槲皮素对异烟肼诱导肝细胞毒性的保护作用及其机制

Protective effects of quercetin on isoniazid-induced hepatotoxicity in vitro

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50μmol/L槲皮素)、谷胱甘肽(GSH)预处理组(20 mg/mL GSH、10 mmol/L INH和50μmol/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。

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 50μmol/L quercetin),glutathione(GSH)pretreated(20 mg/mL GSH,10 mmol/L INH and 50μmol/L quercetin),glutathione(20 mg/mL GSH)and negative control(equal volume of serumfree 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 quercetintreated 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 INHinduced mitochondrial oxidative damage and the mechanism was related to its inhibition of ROS activities.