摘要
目的探索H_(2)O_(2)诱导的氧化损伤时MDM2的功能及与p53的关系。方法使用0.5 mmol/L H_(2)O_(2)建立MLE-12 HALI细胞模型,分为:健康对照组、H_(2)O_(2)损伤组、H_(2)O_(2)+MDM2 overexpressed组、H_(2)O_(2)+MDM2-shRNA组。通过腺病毒载体感染MLE-12细胞过表达、沉默MDM2;采用免疫共沉淀(Co-IP)分析MDM2与p53的相互作用;采用Western blotting检测HALI建模后MDM2、p53、Bcl-2、Bax和cleaved caspase-3蛋白表达水平;检测各组细胞凋亡率。结果通过转录组测序后发现p53信号通路与HALI密切相关。与正常组相比,H_(2)O_(2)损伤组MDM2表达较低(P<0.05);与H_(2)O_(2)损伤组相比,MDM2过表达后MLE-12细胞凋亡率降低(P<0.05),p53、Bax、cleaved caspase-3蛋白表达水平下降,MDM2、Bcl-2蛋白表达上调(P<0.05)。与H_(2)O_(2)损伤组相比,当MDM2沉默时,细胞凋亡率增加(P<0.05),p53、Bax、cleaved caspase-3蛋白表达水平上调,MDM2、Bcl-2蛋白表达下降(P<0.05)。Co-IP实验显示MDM2与p53蛋白结合。结论这些结果表明,MDM2可通过p53/Bcl-2/Bax轴抑制MLE-12细胞凋亡从而发挥对HALI的保护作用。
Objective To explore the function of MDM2 and its relationship with p53 at the cellular level during H_(2)O_(2)induced oxidative damage.Methods MLE-12 HALI cell models were established using 0.5 mmol/L H_(2)O_(2),and were divided into three groups:normal control group,H_(2)O_(2)injury group,H_(2)O_(2)+MDM2 overexpressed group,and H_(2)O_(2)+MDM2 shRNA group.Infection of MLE-12 cells with adenovirus vector overexpressing and silencing MDM2;Using immunoprecipitation(Co-IP)to analyze the interaction between MDM2 and p53;Western blotting was used to detect the protein expression levels of MDM2,p53,Bcl-2,Bax,and cleared caspase-3 after HALI modeling;Measure the apoptosis rate of cells in each group.Results After transcriptome sequencing,the p53 signaling pathway closely related to HALI.Compared with the normal group,the expression of MDM2 in the H_(2)O_(2)injury group was lower(P<0.05);Compared with the H_(2)O_(2)injury group,overexpression of MDM2 resulted in a decrease in the apoptosis rate of MLE-12 cells(P<0.05),a decrease in the expression levels of p53,Bax,and cleared caspase-3 proteins,and an upregulation of MDM2 and Bcl-2 protein expression(P<0.05).Compared with the H_(2)O_(2)injury group,when MDM2 was silenced,the cell apoptosis rate increased(P<0.05),and the expression levels of p53,Bax,and cleared caspase-3 proteins were upregulated,while the expression levels of MDM2 and Bcl-2 proteins decreased(P<0.05).Co-IP experiments showed that MDM2 binds to p53 protein.Conclusions MDM2 can exert a protective effect on HALI by inhibiting MLE-12 cell apoptosis through the p53/Bcl-2/Bax axis.
作者
郑杰
陈博文
梅鸿
刘鑫鑫
廖贞亮
余琨
余虹
冯帮海
陈淼
傅小云
覃松
Zheng Jie;Chen Bowen;Mei Hong;Liu Xinxin;Liao Zhenliang;Yu Kun;Yu Hong;Feng Banghai;Chen Miao;Fu Xiaoyun;Qin Song(Department of Critical Care Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563000;Department of Critical Care Medicine,Affiliated Hospital of Guizhou Medical University,Guiyang 550004;Department of Critical Care Medicine,Zunyi Hospital of Traditional Chinese Medicine,Zunyi 563000)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2024年第8期1110-1116,共7页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金(81960362)
贵州省科技计划(ZK-2023-544)
贵州省卫生健康委科学技术基金(gzwkj2024-310,gzwjkj2019-1-068)
遵义市科技与大数据局科学技术项目:(2023-221,2023-199)。
