香烟烟雾提取物通过氧化应激下调组蛋白去乙酰化酶2诱导小鼠骨骼肌细胞早衰

Cigarette smoke extract induces senescence of murine skeletal muscle cells by oxidative stress-induced down-regulation of histone deacetylase 2

目的探讨香烟烟雾提取物(CSE)是否通过氧化应激减少抗衰老分子组蛋白去乙酰化酶2(HDAC2)的表达而诱导骨骼肌细胞早衰。方法诱导C2C12成肌细胞分化成骨骼肌细胞,观察CSE处理对骨骼肌细胞氧化应激、衰老和HDAC2表达的影响,应用氧化剂H2O2和抗氧化剂N-乙酰半胱氨酸(NAC)为工具药,检测细胞中丙二醛(MDA)含量、活性氧(ROS)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性的变化,应用β半乳糖苷酶染色检测衰老细胞百分率,采用实时荧光定量PCR检测HDAC2 mRNA水平,Western blot法检测HDAC2蛋白水平。结果与对照组相比,CSE和H2O2可增加小鼠骨骼肌细胞MDA含量和ROS水平,而降低SOD、GSH-Px的活性,同时伴有β半乳糖苷酶的水平增加和抗衰老分子HDAC2 mRNA和蛋白水平的降低。NAC预处理后,可降低CSE诱导的MDA含量和ROS活性,提高SOD、GSH-Px的活性,同时β半乳糖苷酶表达降低,HDAC2的水平升高。结论 CSE可能是通过氧化应激降低HDAC2诱导小鼠骨骼肌细胞早衰。

Objective To investigate whether cigarette smoke extract （CSE） induces the senescence of skeletal muscle cells by oxidative stress-induced down-regulation of histone deacetylase 2 （HDAC2）. Methods C2C12 myoblasts were induced to differentiate into skeletal muscle cells. H202 and N-acetyl cysteine （NAC） were used to investigate the effects of CSE on oxidative stress, cell senescence and the expression of HDAC2 in skeletal muscle cells, and detect the changes of the activities of malondialdehyde （MDA）, superoxide dismutase （SOD）, reactive oxygen species （~ ROS） and glutathione peroxidase （GSH-Px）. Cell senescence was identified by senescence-associated [3-galactosidase staining. The expressions of HDAC2 mRNA and protein were measured by real-time quantitative PCR and Western blotting, respectively. Results Compared with the control group, MDA concentration and ROS activity significantly increased, the activities of SOD and GSH-Px significantly decreased, the expression of [3-galactosidase was up-regulated, and the expressions of HDAC2 mRNA and protein were down-regulated in the CSE group and the H202 group. However, the changes in the CSE group were reversed after the pretreatment of NAC. Conclusion CSE may lead to the senescence of murine skeletal muscle cells by oxidative stress-induced down-regulation of HDAC2.