mTOR/P70S6K/HIF-1α信号通路在脂多糖诱导Caco-2细胞屏障损伤中的作用及机制  

Role of mTOR/P70S6K/HIF-1α signaling pathway in Caco-2 cell barrier injury induced by lipopolysaccharide

作　　者：姚刘旭 滕文彬 黄素琴 李玉红 蒋宗明 YAO Liu-xu;TENG Wen-bin;HUANG Su-qin;LI Yu-hong;JIANG Zong-ming(Shaoxing University School of Medicine,Shaoxing 312000,China;Department of Anesthesiology,The First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310000,China;Department of Anesthesiology,Shulan(Hangzhou)Hospital,Shulan International Medical College,Shuren University,Hangzhou 310004,China;Department of Anesthesiology,The First Affiliated Hospital of Shaoxing University,Shaoxing 312000,China)

机构地区：[1]绍兴文理学院医学院,浙江绍兴312000 [2]浙江大学医学院附属第一医院麻醉科,浙江杭州310000 [3]树人大学树兰国际医学院附属树兰(杭州)医院麻醉科,浙江杭州310004 [4]绍兴文理学院医学院附属第一医院麻醉科(绍兴市人民医院),浙江绍兴312000

出　　处：《中国病理生理杂志》2022年第1期122-129,共8页Chinese Journal of Pathophysiology

基　　金：浙江省科学技术厅公益项目(No.LGF19H030011,No.LY21H150001);浙江省医药卫生科技计划项目(No.2020KY329);绍兴市科技项目(No.2020A13014);绍兴市麻醉学重点学科(No.2019SZD04)。

摘　　要：目的:探讨缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)在脂多糖(lipopolysaccharide,LPS)诱导的Caco-2细胞屏障损伤中的作用,并探究哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)/P70核糖体蛋白S6激酶(P70 ribosomal protein S6 kinase,P70S6K)信号通路的调控机制。方法:实验分成两部分,第一部分通过使用HIF-1α激活剂、抑制剂分别处理LPS作用的Caco-2细胞,探讨HIF-1α在LPS诱导的Caco-2细胞屏障损伤中的作用;第二部分则分别采用mTOR抑制剂、激活剂与HIF-1α激活剂、抑制剂联合处理LPS作用的Caco-2细胞,探讨HIF-1α影响LPS诱导的Caco-2细胞屏障损伤的分子机制。分别采用ERS-2电阻仪测量跨膜电阻,荧光法测量荧光右旋糖苷(FD4)浓度,Western blot检测HIF-1α、p-mTOR、p-P70S6K和紧密连接结构蛋白(ZO-1、occludin及claudin-1)的表达。结果:LPS诱导Caco-2细胞损伤,表现为跨上皮细胞电阻值(transepithelial cell resistance,TEER)降低,FD4浓度上升;HIF-1α激活剂DMOG可增加LPS作用的Caco-2细胞TEER、降低FD4浓度,同时上调HIF-1α和紧密连接结构蛋白的表达(P<0.05),而HIF-1α抑制剂BAY87-2243的作用则与DMOG作用相反;mTOR抑制剂雷帕霉素可降低LPS作用的Caco-2细胞TEER、增加FD4浓度,同时下调p-mTOR、p-P70S6K、HIF-1α及紧密连接结构蛋白的表达(P<0.05),而mTOR激活剂MHY1485则可增加LPS作用的Caco-2细胞TEER、降低FD4浓度,上调Caco-2细胞中p-mTOR和p-P70S6K表达水平(P<0.05),但不影响HIF-1α和紧密连接结构蛋白的表达水平。在雷帕霉素基础上加入DMOG,雷帕霉素对LPS处理的Caco-2细胞的效应被逆转;而在MHY1487基础上加入BAY87-2243,MHY1487对LPS处理的Caco-2细胞的效应被逆转。结论:HIF-1α可减轻LPS诱导的Caco-2细胞屏障损伤,其作用受mTOR/P70S6K信号通路调节。AIM:To investigate the effect of hypoxia-inducible factor-1α(HIF-1α)on the barrier damage of Caco-2 cells induced by lipopolysaccharide(LPS),which is regulated by mammalian rapamycin target protein(mTOR)/P70 ribosomal protein S6 kinase(P70 S6 K)signaling pathway.METHODS:Caco-2 cells were selected and experiment was divided into 2 parts. Part I:HIF-1α activator(DMOG)and inhibitor(BAY87-2243)were used to treated LPS-stimulated Caco-2 cells respectively,to explore the effect of HIF-1α on LPS induced Caco-2 cell barrier injury;Part II:mTOR inhibitor(rapamycin),activator(MHY1485)combined with or without HIF-1α activator,inhibitor to treated LPS-stimulated Caco-2 cells respectively,and then the molecular mechanism of HIF-1α reducing LPS induced Caco-2 cell barrier damage was detected. The transmembrane resistance was measured by ERS-2 resistance meter,the permeation of fluorescein-labeled dextran(FD4)was measured by fluorescence method,and the protein levels of HIF-1α,Zona occludens 1(ZO-1),occludin,claudin-1,p-mTOR and p-P70 S6 K were determined by Western blot.RESULTS:LPS induced Caco-2 cell injury,showing lower transepithelial cell resistance(TEER)and higher FD4 concentration. DMOG increased the TEER value of Caco-2 cells,decreased the concentration of FD4,and up-regulated the expression of the HIF-1α and ZO-1,occludin and claudin-1(P<0. 05). BAY87-2243 had the opposite effect with DMOG. Rapamycin decreased TEER,increased FD4 concentration and decreased the protein levels of p-mTOR,p-P70 S6 K,HIF-1ɑ and ZO-1,occludin and claudin-1 in Caco-2 cells(P<0. 05). MHY1487 increased TEER,decreased FD4 concentration,increased the protein levels of p-mTOR and p-P70 S6 K in Caco-2 cells(P<0. 05),and the expression of HIF-1α and HIF-1α and ZO-1,occludin and claudin-1 remained unchanged. DMOG offset the effect of rapamycin on Caco-2 cells,while BAY87-2243 offset the effect of MHY1487 on Caco-2 cells.CONCLUSION:HIF-1α attenuates the damage of Caco-2 cell barrier induced by LPS,which is regulated by mTOR/P70 S6 K signal

关 键 词：脓毒症 肠道上皮损伤 低氧诱导因子1Α mTOR/P70S6K信号通路 

分 类 号：R459.7[医药卫生—急诊医学] R363.2[医药卫生—治疗学]