异烟肼通过ROS/Caspase-3信号通路诱导L-02细胞凋亡及槲皮素的保护作用

目的：探讨ROS/Caspase-3信号通路在异烟肼（INH）诱导人正常肝细胞（L-02细胞）凋亡中的作用及槲皮素的保护效应。方法：将L-02细胞随机分为对照组、INH组（10 mmol/L INH）、25 μmol/L槲皮素组（10 mmol/L INH和25 μmol/L槲皮素）、50 μmol/L槲皮素组（10 mmol/L INH和50 μmol/L槲皮素）。各组均处理24 h后，应用MTT法检测L-02细胞存活率，分光光度法检测细胞上清液中LDH活性，Hoechst33258荧光染色法检测细胞凋亡率；制备L-02细胞线粒体，利用DCFH-DA和Rho-123荧光探针检测活性氧（ROS）水平及线粒体膜电位（△Ψm），应用Western blot检测Caspase-3蛋白的表达。结果：与对照组比较，INH组细胞存活率显著降低（P < 0.01），与INH组比较，50 μmol/L槲皮素处理组细胞存活率明显增加（P < 0.01）；INH组细胞LDH活性、细胞凋亡率、线粒体ROS水平较对照组显著升高（P < 0.01），25和50 μmol/L槲皮素组细胞LDH活力、细胞凋亡率、线粒体ROS水平较INH组明显降低（P < 0.05或P < 0.01），且50 μmol/L槲皮素组作用更显著；INH组细胞线粒体△Ψm较对照组显著降低（P < 0.01），25和50 μmol/L槲皮素组细胞线粒体△Ψm较INH组明显升高（P < 0.05或P < 0.01），50 μmol/L槲皮素组的作用更加明显；与对照组比较，INH组细胞Caspase-3蛋白表达显著增加（P < 0.01），与INH组比较，25和50 μmol/L槲皮素组细胞Caspase-3蛋白表达明显减少（P < 0.05或P < 0.01），且50 μmol/L槲皮素组作用更明显。结论：INH可以诱导L-02细胞发生凋亡，ROS/Caspase-3信号通路参与了其凋亡的过程；槲皮素对INH诱导的细胞凋亡具有保护效应，机制可能与其减少ROS释放，抑制ROS/Caspase-3信号通路有关。

Involvement of the ROS/Caspase-3 signaling pathway in isoniazid-induced apoptosis in L-02 cells and the protective effect of quercetin

OBJECTIVE:To investigate involvement of the ROS/Caspase-3 signaling pathway in INH-induced apoptosis in L-02 cells,and the protective effect of quercetin. METHODS:L-02 cells were randomly divided into control and treatment groups. The latter groups were treated with INH (10 mmol/L INH),25 μmol/L quercetin (10 mmol/L INH and 25 μmol/L quercetin) and 50 μmol/L quercetin group (10 mmol/L INH and 50 μmol/L quercetin). Vitality of L-02 cells was detected by the MTT method. LDH activity in supernatant fluid was measured by colorimetric method. Apoptosis of L-02 was determined by Hoechst 33258 fluorescent staining. Mitochondria of L-02 cells,reactive oxygen species (ROS) level and mitochondrial membrane potential (△Ψm) were analyzed with fluorescent probe DCFH-DA and Rho-123. Protein expression of Caspase-3 was analyzed with western blot method. RESULTS:Compared with the controls and with L-02 cells treated with INH and quercetin,cell vitality of the INH group was significantly declined (P < 0.01). Compared with the INH group,cell vitality of the 50 μmol/L quercetin group was markedly increased (P < 0.01). Activities of LDH,cell apoptosis rate and level of mitochondrial ROS in cells of the INH group were significantly increased over the control group (P < 0.01). Activities of LDH,cell apoptosis rates and levels of mitochondrial ROS in the 25 μmol/L and 50 μmol/L quercetin groups were significantly decreased compared with that of the INH group (P < 0.05 or P < 0.01,respectively),and the effects in the 50 μmol/L quercetin group were more obvious. The mitochondrial membrane potential of the INH group was significantly lower than that of the control group (P < 0.01). The mitochondrial membrane potential of the 25 μmol/L and 50 μmol/L quercetin groups were markedly higher than that of the INH group (P < 0.05 or P < 0.01,respectively),and the 50 μmol/L quercetin group was more effective compared with the control group. Protein expression of Caspase-3 in the INH group was markedly increased (P < 0.01),compared with the INH group. Protein expressions of Caspase-3 of the 25 μmol/L and 50 μmol/L quercetin groups were significantly reduced (P < 0.05 or P < 0.01,respectively),and the 50 μmol/L quercetin group had more obvious effect. CONCLUSION:INH induced cell apoptosis in the L-02 cells and ROS/Caspase-3 signaling pathway was involved in this process.Quercetin had a protective effect against the INH-induction of cell apoptosis,the mechanism may be related to reducing ROS release,and inhibiting the ROS/Caspase-3 signaling pathway.