白藜芦醇激活沉默信息调节子1抑制NLRP3炎症小体对脓毒症肠道黏膜屏障功能损伤的研究  

Effects of resveratrol-mediated inhibition of NOD-like receptor protein 3 inflammasomevia activating silent information regulator 1 on the injury of intestinal mucosal barrier function after sepsis

作　　者：古丽菲热·塔依尔 杨春波[1] 李祥 王毅[1] 于湘友[1] Gulifeire Tayier;Yang Chunbo;Li Xiang;Wang Yi;Yu Xiangyou(Department of Critical Care Medicine,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]新疆医科大学第一附属医院重症医学科,乌鲁木齐830054

出　　处：《中华危重病急救医学》2021年第5期535-540,共6页Chinese Critical Care Medicine

基　　金：新疆维吾尔自治区高校科研计划项目(XJEDU2018I011)

摘　　要：目的探讨白藜芦醇(RSV)能否通过激活沉默信息调节子1(SIRT1)调控NOD样受体蛋白3(NLRP3)炎症小体活化,进而改善脓毒症肠道炎症反应及细胞凋亡,从而发挥肠道屏障功能保护作用。方法①体外实验:培养人结直肠腺癌细胞(Caco-2),并分为正常组(完全培养基培养48 h),脂多糖(LPS)组(完全培养基培养24 h后加入2 mg/L的LPS处理12 h),RSV低、中、高浓度组及SIRT1抑制剂(EX-527)组(完全培养基培养24 h后加入2 mg/L的LPS处理6 h,随后再分别加入RSV 10、20、40μmol/L或EX-52710μmol/L处理6 h)。采用酶联免疫吸附试验(ELISA)检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-18、IL-1β)等炎性因子水平;用流式细胞仪检测细胞凋亡水平;用蛋白质免疫印迹试验(Western blotting)检测NLRP3、SIRT1、天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)及凋亡相关点样蛋白(ASC)的蛋白表达。②体内实验:将24只雄性Wistar大鼠按随机数字表法分为假手术(Sham)组、盲肠结扎穿孔术(CLP)6 h组、CLP 24 h组及RSV干预组(CLP术后6 h、12 h腹腔注射RSV 20 mg/kg),每组6只。采用免疫组化法检测大鼠肠道中NLRP3、caspase-1及ASC表达情况。结果①与正常组比较,经LPS处理后细胞上清液中炎性因子水平升高,SIRT1蛋白表达降低,NLRP3、caspase-1及ASC蛋白表达升高。与LPS组相比,不同浓度RSV可降低炎性因子水平,增加SIRT1活性,抑制NLRP3炎症小体下游产物caspase-1、ASC的表达,其中40μmol/L高浓度RSV作用最为明显〔TNF-α(ng/L):8.77±0.43比12.66±0.81,IL-6(ng/L):1.35±0.20比1.93±0.09,IL-1β(ng/L):1.05±0.04比1.31±0.07,IL-18(ng/L):519.50±11.16比622.70±30.69,SIRT1/β-actin:0.80±0.05比0.58±0.02,caspase-1/β-actin:0.55±0.06比0.78±0.06,ASC/β-actin:0.78±0.08比1.04±0.15,均P<0.05〕,而SIRT1抑制剂EX-527的作用则相反。正常组、LPS组及RSV低、中、高浓度组和EX-527组间细胞凋亡率差异并无统计学意义〔(7.03±0.57)%、Objective To explore whether resveratrol(RSV)could activate silent information regulator 1(SIRT1)to regulate the activation of NOD-like receptor protein 3(NLRP3)inflammasome in sepsis induced intestinal injury model,and then reduce intestinal inflammation and cell apoptosis,so as to play a protective role in intestinal barrier function.Methods①In vitro experiment:human Colorectal adenocarcinoma cells(Caco-2)were cultured,which were divided into normal group(normal culture on complete medium for 48 hours),lipopolysaccharide(LPS)group(normal culture on complete medium for 24 hours,then LPS containing 2 mg/L complete medium intervention for 6 hours),RSV low,medium and high concentration groups and SIRT1 inhibitor(EX-527)group(complete medium normal culture for 24 hours,LPS containing 2 mg/L complete medium intervention for 6 hours,followed by RSV 10,20,40μmol/L or EX-52710μmol/L intervention for 6 hours,respectively).The levels of tumor necrosis factor-α(TNF-α)and interleukins(IL-6,IL-18,IL-1β)in the cell supernatant were determined by enzyme linked immunosorbent assay(ELISA).The apoptosis level of the cells was detected by flow cytometry.Western blotting was used to detect the protein levels of NLRP3,SIRT1,caspase-1 and apoptosis-associated speck-like protein containing a CARD(ASC).②In vivo experiment:according to random number table method,24 male Wistar rats were divided into sham operation group(Sham group),cecal ligation and perforation(CLP)6 hours group(CLP 6 h group),CLP 24 h group and RSV intervention group[RSV(20 mg/kg)was intraperitoneally injected 6 hours and 12 hours after CLP],with 6 rats in each group.The levels of NLRP3,caspase-1 and ASC in the intestine of rats were detected by immunohistochemistry.Results①Compared with the normal group,the levels of inflammatory factors in the cell supernatant of the LPS group were increased and the expression of SIRT1 protein was decreased,while the protein expressions of NLRP3,caspase-1 and ASC were increased.Compared with LPS group,different concentrat

关 键 词：脓毒症 肠道 NLRP3炎症小体 沉默信息调节子1 白藜芦醇 

分 类 号：R285.5[医药卫生—中药学]