乙醛脱氢酶2减轻多柔吡星所致细胞毒性作用的机制

Mechanisms for inhibitory effect of ALDH2 on doxorubicininduced cytotoxicity in C2C12 myogenic cell line

目的:探讨乙醛脱氢酶2(aldehyde dehydrogenase 2,ALDH2)对多柔吡星(doxorubicin,DOX)所致肌原细胞毒性的保护作用及其机制。方法:以小鼠C2C12肌原细胞为研究对象,通过基因转染方式调节细胞内ALDH2表达水平,以流式细胞术检测细胞凋亡率,MTT法检测细胞增殖抑制率,化学荧光法检测组织活性氧(reactive oxygen species,ROS)、4羟壬烯醛(4-hydroxynonenal,4-HNE)蛋白加合物含量及caspase-3/7活性,Western印迹检测Bcl-2,NADPH氧化酶2(NADPH oxidase 2,NOX2)、胞浆调节亚单位p-p47PHOX水平。结果:ALDH2过表达可显著降低DOX所致C2C12细胞凋亡及增殖抑制,而抑制ALDH2的表达则增加DOX对C2C12细胞凋亡诱导及增殖抑制作用;ALDH2过表达可下调p47PHOX磷酸化水平,抑制NOX2活化及ROS生成,而ALDH2低表达则上调p47PHOX磷酸化水平,促进NOX2活化及ROS生成;此外,NOX2活性抑制剂apocynin可抑制p47PHOX磷酸化、ROS生成及caspase-3/7的酶活性,同时增加ALDH2酶活性及其m RNA水平。结论:DOX所致的肌原细胞毒性与NOX2依赖性细胞氧化应激增强、ALDH2酶活性及表达降低有关,而ALDH2通过抑制上述NOX2信号,减轻细胞凋亡而发挥保护细胞作用。

Objective： To investigate the mechanisms for inhibitory effect of aldehyde dehydrogenase 2（ALDH2） on doxorubicin（DOX）-induced cytotoxicity in C2C12 myogenic cell line.Methods： Cell apoptosis was evaluated by flow cytometry and the activity of capase-3/7. The relative content of reactive oxygen species（ROS） and 4-hydroxynonenal（4-HNE） were detected by chemical fluorometric enzyme immunoassay. The protein and m RNA expression of ALDH2, Bcl-2, NADPH oxidase 2（NOX2） and the cytoplasmic subunit p-p47 PHOX were evaluated by Western blot and quantitative PCR, respectively. Results： Overexpression of ALDH2 at t enuated DOX-induced cell toxicity（increase in apoptosisand inhibition of proliferation）, which were reversed by downregulation of ALDH2. Overexpression of ALDH2 reduced p47 PHOX phosphorylation levels, and suppressed activation of NOX2 and ROS production, which were reversed by downregulation of ALDH2. Moreover, apocynin, an inhibitor of NOX, reduced the cytotoxicity of DOX concomitantly with a decrease in phosphorylation of p47 PHOX, ROS production and caspase-3/7 activity, and an increase in the activity and expression of ALDH2. Conclusion： DOX-induced cytotoxicity is related to increase of intracellular oxidative stress, which is involved in unregulation of NOX2 and downregulation of ALDH2. Activation of ALDH2 could exert cytoprotection via inhibiting NOX2-dependent ROS production.