摘要
目的 探讨炎症环境下Wnt/β-catenin通路对肺微血管内皮屏障通透性的影响。方法 分别用浓度为0、25、50、100、200 ng·mL^(-1)的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)处理人肺微血管内皮细胞(human pulmonary microvascular endothelial cells,HPMVECs)24 h,采用CCK-8检测细胞存活率,实时荧光定量PCR(RT-qPCR)检测闭合蛋白-5(claudin-5)、闭锁小带蛋白1(zonula occludens 1,ZO-1)的表达,选取TNF-α最佳干预浓度。将细胞分为control组、TNF-α 100 ng·mL^(-1)组、Wnt通路抑制对照组(XAV-NC组)、Wnt通路抑制组(XAV组),应用Wnt/β-catenin通路抑制剂XAV-939(10μmol·L^(-1),24 h)后,采用CCK-8法检测细胞存活率,细胞划痕实验检测细胞迁移,FITC-葡聚糖检测微血管内皮屏障通透性,RT-qPCR和蛋白免疫印迹(Western blot)测定各实验组β-连环蛋白(β-catenin)、细胞周期蛋白D1(Cyclin D1)、claudin-5、ZO-1的表达。结果 随TNF-α浓度增加,HPMVECs存活率逐渐降低,claudin-5、ZO-1 mRNA表达逐渐减少(P均<0.05),筛选出TNF-α作用的最适剂量为100 ng·mL^(-1)。应用XAV-939后,与control组相比,TNF-α 100 ng·mL^(-1)组细胞存活率及迁移率降低,Transwell小室通过FITC-葡聚糖增多,屏障相对通透性系数增大,β-catenin及Cyclin D1表达增加,claudin-5、ZO-1的表达减少(P均<0.05);与XAV-NC组比,XAV组细胞存活率及迁移率增加,Transwell小室通过FITC-葡聚糖减少,屏障相对通透性系数减小,β-catenin及Cyclin D1表达减少,claudin-5、ZO-1的表达增加(P均<0.05)。结论 TNF-α可通过激活Wnt/β-catenin通路,使紧密连接蛋白claudin-5、ZO-1的表达减少,增加肺微血管内皮屏障通透性。
Objective To investigate the effect of Wnt/β-catenin pathway on the permeability of pulmonary microvascular endothelial barrier in inflammatory environment.Methods Human pulmonary microvascular endothelial cells(HPMVECs)were treated with TNF-α at the concentrations of 0 ng·mL^(-1),25 ng·mL^(-1),50 ng·mL^(-1),100 ng·mL^(-1)and 200 ng·mL^(-1)for 24 h.The cell survival rate was measured by CCK-8,and the expression of claudin-5 and ZO-1 was detected by RT-q PCR.The best intervention concentration of TNF-αwas selected.The cells were divided into control group,TNF-α 100 ng·mL^(-1) group,Wnt pathway inhibited control group(XAV-NC group)and Wnt pathway inhibitor group(XAV group).After treated with Wnt/β-catenin pathway inhibitor XAV-939(10 μmol·L^(-1),24 h),the cell survival rate was detected by CCK-8,cell migration was detected by scratch test,microvascular endothelial barrier permeability was detected by FITC-glucan,and the expression of β-catenin,Cyclin D1,claudin-5 and ZO-1 in each experimental group was determined by RT-q PCR and Western blot.The data were analyzed by Image J and Prism 5.0 software.Results With the increase of TNF-α concentration,the survival rate of HPMVECs and the expression of claudin-5 and ZO-1 m RNA decreased gradually(P < 0.05).The optimal dose of TNF-α was screened as 100 ng·mL^(-1).After the administration of XAV-939,compared with control group,the cell survival rate and migration rate of TNF-α 100 ng·mL^(-1) group decreased,the amount of Transwell chamber passing through FITC-glucan increased,the relative permeability coefficient of barrier increased,the expression of β-catenin and Cyclin D1 increased,and the expression of claudin-5 and ZO-1 decreased(P<0.05).Compared with XAV-NC group,the cell survival rate and migration rate of XAV group increased,the amount of Transwell chamber passing through FITC-glucan decreased,the relative permeability coefficient of barrier decreased,the expression of β-catenin and Cyclin D1 decreased,and the expression of claudin-5 and ZO-1 increased(P all<0.05).Conclusion TNF-α can decrease the expression of tight junction proteins claudin-5 and ZO-1 and increase the permeability of pulmonary microvascular endothelial barrier by activating Wnt/β-catenin pathway.
作者
吴彦立
周文杰
马希刚
WU Yanli;ZHOU Wenjie;MA Xigang(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Department of Intensive Care Unit,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2022年第3期239-245,266,共8页
Journal of Ningxia Medical University
基金
宁夏自然科学基金项目(2022AAC02064)
宁夏回族自治区重点研发项目(2020BEG03012)。
作者简介
吴彦立(1997—),在读硕士研究生,研究方向:脓毒症与急性肺损伤;通信作者:马希刚,男,教授,主任医师,博士研究生导师,从事重症医学研究。E-mail:nyfyicu@163.com。
