the mechanism of CYP450 enzymatic selectivity Guide OLA-ROS-apoptotic pathway. Methods (1) Human renal tubular epithelial cells (HK-2) cultured in vitro were used as the cell model for detecting the renal tubular toxicity induced by OLA. The somatic cell mixed enzyme system (S9) was added to HK-2 cells to simulate the in vivo metabolism Environment, the chemical inhibitors of CYP450 isoenzymes (CYP2D6, NADPH: P450 reductase, CYP2A, CYP3A, CYP2C, CYP2E1 and CYP1A1 / CYP1A2) were separately added to the culture system to cause inhibition of different P450 isoenzyme activities, The proliferation inhibition rate (MTT) assay was used to examine the cytotoxicity of OLA alone or in combination with inhibitors of OLA. (2) The effect of CYP450 isoenzyme inhibitor on the production of ROS in HK-2 cells induced by OLA was detected by flow cytometry (DCF), and the main CYP450 isoenzymes in HK-2 cells that affected the effect of OLA were screened. Metabolic path Results (1) The results of MTT assay showed that there was significant difference in the cell viability between OLA + S9 + α-naphthoflavone group and OLA + S9 group and between OLA + 4-methylpyrazole + S9 group and OLA + S9 group (P <0.05), indicating that the cytotoxicity of OLA can be alleviated by inhibiting CYP4501A enzyme and CPY2E1 activity. (2) OLA induced a dose-dependent increase in intracellular ROS level, and the addition of CYP1A inhibitor and CPY2E1 inhibitor significantly reduced ROS production in HK-2 cells. Conclusion OLA induces ROS production in HK-2 cells through the metabolism of CYP1A and CYP2E1, which may induce cytotoxicity and nephrotoxicity in HK-2 cells.