鞘氨醇激酶-1对热联合内毒素打击小鼠肺微血管内皮细胞通透性的影响  

Effect of SphK1 on the permeability of mice lung microvascular endothelial cells induced by double insults of heat and endotoxin

作　　者：易万华 古正涛[2] 文强 苏磊 YI Wan-hua;GU Zheng-tao;WEN Qiang;SU Lei(Graduate School,Guangdong Pharmaceutical University,Guangzhou 510010,China;Trauma Center,the Third Affiliated Hospital of Southern Medical University,Guangzhou 510630,China;Department of Intensive Care Unit,key Laboratory of Hot Zone Trauma Care and Tissue Repair of Chinese PLA,General Hospital of Southern Theater Command,Guangzhou 510010,China)

机构地区：[1]广东药科大学研究生院,广州510080 [2]南方医科大学附属第三医院创伤中心,广州510630 [3]南部战区总医院重症医学科/全军热区创伤救治与组织修复重点实验室,广州510010

出　　处：《解放军医学杂志》2019年第12期1018-1023,共6页Medical Journal of Chinese People's Liberation Army

基　　金：军事医学创新工程专项(17CXZ008);广东省自然科学基金(2014A 030313599)~~

摘　　要：目的探讨鞘氨醇激酶-1(SphK1)对热联合内毒素(LPS)打击小鼠肺微血管内皮细胞(PMVECs)通透性的影响。方法分离培养并鉴定PMVECs,建立热联合LPS双重打击PMVECs模型。将PMVECs细胞分为对照组、热打击组(HS组)、内毒素打击组(LPS组)及热联合内毒素打击组(HS+LPS组),采用Western blotting检测各组细胞2种鞘氨醇激酶亚型(SphK1/2)、3种1-磷酸鞘氨醇膜受体(S1PR1/2/3),以及相关紧密连接蛋白如闭合蛋白(occludin)、闭锁小带蛋白1(ZO-1)的表达,荧光定量PCR检测SphK及S1PR mRNA的表达。将细胞分为对照组、HS+LPS组及DMS预处理热联合内毒素打击组(HS+LPS+DMS组),采用SphK活性检测试剂盒检测酶活性,CCK-8试剂盒检测细胞活力,EVOM跨膜电阻测量仪检测细胞跨上皮电阻(TEER),并观察特异性抑制SphK1表达对细胞活力和通透性的影响。结果热联合内毒素打击后,细胞SphK1和S1PR3蛋白、mRNA表达及酶活性水平与正常对照组比较明显上升(P<0.01),细胞活力、TEER值、紧密连接蛋白(ZO-1、occludin)表达较对照组下降;采用SphK1特异性抑制剂DMS预处理PMVECs细胞后,明显提升了细胞活力[63.3%±7.1%vs.41.3%±4.5%,P<0.05]、TEER值[(154.3±16.0)(Ω·cm2)vs.(84.7±9.6)(Ω·cm2)],以及紧密连接蛋白的表达(P<0.05)。结论SphK1的激活可以促进热联合LPS打击小鼠PMVECs通透性的增加,主要参与的下游1-磷酸鞘氨醇(S1P)细胞膜表面受体可能为S1PR3,特异性抑制SphK1表达可减轻PMVECs损伤。Objective To investigate the effects of sphingosine kinase-1(SphK1)on the permeability of heat shock(HS)-combined endotoxin(LPS)treated microvascular endothelial cells(PMVECs)in mice.Methods PMVECs were isolated and characterized followed by treatment of HS and LPS.To examine the expression levels of SphK1 associated signaling,Sphk1/2 and 1-phosphate pyridoxal membrane receptors(S1PR1/2/3)were quantified using Western blotting and fluorescence quantitative PCR.Besides,SphK enzyme activity was quantified using SphK kits.To evaluate effects of HS and LPS on cells,cell tight junctions,such as closed protein(occludin)and zonula occludens-1(ZO-1)were quantified using Western blotting;cell vitality was determined using CCK-8 kit;cell permeability was assessed through transepithelial electrical resistance(TEER)using the EVOM crossmembrane resistance meter.Results After cells were treated with HS and LPS,the expression levels SphK1 and S1PR3 increased significantly(P<0.01).As expected,SphK1 enzyme activity also elevated significantly(P<0.01).However,TEER value and ZO-1/occludin expression decreased compared to the control group.These phenomena could be rescued by pretreat PMVECs with SphK1-specific inhibitor DMS pre-treatment of PMVECs cells[cell vitality:63.3%±7.1%vs.41.3%±4.5%,P<0.05;TEER value;(154.3±16.0)(Ω·cm2)vs.(84.7±9.6)(Ω·cm2)].Conclusions SphK1 activation,possibly through S1P-S1PR3 signaling,can increase the permeability of HS and LPS treated PMECVs.Inhibiting SphK1 can reduce the damage to endothelial cells in the lung microvascular.

关 键 词：重症中暑 肺损伤 SphK1 通透性 

分 类 号：R594.12[医药卫生—内科学]