槲皮素对异烟肼诱导肝细胞毒性的保护作用及其机制

目的： 探讨活性氧（ROS）介导的线粒体氧化损伤在异烟肼（INH）诱导肝细胞毒性中的作用及槲皮素对INH肝细胞毒性的保护效应及机制。方法： 将L-02细胞随机分为5组：异烟肼组（10 mmol/L INH）、槲皮素处理组（10 mmol/L INH和50 μmol/L槲皮素）、谷胱甘肽（GSH）预处理组（20 mg/mL GSH、10 mmol/L INH和50 μmol/L槲皮素）、谷胱甘肽组（20 mg/mL GSH）、对照组（等体积的无血清培养基），作用24 h后，采用差速离心法制备细胞线粒体，荧光探针DCFH-DA和Rho-123检测细胞线粒体ROS水平及膜电位；TBA比色法测定丙二醛（MDA）含量；DPNH比色法测定蛋白质羰基含量；ELISA法检测8-羟基脱氧鸟嘌呤核苷（8-OHdG）含量。结果： 与对照组相比，INH处理细胞后细胞线粒体ROS水平升高（P < 0.01），膜电位降低（P < 0.01）。与异烟肼组相比，槲皮素处理组细胞线粒体ROS水平降低（P < 0.01），膜电位升高（P < 0.05）；谷胱甘肽预处理组细胞线粒体ROS水平低于槲皮素处理组（P < 0.05），线粒体膜电位高于槲皮素处理组（P < 0.05）。与对照组相比，细胞经INH处理后MDA、蛋白质羰基及8-OhdG的含量增加（P < 0.01）。与异烟肼组比较，应用槲皮素后可使MDA、蛋白羰基、8-OHdG的含量减少（P < 0.01）；谷胱甘肽预处理组MDA、蛋白羰基、8-OHdG含量低于槲皮素处理组（P < 0.05）。结论： 在本实验条件下，INH可诱导L-02细胞线粒体氧化损伤，且ROS参与了此过程；槲皮素对INH诱导的线粒体氧化损伤具有保护效应，其机制可能与其抑制ROS水平有关。

Protective effects of quercetin on isoniazid-induced hepatotoxicity in vitro

OBJECTIVE: To investigate the role of ROS-mediated mitochondrial oxidative damage in isoniazid (INH)-induced hepatotoxicity in vitro and the protective effects from quercetin. METHODS: L-02 hepatocytes in cultures were randomly divided into five groups:isoniazid (10 mmol/L INH), INH + quercetin treatment (10 mmol/L INH and 50 μmol/L quercetin), glutathione (GSH) pretreated (20 mg/mL GSH, 10 mmol/L INH and 50 μmol/L quercetin), glutathione (20 mg/mL GSH) and negative control (equal volume of serum-free medium). After 24 hours of treatment, cell mitochondria were prepared by differential centrifugation, and fluorescent probes DCFH-DA and Rho-123 were used to detect mitochondrial ROS level and membrane potential, Contents of malondialdehyde (MDA) were determined using TBA colorimetry. Contents of protein carbonyl were determined using DPNH colorimetry. Contents of 8-hydroxydeoxyguanine nucleoside (8-OHdG) were determined using ELISA. RESULTS: Compared with the control group, INH-treated cells showed the followings:levels of ROS in mitochondria were elevated (P < 0.01), and membrane potentials were declined (P < 0.01). Compared with the INH group, the quercetin-treated cells showed the followings:mitochondrial ROS levels were reduced (P < 0.01) and membrane potentials were elevated (P < 0.05). Compared with the quercetin-treated group, the glutathione-pretreated cells showed the followings:levels of ROS were lower and the mitochondrial membrane potentials were higher (P < 0.05). Compared with the control group, contents of MDA, protein carbonyl and 8-OHdG were increased after the INH treatment (P < 0.01). Compared with isoniazid group, contents of MDA, protein carbonyl and 8-OHdG in quercetin treatment group were decreased (P < 0.01).Contents of MDA, protein carbonyl and 8-OHdG in the GSH pretreatment group were lower than those in quercetin treatment group (P < 0.05). CONCLUSION: Under the experimental conditions, INH induced oxidative damage of mitochondria in L-02 cells. Quercetin was shown to provide protective effect on INH-induced mitochondrial oxidative damage and the mechanism was related to its inhibition of ROS activities.