雌激素相关受体α对脂多糖诱导的内皮细胞凋亡及连接蛋白降解的影响

Effect of estrogen-related receptorαon lipopolysaccharide-induced vascular endothelial apoptosis and tight junction protein degradation

目的探讨雌激素相关受体α(ERRα)对脂多糖(LPS)诱导的内皮细胞凋亡及连接蛋白降解的影响。方法体外培养ERRα敲低的稳转细胞株,并通过LPS处理,将细胞分为四组:正常对照组(Ctr组);shERRα敲低组(shERRα组);正常细胞+LPS处理组(LPS组):六孔板中的细胞用无血清的培养基饥饿培养12 h后,用20μg/mL LPS处理12 h;shERRα+LPS组:ERRα敲低的细胞按LPS组处理。使用ROS荧光试剂盒检测细胞内ROS的水平;利用TUNEL、AnnexinV-FITC和PI双染检测细胞凋亡情况;细胞荧光检测连接蛋白ZO-1在细胞膜表达情况,Western Blot检测凋亡相关蛋白Bcl-2、Bax、Smac、细胞色素c和连接蛋白ZO-1,Occludin、JAM-A及E-Ca在分子水平的表达情况。结果与Ctr组及shERRα组相比,LPS组ROS水平增加,细胞凋亡率增加(TUNEL检测为16.44±2.55,流式细胞术检测结果为23.56±2.22),促凋亡蛋白Bax、Smac及细胞色素c表达量增高,而抗凋亡蛋白Bcl-2和紧密连接蛋白表达量降低,细胞荧光结果显示连接蛋白ZO-1在包膜发生降解,网状结构断裂。而与LPS组相比,在shERRα+LPS组中,抑制ERRα的表达加剧上诉细胞损伤。结论ERRα能够通过负性调节肺微血管内皮细胞内的细胞凋亡,影响肺微血管内皮的功能,从而调控脓毒症诱导的急性肺损伤。

Objective To explore the effect of estrogen-related receptorα(ERRα)on lipopolysaccharide(LPS)-induced vascular endothelial cell apoptosis and tight junction protein degradation.Methods RPMVECs transfected with shERRαwere cultured in vitro and divided into four groups:Normal control group(Ctr group);shERRαknockdown group(shERRαgroup);normal cells+LPS treated group(LPS group):The cells in the six-well plates were cultured in serum-free medium for 12 h,and then treated with 20μg/mL LPS for 12 h;and shERRα+LPS group:ERRαknockdown cells were treated as the LPS group.ROS fluorescence kit was used to detect the intracellular ROS levels.Apoptosis ratio was detected by TUNEL staining,AnnexinV-FITC and PI.Cell membrane ZO-1 expression was detected by cellular immunofluorescence,and the levels of apoptosis-related proteins Bcl-2,Bax,Smac,Cytochrome c,and tight junction protein ZO-1,as well as the expression of Occludin,JAM-A and E-Ca at molecular level were detected by Western blot.Results Compared with the Ctr group and the shERRαgroup,the ROS level,apoptosis rate(TUNEL test:16.44±2.55;and flow cytometry test:23.56±2.22),the expression of pro-apoptotic proteins Bax,Smac and Cytochrome c were increased in the LPS group,while the expression of anti-apoptotic proteins Bcl-2 and tight junction protein were decreased.In the LPS group.Cellular immunofluorescence results showed that the ZO-1 was degraded in the cell membrane and the network structure was broken.Compared with the LPS group,inhibition of ERRαin the shERRα+LPS group increased cell damage.Conclusions ERRαcan negatively regulate the apoptosis and affect the function of pulmonary microvascular endothelial cells,thereby regulating sepsis-induced acute lung injury.