异烟肼和利福平致小鼠原代肝细胞损伤时CYP2E1和CYP3A酶活性的变化

The Hepatocyte Toxicity and the Effect of Isoniazid and Rifampicin on the Activities of CYP2E1 and CYP3A in Mouse Primary Hepatocytes

目的研究异烟肼和利福平对小鼠原代肝细胞的损伤作用及对CYP450同工酶CYP2E1和CYP3A酶活性的影响。方法从小鼠肝脏中分离出肝细胞,原代培养3 d后,加入药物后孵育2 d。收集细胞培养上清液,测定其中的乳酸脱氢酶(LDH)释放量以反映药物对肝细胞的损伤作用。测定CYP450酶活性的细胞分别加入CYP2E1和CYP3A的特异性底物(对硝基酚和咪达唑仑)孵育2 h,反应终止后用高效液相色谱-质谱法测定特异性底物的浓度,浓度减少程度代表CYP2E1和CYP3A活性的变化。结果与对照组比,异烟肼中、高浓度组,利福平高浓度组,合用中、高浓度组LDH的释放量显著增加。与对照组比较,异烟肼组与合用组CYP2E1底物对硝基酚浓度均显著降低;利福平组与合用组CYP3A底物咪达唑仑浓度均显著降低。结论高浓度的异烟肼和利福平均对小鼠原代肝细胞有损伤作用,两药合用时,低浓度能引起肝细胞损伤。异烟肼和利福平对CYP2E1和CYP3A酶活性的诱导可能是其导致肝损伤的机制之一。

Objective To study the hepatocyte toxicity and the effect of isoniazid and rifampicin on the activities of CYP2E1 and CYP3A in mouse primary hepatocytes.Methods The primary hepatocytes were isolated from mice and cultured for 3 days.Then the cells were treated with isoniazid and rifampicin.After two-day culture,culture media were collected for lactate dehydrogenase assay and the level of lactate dehydrogenase leakage was selected as an indicator of hepatocyte damage.Substrates（4-nitrophenol for CYP2E1,midazolam for CYP3A） were added,the concentration of which was measured with high performance liquid chromatography-MS to assess the activities of CYP2E1 and CYP3A.Results compared to the negative control group,the LDH leakage of hepatocytes exposed to rifampicin,isoniazid and their combination was significantly increased.While the concentration of 4-nitrophenol in isoniazid group and combined group was low.The concentration of midazolam in rifampicin group and combined group was significantly different form that in the negative control group.Conclusions Rifampicin or isoniazid at an high concentration causes hepatotoxicity.The combination of both drugs at a low concentration causes hepatotoxicity.The hepatotoxicity may be caused by the activity of CYP2E1 and CYP3A induced by isoniazid and rifampicin.