基于自噬角度探究A2AR对内毒素诱导的Caco-2肠上皮细胞炎性损伤的影响  

To explore the effect of A2AR on endotoxin-induced inflammatory injury of Caco-2 intestinal epithelial cells from the perspective of autophagy

作　　者：陈禺 王小吉 李合 Chen Yu;Wang Xiaoji;Li He(Dept of General Surgery,2Dept of Hepatobiliary Surgery,The First Affiliated Hospital of Hainan Medical College,Haikou 570000)

机构地区：[1]海南医学院第一附属医院普通外科,海口570000 [2]海南医学院第一附属医院肝胆外科,海口570000

出　　处：《安徽医科大学学报》2024年第9期1636-1642,共7页Acta Universitatis Medicinalis Anhui

基　　金：海南省卫生健康行业科研项目(编号:22A200079)。

摘　　要：目的探究腺苷A2A受体(A2AR)对内毒素脂多糖(LPS)诱导的Caco-2肠上皮细胞炎性损伤的影响及机制。方法首先将Caco-2细胞分为对照组、LPS组(10μg/ml LPS处理12 h)、A2AR激动剂(CGS21680)组(10μmol/L CGS21680预处理10 min)、CGS21680+LPS组(10μmol/L CGS21680预处理10 min、10μg/ml LPS处理12 h),CCK-8法测定各组细胞活力,ELISA法测定各组细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)的分泌水平,实时荧光定量PCR检测各组细胞中TNF-α、IL-1β、IL-6的mRNA表达水平,Western blot分析各组细胞中自噬相关蛋白微管相关轻链蛋白3(LC3)-Ⅱ/LC3-Ⅰ、自噬相关蛋白(Beclin1)的蛋白表达水平;再将Caco-2细胞分为对照组、LPS组(10μg/ml LPS处理12 h)、CGS21680+LPS组(10μmol/L CGS21680预处理10 min、10μg/ml LPS处理12 h)、CGS21680+LPS+雷帕霉素(Rapa)组(10μmol/L CGS21680预处理10 min、10μg/ml LPS与5μmol/L Rapa处理12 h),CCK-8法测定各组细胞活力,ELISA法测定各组细胞上清液中TNF-α、IL-1β、IL-6的分泌水平。结果与对照组比较,LPS组Caco-2细胞活力显著降低(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平显著升高(P<0.05),细胞中TNF-α、IL-1β、IL-6的mRNA相对表达量均显著上调(P<0.05),并检测到LC3-Ⅱ/LC3-Ⅰ比值与Beclin1蛋白相对表达量显著上调(P<0.05);与LPS组比较,CGS21680+LPS组Caco-2细胞活力显著升高(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平显著降低(P<0.05),细胞中TNF-α、IL-1β、IL-6的mRNA相对表达量均显著下调(P<0.05),且LC3-Ⅱ/LC3-Ⅰ比值与Beclin1蛋白相对表达量显著下调(P<0.05);此外,与CGS21680+LPS组比较,CGS21680+LPS+Rapa组Caco-2细胞活力又显著降低(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平也显著升高(P<0.05)。结论使用A2AR激动剂能够减轻内毒素LPS诱导的Caco-2肠上皮细胞炎性损伤,提高细胞活力,这可能与其抑制自噬水平有关。Objective To explore the effect and mechanism of adenosine A2A receptor(A2AR)on lipopolysaccharide(LPS)-induced inflammatory injury of Caco-2 intestinal epithelial cells.Methods Caco-2 cells were divided into control group,LPS group(treated with 10μg/ml LPS for 12 h),A2AR agonist(CGS21680)group(pretreated with 50 nmol/L CGS21680 for 10 min),CGS21680+LPS group(pretreated with 50 nmol/L CGS21680 for 10 min,treated with 10μg/ml LPS for 12 h),cell viability was determined using CCK-8 assay,the secretion levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in cell supernatant of each group were determined using ELISA.mRNA expression levels of TNF-α,IL-1βand IL-6 in cells of each group were detected by real-time fluorescence quantitative PCR,the protein expression levels of microtubule associated light chain protein 3(LC3)-Ⅱ/LC3-Ⅰand autophagy associated protein(Beclin1)in cells of each group were analyzed using Western blot analysis.Caco-2 cells were then divided into control group,LPS group(pretreated with 50 nmol/L CGS21680 for 10 min),CGS21680+LPS group(pretreated with 50 nmol/L CGS21680 for 10 min,treated with 10μg/ml LPS for 12 h),CGS21680+LPS+Rapa group(pretreated with 50 nmol/L CGS21680 for 10 min,treated with 10μg/ml LPS and 5μmol/L Rapa for 12 h),cell viability was determined using CCK-8 assay,the secretion levels of TNF-α,IL-1βand IL-6 in cell supernatant of each group were determined using ELISA.Results Compared with the control group,the viability of Caco-2 cells in LPS group significantly decreased(P<0.05),the levels of TNF-α,IL-1βand IL-6 in supernatant significantly increased(P<0.05),the mRNA relative expressions of TNF-α,IL-1β,IL-6 in cells significantly increased(P<0.05),the LC3-Ⅱ/LC3-Ⅰratio and the relative expression of Beclin1 protein were significantly up-regulated(P<0.05).Compared with LPS group,the viability of Caco-2 cells in CGS21680+LPS group significantly increased(P<0.05),the levels of TNF-α,IL-1βand IL-6 in supernatant significantl

关 键 词：腺苷A2A受体 Caco-2肠上皮细胞 炎症性肠病 脂多糖 细胞损伤 自噬 

分 类 号：R574[医药卫生—消化系统]