组织蛋白酶S对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞损伤和内皮间质转化的影响和机制研究  

Effect and mechanism of cathepsin S on oxidized low-density lipoproteininduced human umbilical vein endothelial cell injury and endothelialmesenchymal transition

作　　者：葛曙雄 王辉[1] 许中友[1] GE Shuxiong;WANG Hui;XU Zhongyou(Department of Vascular,People's Hospital Affiliated to Ningbo University,Ningbo 315000,China)

机构地区：[1]宁波大学附属人民医院血管外科,宁波315000

出　　处：《中国免疫学杂志》2023年第1期43-48,共6页Chinese Journal of Immunology

摘　　要：目的:探究组织蛋白酶S(CTSS)对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)凋亡、炎症和内皮间质转化(EndMT)的影响和机制。方法:将HUVECs细胞分成4组:对照组、ox-LDL组、ox-LDL+si-CTSS组、oxLDL+si-NC组。ox-LDL组加入100μg/ml ox-LDL孵育24 h,ox-LDL+si-CTSS组和ox-LDL+si-NC组先用转染试剂将CTSS siRNA或对照siRNA转染至细胞中48 h再加入100μg/ml ox-LDL孵育24 h,对照组不进行处理。CCK-8试剂盒检测细胞增殖,流式细胞术检测细胞凋亡,ELISA测定实验检测细胞因子IL-1β和TNF-α表达,Western blot检测CTSS、EndMT相关蛋白(内皮细胞标志物CD31、VE-cadherin和间质细胞标志物α-SMA、Vimentin)、磷酸化p38 MAPK(p-p38)、总的p38 MAPK(t-p38)、p-Akt、Akt、pERK1/2和ERK1/2蛋白含量,显微镜观察细胞形态。此外,使用p38 MAPK激活剂茴香霉素(AM)进一步验证下调CTSS对oxLDL诱导的内皮细胞损伤的作用机制。结果:与对照组相比,ox-LDL组中CTSS表达增加,细胞活力降低,凋亡细胞数目增多,IL-1β和TNF-α含量增加,多数细胞变成纺锤样,CD31和VE-cadherin表达下调,α-SMA和Vimentin表达上调,p-p38/t-p38、pAkt/Akt和p-ERK1/2/ERK1/2增加,差异均有统计学意义(P<0.05);与ox-LDL组相比,ox-LDL+si-CTSS组中CTSS表达降低,细胞活力增加,凋亡细胞数目减少,IL-1β和TNF-α含量降低,纺锤样细胞数目减少,CD31和VE-cadherin表达上调,α-SMA和Vimentin表达下调,p-p38/t-p38、p-Akt/Akt和p-ERK1/2/ERK1/2减少,差异均有统计学意义(P<0.05)。与ox-LDL+si-CTSS组相比,ox-LDL+si-CTSS+AM组细胞活力减弱,TNF-α含量增多,CD31的表达降低,而α-SMA表达升高,差异均具有统计学意义(P<0.05)。结论:下调CTSS可减弱ox-LDL诱导的内皮细胞凋亡、炎症和EndMT,这很大程度上是通过抑制p38 MAPK、Akt和ERK1/2通路的活化发挥作用的。Objective:To investigate the role and mechanism of cathepsin S(CTSS)on low-density lipoprotein(ox-LDL)-induced apoptosis,inflammation and endothelial-mesenchymal transition(EndMT)in human umbilical vein endothelial cells(HUVECs).Methods:HUVECs cells were divided into four groups:control group,ox-LDL group,ox-LDL+si-CTSS group and ox-LDL+si-NC group.Cells in ox-LDL group were treated with 100μg/ml ox-LDL for 24 h.Cells in ox-LDL+si-CTSS and ox-LDL+si-NC groups were transfected with CTSS siRNA or control siRNA for 48 h and then incubated with 100μg/ml ox-LDL for 24 h.And cells in control group did not perform any treatment.Cell proliferation was detected by CCK-8 assay kit.Flow cytometry was used to analyze cell apoptosis.ELISA was used to assay the contents of cytokines,including IL-1βand tumor necrosis factor(TNF)-α.Western blot was used to measure the levels of CTSS,EndMT related proteins(endothelial cell markers CD31 and VE-Cadherin;mesenchymal cell markersα-SMA and Vimentin),phosphorylated p38 MAPK(p-p38),total p38 MAPK(t-p38),p-Akt,Akt,p-ERK1/2 and ERK1/2.Cell morphology was observed under a microscope.Additionally,anisomycin(AM),a p38 MAPK activator,was used to further confirm the mechanism of silencing CTSS on ox-LDL-induced endothelial cell injury.Results:Compared with control group,CTSS expression,the number of apoptotic cells and the contents of IL-1βand TNF-αwere increased,cell viability was decreased,most cells expressed spindle-like morphology,the expression of CD31 and VE-cadherin was downregulated,the expression ofα-SMA and Vimentin were upregulated,and levels of p-p38/t-p38,p-Akt/Akt and p-ERK1/2/ERK1/2 were elevated in the ox-LDL group(all P<0.05).Compared with ox-LDL group,CTSS expression,the number of apoptotic cells and the contents of IL-1βand TNF-αwere decreased,cell viability was increased,the number of cells with spindle-like morphology was reduced,the expressions of CD31 and VE-cadherin were upregulated,the expressions ofα-SMA and Vimentin were downregulated,and levels of p-p38/

关 键 词：组织蛋白酶S 氧化型低密度脂蛋白 人脐静脉内皮细胞 内皮间质转化 信号通路 

分 类 号：R541.4[医药卫生—心血管疾病]