阿奇霉素诱导衰老细胞凋亡研究  

Azithromycin-induced Apoptosis in Senescent Cells

作　　者：谭悦浩 李灿[1] 刘漪沦 孔德华 胡康安 张迪 孙静[1] 王航宇[3] 邓峰美[1] Tan Yuehao;Li can;Liu Yilun;Kong Dehua;Hu Kang'an;Zhang Di;Sun Jing;Wang Hangyu;Deng Fengmei(School of Basic Medicine,Chengdu Medical College,Chengdu 610500;Sichuan Institute of Geriatrics Clinical Medical Research,Chengdu 610500;Shihezi University,Shihezi 832000)

机构地区：[1]成都医学院基础医学院,成都610500 [2]四川省老年医学临床医学研究中心,成都610500 [3]石河子大学,石河子832000

出　　处：《国际老年医学杂志》2021年第4期193-198,共6页International Journal of Geriatrics

基　　金：国家自然科学基金项目(81560566);四川省科技厅项目(2017JY0150,2018054);四川省教育厅项目(17ZA0113);发育与再生四川省重点实验室基金项目(SYS15-004,SYS15-008);成都医学院第一附属医院专项科研基金(CYFY2017YB01)。

摘　　要：目的探究阿奇霉素(AZM)对H_(2)O_(2)诱导的小鼠胚胎成纤维细胞系(NIH/3T3)衰老模型的效果及相关机制。方法采用H_(2)O_(2)处理NIH/3T3细胞,构建细胞衰老模型,利用ELISA检测相关标志物水平,分析衰老相关的β-半乳糖苷酶(SA-β-GAL)活性以检测模型的相关表型;运用Western blot检测其衰老相关分泌表型(SASP)的蛋白标志物表达水平,qPCR分析其相关mRNA水平;使用AZM处理正常细胞及衰老细胞,并运用流式细胞术、TUNEL、Western blot等检测AZM作用下相关表型并验证相关表型的标志物。结果H_(2)O_(2)处理后的NIH/3T3细胞核体积变大;β-半乳糖苷酶染色蓝染比率升高(P<0.05);衰老相关细胞炎性因子IL-6、IL-8/CXCL8分泌量显著上升(P<0.05);衰老标志性蛋白P53、P21、P16水平均显著升高(P<0.05),同时mRNA呈相同趋势(P<0.05)。衰老细胞在使用不同浓度AZM处理后,发生凋亡,并具有浓度依赖性。使用AZM处理不同实验组细胞24h后,Western blot结果显示衰老细胞cleaved PARP/PARP、Bax上调、Bcl-2下调(P<0.05);经Annexin V-PE/7-AAD双染,Annexin V阳性比率上升(P<0.05);正常细胞在AZM的处理下凋亡比例未见明显差异(P>0.05);TUNEL实验结果与上述一致。结论AZM可能特异性诱导衰老细胞凋亡,为临床上抗衰老药物的选择与应用提供了新的策略。Objective To investigate the effect of azithromycin(AZM)on aging model of H2 O2-induced mouse embryon-ic fibroblast cell line(NIH/3T3)and its possible mechanism.Methods Cell aging model was established by treating NIH/3T3 cell with H_(2)O_(2).The cytokines were detected by ELISA.The senescence-associatedβ-galactosidase(SA-β-GAL)was analyzed by a kit.The protein expression of senescence-associated secretory phenotype(SASP)was detected by western blot and mRNA level was analyzed by qPCR.The normal cells and the senescent cells were treated with AZM.Flow cytometry,TUNEL and western blot were used to detect the relevant phenotypes.Results Compared with the negative control group,after treatment with H2O2,the nu-clear size of NIH/3T3 cells and the rate of SA-βGal positive cells were significantly increased(P<0.05).The levels of senes-cence-associated inflammatory cytokines including IL-6 and IL-8/CXCL8 were significantly increased(P<0.05).The levels of senescence-associated protein including p53,p21 and p16 were significantly increased(P<0.05),and mRNA levels showed a same trend(P<0.05).The apoptosis appeared in the cells treated with AZM and showed concentration-dependent pattern.After treatment with AZM for 24 h,western blot showed up-regulation of the cleaved PARP/PARP and Bax,and down-regulation of Bcl-2 in senescent cells(P<0.05).Staining with Annexin V-PE/7-AAD showed an increased positive ratio of Annexin V(P<0.05).However,there was no significant difference in the apoptosis rate of normal cells after AZM administration(P>0.05).TUNEL staining revealed a similar trend.Conclusion AZM may specifically induce apoptosis in senescent cells.

关 键 词：阿奇霉素 NIH/3T3细胞 衰老细胞 凋亡 

分 类 号：R965[医药卫生—药理学]