丹那唑抑制大鼠线粒体呼吸链复合酶I 导致原代肝细胞坏死

摘要:目的　研究丹那唑对大鼠原代肝细胞的损伤作用与机制。方法　原代大鼠肝细胞贴壁培养4 h，加入丹那唑0，50，100，200 μmol/L，4 h和24 h后采用乳酸脱氢酶法测定细胞活力并检测三磷酸腺苷（ATP）水平，同时提取线粒体，分别测5个呼吸链复合酶（complex I～V）活性。培养液中加入20 mmol/L果糖促进糖酵解，观察果糖对丹那唑细胞毒性的影响。结果　丹那唑50，100 μmol/L处理4 h和24 h对细胞几乎没有杀伤作用，但可致ATP显著降低；200 μmol/L处理4 h和24 h均可导致细胞坏死。果糖可对抗丹那唑4 h具杀伤作用，但对24 h杀伤作用无效。丹那唑（50～200 μmol/L）显著抑制线粒体呼吸链复合酶I活性，但不影响complex Ⅱ～V活性。结论　丹那唑选择性抑制线粒体呼吸链复合酶I活性而导致ATP生成减少并诱发细胞坏死。

Danazol induces necrosis of primary cultured rat hepatocytes by inhibition of mitochondrial respiratory chain complex I

Abstract: Objective This work was to explore the cytotoxicity and mechanisms of danazol on primary cultured rat hepatocytes. Methods Primary rat hepatocytes were cultured for 4h to establish the monolayer, and danazol was added at final concentrations of 0, 50, 100 and 200μmol/L. Cell viability was measured by the release of lactate dehydrogenase. The level of adenosine triphosphate (ATP) level was measured in parallel. Mitochondria were isolated and the activities of the five respiratory chains (complex I～V) were determined. Fructose, a more efficient substrate of glycolysis than glucose, was added at a final concentration of 20mmol/L to promote glycolysis, and the effect of fructose on danazol cytotoxicity was observed. Results At 50μmol/L and 100μmol/L, danazol showed barely detectable cytotoxicity at 4h and 24h, but ATP level was significantly decreased. At 200μmol/L, danazol caused remarkable necrosis, which was decreased by fructose at 4h but not at 24h. Danazol strongly inhibited the activity of complex I at a concentration of 50～200μmol/L, but did not affect the activities of complex Ⅱ～V. Conclusion Danazol selectively inhibits mitochondrial respiratory chain complex I, and consequently depletes cellular ATP, leading to the necrosis of primary cultured rat hepatocytes.