脓毒症肠道损伤模型中NLRP3炎症小体活化介导炎症反应及细胞凋亡  

Activation of NOD-like receptor protein 3 inflammasome mediates inflammatory response and apoptosis in septic intestinal injury model

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7期855-860,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：目的探讨不同严重程度脓毒症肠道损伤模型中NOD样受体蛋白3(NLRP3)炎症小体表达及其介导的炎症反应和凋亡。方法体外培养人结直肠腺癌细胞株(Caco-2),取对数生长期细胞分为空白对照组(用完全培养基正常培养)及脂多糖(LPS)1、2、4 mg/L组(用含1、2、4 mg/L LPS的完全培养基培养)。分别于6、12、24 h收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-1β、IL-18)水平;采用流式细胞仪检测细胞凋亡水平。收集细胞,采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测NLRP3、沉默信息调节因子1(SIRT1)的mRNA表达;采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)以及凋亡相关点样蛋白(ASC)的蛋白表达。结果ELISA结果显示,在同一干预时间点,与空白对照组相比,LPS各组细胞上清液中IL-6、TNF-α、IL-1β及IL-18水平均呈剂量依赖性增加,并呈一定时间依赖性,其中24 h时LPS 4 mg/L组升高最为显著〔IL-6(ng/L):3.55±0.06比0.67±0.09,TNF-α(ng/L):15.37±0.19比5.04±0.14,IL-1β(ng/L):2.26±0.10比0.56±0.09,IL-18(ng/L):433.92±22.55比93.55±21.13,均P<0.05〕。细胞凋亡检测结果显示,与空白对照组相比,LPS各组细胞凋亡率均有增加趋势,并呈一定剂量依赖性和时间依赖性,以24 h时LPS 4 mg/L组细胞凋亡率升高最为显著〔(14.83±3.73)%比(5.87±1.17)%,P<0.05〕。RT-qPCR结果显示,随LPS剂量增加和干预时间延长,细胞NLRP3 mRNA表达逐渐升高,而SIRT1 mRNA表达则呈下降趋势,24 h时LPS 4 mg/L组与空白对照组比较差异均有统计学意义〔NLRP3 mRNA(2-ΔΔCt):8.20±2.82比1.00±0.36,SIRT1 mRNA(2-ΔΔCt):0.58±0.01比1.03±0.06,均P<0.05〕。Western blotting显示,与空白对照组相比,LPS各组NLRP3、caspase-1、ASC的蛋白表达均明显升高,SIRT1蛋白表达明显降低;在各干预时间点,随LPS剂量增加,NLObjective To investigate the expression of NOD-like receptor protein 3(NLRP3)inflammasome in intestinal injury models with different severity of sepsis and the inflammatory response and apoptosis mediated by NLRP3 inflammasome.Methods Human colorectal adenocarcinoma cells(Caco-2)were cultured in vitro.The logarithmic growth phase cells were divided into blank control group(normal culture in complete medium)and lipopolysaccharide(LPS)1,2 and 4 mg/L groups(complete medium containing 1,2 and 4 mg/L LPS,respectively).The supernatant were collected at 6,12 and 24 hours,and the levels of tumor necrosis factor-α(TNF-α),interleukins(IL-6,IL-1β,IL-18)were detected by enzyme linked immunosorbent assay(ELISA).The apoptotic level of cells was detected by flow cytometry.The cells were harvested,and the real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect the mRNA expressions of NLRP3 and silent information regulator 1(SIRT1).Western blotting was used to detect the protein expressions of NLRP3,SIRT1,caspase-1 and apoptosis-associated speck-like protein(ASC).Results ELISA results showed that the levels of IL-6,TNF-α,IL-1β,and IL-18 in cell supernatant of LPS groups increased in a dose-dependent and time-dependent manner as compared with the blank control group during the same intervention period.The increase was most significant in LPS 4 mg/L group at 24 hours[IL-6(ng/L):3.55±0.06 vs.0.67±0.09,TNF-α(ng/L):15.37±0.19 vs.5.04±0.14,IL-1β(ng/L):2.26±0.10 vs.0.56±0.09,IL-18(ng/L):433.92±22.55 vs.93.55±21.13,all P<0.05].The results of the apoptotic test showed that,compared with the blank control group,the apoptotic rate of LPS groups increased in a dose-dependent and time-dependent manner,and the apoptotic rate of LPS 4 mg/L group increased most significantly at 24 hours[(14.83±3.73)%vs.(5.87±1.17)%,P<0.05].RT-qPCR results showed that the expression level of NLRP3 mRNA was increased,while the expression level of SIRT1 mRNA was decreased with the increase of L

关 键 词：脓毒症 肠道 NOD样受体蛋白3炎症小体 炎症反应 凋亡 

分 类 号：R459.7[医药卫生—急诊医学]