LPS致PMVECs损伤时CPNE1/CAV-1相互作用机制研究  

The study on the interaction mechanism between CPNE1 and CAV-1 in LPS-stimulated PMVECs

作　　者：吴翔晖 李琪琪 李帅[1] 陆宗庆 方普 庞晴晴 汪博 尤青海[1] WU Xianghui;LI Qiqi;LI Shuai;LU Zongqing;FANG Pu;PANG Qingqing;WANG Bo;YOU Qinghai(Department of Respiratory and Critical Care Medicine,the First Affiliated Hospital of Anhui Medical University,Hefei,Anhui 230032,China)

机构地区：[1]安徽医科大学第一附属医院呼吸与危重症医学科,安徽合肥230032

出　　处：《临床肺科杂志》2025年第5期692-698,共7页Journal of Clinical Pulmonary Medicine

基　　金：安徽省自然科学基金项目(2208085MH195)。

摘　　要：目的探讨脂多糖(LPS)刺激肺微血管内皮细胞(PMVECs)时Copine1(CPNE1)与小窝蛋白-1(CAV-1)的相互作用及SHH信号通路对其调控作用。方法体外培养PMVECs,Western Blot(WB)检测LPS刺激时CPNE1、CAV-1和GLI1表达变化,随机分为LPS时效组(LPS 10mg/L,0、3、6、12、18、24h)和量效组(0、1、5、10、20、40mg/L);siRNA沉默CPNE1,WB和免疫荧光检测沉默CPNE1后PMVECs表达CAV-1变化,随机分为Control组、LPS组、siCPNE1组、siCPNE1+LPS组;10mg/L LPS刺激PMVEC时给予SHH信号通路抑制剂(Vismodegib)干扰,随机分为Control组、LPS组、Vismodegib组、Vismodegib+LPS组;siRNA沉默CPNE1后给予SHH信号通路激动剂(SAG)干扰,分为Control组、si-CPNE1组、SAG组、si-CPNE1+SAG组。结果(1)LPS刺激PMVECs12h后,与0h组比较,CPNE1、CAV-1和GLI1蛋白的表达均显著增加(P<0.05)。(2)共聚焦免疫荧光技术显示LPS刺激PMVECs时CAV-1和CPNE1的荧光信号增强;与Control组比较,si-CPNE1组的荧光信号降低;与LPS组比较,si-CPNE1+LPS组的荧光信号降低。WB检测提示与LPS组比较,siCPNE1+LPS组CAV-1蛋白表达显著下降(P<0.05)。(3)Vismodegib显著下调LPS诱导增加的GLI1、CPNE1和CAV-1蛋白表达水平(P<0.05);SAG显著上调CPNE1、CAV-1和GLI1蛋白表达水平(P<0.05);沉默CPNE1后给予SAG干扰,CAV-1和CPNE1蛋白表达水平并未出现显著上升(P>0.05)。结论LPS刺激时CPNE1与CAV-1在PMVECs中协同响应,且均受SHH信号通路调控。Objective To explore the interaction between CPNE1 and Caveolin-1(CAV-1)in pulmonary microvascular endothelial cells(PMVECs)stimulated by lipopolysaccharide(LPS)and the role of the SHH signaling pathway in its regulation.Methods PMVECs were cultured in vitro.The expression of CPNE1,CAV-1,and GLI1 in LPS-stimulated PMVECs were detected by Western blot(WB),and the cells were randomly divided into LPS time-dependent groups(LPS 10mg/L,0,3,6,12,18,24h)and quantitatively effective groups(0,1,5,10,20,40mg/L).WB and immunofluorescence detection in expression of CAV-1 were used after silencing of CPNE1 in PMVECs,and cells were randomly divided into control,LPS,siCPNE1,siCPNE1+LPS groups;10mg/L LPS stimulation of PMVECs was given with SHH signaling pathway inhibitor(Vismodegib)interference.Cells were randomly divided into Control,LPS,Vismodegib,Vismodegib+LPS groups;SHH signaling pathway agonist(SAG)interference was given after silencing CPNE1 by siRNA,Cells were divided into control,siCPNE1,SAG,si-CPNE1+SAG groups.Results(1)After LPS stimulation of PMVECs for 12h,the expression of CPNE1,CAV-1 and GLI1 proteins were all significantly increased compared with the unstimulated group(P<0.05),and positively correlated with the concentration trend.(2)Confocal immunofluorescence technique showed that the fluorescence signals of both CAV-1 and CPNE1 were significantly enhanced by LPS stimulation.Compared with the control group,the fluorescence signal in the si-CPNE1 group was significantly reduced.Compared with the LPS group,the fluorescence signal of the si-CPNE1+LPS group was significantly reduced.WB assay suggested that CAV-1 protein expression was significantly decreased in the siCPNE1+LPS group compared with the control-siRNA+LPS group(P<0.05).(3)Vismodegib significantly down-regulated the LPS-induced increase in GLI1,CPNE1 and CAV-1 protein expression levels(P<0.05).SAG significantly up-regulated CPNE1,CAV-1 and GLI1 protein expression levels(P<0.05).SAG interference given after silencing CPNE1 did not show significant incre

关 键 词：脂多糖 肺微血管内皮细胞 Copine1 小窝蛋白-1 Sonic Hedgehog信号通路 

分 类 号：R563[医药卫生—呼吸系统]