摘要
目的:探讨miR-205对体外脓毒症心肌细胞模型细胞凋亡和炎症因子分泌的影响。方法:qRT-PCR方法分析脓毒症大鼠心肌组织中miR-205表达变化,TUNEL法检测细胞凋亡,ELISA法检测TNF-α、IL-6水平。心肌细胞分成Control组、LPS组(LPS诱导)、miR-NC+LPS组(转染mimics control,LPS诱导)、miR-205+LPS组(转染miR-205 mimics,LPS诱导),CCK-8方法检测细胞增殖,流式细胞术检测细胞凋亡变化,Western blot检测C-Caspase-3、NF-κBp65蛋白表达,二硝基苯肼法检测LDH水平,硫代巴比妥酸法检测MDA水平,黄嘌呤法检测SOD水平,比色法检测GSH-Px水平,ELISA法检测TNF-α、IL-6水平。用NF-κB信号激活剂处理上调miR-205的心肌细胞,同样检测细胞增殖、凋亡、氧化损伤以及细胞炎症因子分泌水平。结果:脓毒症大鼠心肌组织中miR-205表达水平降低,心肌组织中细胞凋亡指数增加,TNF-α、IL-6水平升高。LPS组心肌细胞中miR-205水平降低,细胞存活率降低,细胞凋亡率升高,细胞中C-Caspase-3蛋白表达增多,培养液上清中LDH、TNF-α、IL-6水平升高,细胞中MDA水平升高,SOD、GSH-Px水平降低,与Control组相比,差异有统计学意义(P<0.05)。miR-205+LPS组心肌细胞中miR-205水平升高,细胞存活率升高,细胞凋亡率降低,细胞中C-Caspase-3蛋白表达减少,培养液上清中LDH、TNF-α、IL-6水平降低,细胞中MDA水平降低,SOD、GSH-Px水平升高,与miR-NC+LPS组相比,差异有统计学意义(P<0.05)。NF-κB信号激活剂逆转miR-205对LPS诱导的心肌细胞损伤作用。结论:miR-205抑制体外脓毒症心肌细胞模型细胞凋亡和炎症因子分泌,机制可能与下调NF-κB信号有关。
Objective:To investigate the effect of miR-205 on apoptosis and secretion of inflammatory factors in an in vitro sepsis cardiomyocyte model.Methods:qRT-PCR method was used to analyze the expression of miR-205 in myocardial tissue of septic rats,apoptosis was detected by TUNEL method,and TNF-α,IL-6 levels were detected by ELISA.Cardiomyocytes were divided into Control group,LPS group(LPS induction),miR-NC+LPS group(transfection mimics control,LPS induction),miR-205 group(transfection miR-205 mimics,LPS induction),CCK-8 method was used to detect proliferation,flow cytometry was used to detect changes in cell apoptosis,Western blot was used to detect the expressions of C-Caspase-3 and NF-κBp65 protein,dinitrophenylhydrazine method was usedto detect LDH level,thiobarbituric acid method was used to detect MDA level,xanthine method was used to detect SOD level,colorimetric method was used to detect GSH-Px level,ELISA method was used to detect TNF-αand IL-6 levels.The cardiomyocytes that up-regulated miR-205 were treated with NF-κB signal activator,and the levels of cell proliferation,apoptosis,oxidative damage and cell inflammatory factor secretion were also detected.Results:Expression level of miR-205 in myocardial tissue of septic rats was decreased,the apoptosis index and TNF-α,IL-6 levels in myocardial tissue were increased.The level of miR-205 in cardiomyocytes of LPS group was decreased,cell survival rate was decreased,cell apoptosis rate was increased,C-Caspase-3 protein expression was increased,and LDH,TNF-α,IL-6 levels in the culture supernatant were increased,the MDA level in the cells was increased,and the SOD and GSH-Px levels were decreased,compared with Control group,the difference was statistically significant(P<0.05).In miR-205+LPS group,the level of miR-205 in cardiomyocytes was increased,cell survival rate was increased,cell apoptosis rate was decreased,C-Caspase-3 protein expression in the cells was decreased,and LDH,TNF-α,IL-6 levels in the culture supernatant were decreased,level of MDA in t
作者
谢斌
邓超
陈栩栩
苏醒
王红梅
XIE Bin;DENG Chao;CHEN Xuxu;SU Xing;WANG Hongmei(Department of Critical Care Medicine,Central South University Xiangya School of Medicine Affliated Haikou Hospital,Haikou 570203,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2022年第8期933-939,共7页
Chinese Journal of Immunology
