槲皮素抑制异烟肼诱导的L-02细胞线粒体氧化应激性DNA损伤

Protective effect of quercetin on L-02 cells by inhibiting DNA damage of INH-induced mitochondrial oxidative stress

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L-02细胞DNA损伤中的作用及槲皮素对细胞的保护作用。方法:建立体外培养INH致肝细胞L-02损伤的模型,将细胞分为对照(control)组、INH组、槲皮素低剂量(Que low)及高剂量(Que high)组。利用彗星试验评价细胞DNA损伤;制备L-02细胞线粒体,应用荧光探针DCFH-DA和rhodamine 123检测细胞线粒体ROS水平及线粒体膜电位(ΔΨm);采用TBA法测定丙二醛(MDA)含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)的活性;采用Western blotting法检测细胞中Bcl-2和Bax蛋白表达,计算Bax/Bcl-2值。结果:INH可诱导L-02细胞DNA损伤,使细胞线粒体ROS水平、细胞MDA含量及Bax/Bcl-2值明显增高,并使细胞ΔΨm值和SOD活性明显下降。而槲皮素能减轻细胞DNA损伤,减少细胞ROS水平,增加细胞ΔΨm值,降低细胞MDA含量,增加SOD活性,减少Bax/Bcl-2值。结论:INH可通过诱导细胞线粒体氧化应激导致L-02细胞DNA损伤。槲皮素能减轻INH诱导L-02细胞的DNA损伤,对L-02细胞具有保护作用,可能与其抑制ROS介导的线粒体氧化损伤有关。

AIM： To investigate the role of reactive oxygen species（ ROS）-mediated mitochondrial oxidative injury in isonicotinyl hydrazide（ INH）-induced DNA damage and the protective effect of quercetin on L-02 cells. METHODS： The injury model of hepatocyte L-02 cells in vitro induced by INH was established. The cells were divided into control group,INH group,low-dose quercetin group and high-dose quercetin group. The DNA damage of L-02 cells was evaluated by the comet test. The mitochondrion was prepared,and the level of mitochondrial ROS and the value of mitochondrial membrane potential（ ΔΨm） were detected by fluorescent probes DCFH-DA and rhodamine 123. The content of MDA was measured by TBA method. The activity of SOD was assessed with the xanthine oxidase method. The protein expression of Bcl-2 and Bax was determined by Western blotting,and the value of Bax / Bcl-2 was calculated. RESULTS：INH induced obvious DNA damage,increased the level of mitochondrial ROS,the content of MDA and the value of Bax /Bcl-2,and markedly reduced the value of ΔΨmand the activity of SOD in the L-02 cells. Quercetin attenuated DNA damage,reduced the level of mitochondrial ROS,elevated the value of ΔΨm,declined the content of MDA,increased the activity of SOD and decreased the value of Bax / Bcl-2 in the L-02 cells. CONCLUSION： INH induces DNA damage in L-02 cells by generation of mitochondrial oxidative stress. Quercetin has a protective effect on L-02 cells to attenuate the INH-induced DNA damage by inhibiting ROS-mediated mitochondrial oxidative damage.