癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (1): 53-57,68.doi: 10.3969/j.issn.1004-616x.2019.01.010

• 论著 • 上一篇    下一篇

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

陈廷玉, 王东伟, 张金波, 王伟, 王景涛, 盛颜良, 卢春凤   

  1. 佳木斯大学, 黑龙江 佳木斯 154007
  • 收稿日期:2018-11-07 修回日期:2018-12-18 出版日期:2019-01-31 发布日期:2019-01-31
  • 通讯作者: 卢春凤,E-mail:luchunfengchen@126.com E-mail:luchunfengchen@126.com
  • 作者简介:陈廷玉,E-mail:chenty123@163.com。
  • 基金资助:
    国家自然科学基金资助项目(81373497)

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

CHEN Tingyu, WANG Dongwei, ZHANG Jinbo, WANG Wei, WANG Jingtao, SHENG Yanliang, LU Chunfeng   

  1. Jiamusi University, Jiamusi 154007, Heilongjiang, China
  • Received:2018-11-07 Revised:2018-12-18 Online:2019-01-31 Published:2019-01-31

摘要: 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。

关键词: 槲皮素, 异烟肼, 活性氧, Caspase-3, 细胞凋亡

Abstract: 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.

Key words: quercetin, isoniazid, ROS, Caspase-3, apoptosis

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