cGAS/STING通过NLRP3炎性小体调控人肺微血管内皮细胞炎症的作用机制  

cGAS/STING regulates inflammation of human pulmonary microvascular endothelial cells via NLRP3 inflammasome

作　　者：姜硕 王梦楠 赵慧颖[1] 郭晓夏[1] 王慧霞 安友仲[1] Jiang Shuo;Wang Mengnan;Zhao Huiying;Guo Xiaoxia;Wang Huixia;An Youzhong(Department of Critical Care Medicine,Peking University People's Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院重症医学科,北京100044

出　　处：《中华重症医学电子杂志》2021年第3期233-240,共8页Chinese Journal Of Critical Care & Intensive Care Medicine(Electronic Edition)

摘　　要：目的探讨鸟嘌呤核苷酸腺嘌呤核苷酸合成酶(cGAS)/干扰素激活蛋白(STING)通过NOD样受体蛋白3(NLRP3)炎性小体调控人肺微血管内皮细胞(HPMVECs)炎症的作用机制。方法原代培养HPMVECs,进行脂多糖(LPS)量效实验及cGAS、STING和NLRP3抑制干预实验。(1)量效实验:以50、100、1000 ng/ml的LPS作用24 h后(分别为50 ng/mlLPS组、100 ng/mlLPS组、1000 ng/ml LPS组),用实时荧光反转录-聚合酶链反应(qRT-PCR)和蛋白质免疫印迹试验(Western Blot)分别检测cGAS、STING和NLRP3表达。(2)cGAS抑制干预实验:使用cGAS(siRNA)转染,再加入100 ng/ml的LPS处理24 h;同时设立空白对照组、LPS刺激组、siRNA单独处理组,采用WesternBlot检测STING、NLRP3,及NLRP3下游因子白介素(IL)-1β和IL-18的表达。(3)STING抑制干预实验:使用STING(siRNA)转染,再加入100 ng/ml的LPS处理24 h;同时设立空白对照组,采用WesternBlot检测cGAS、NLRP3,及NLRP3下游因子IL-1β和IL-18的蛋白表达。(4)NLRP3抑制干预实验:使用NLRP3炎性小体抑制剂MCC950预处理30 min,再加入100 ng/ml的LPS处理24 h;同时设立空白对照组,采用WesternBlot检测cGAS、STING,及NLRP3下游因子IL-1β和IL-18的蛋白表达。结果(1)量效实验:与空白对照组比较,HPMVECs中cGAS、STING、NLRP3的mRNA水平和蛋白表达均显著升高,差异有统计学意义(P<0.05)。(2)cGAS抑制干预实验:与空白对照组比较,LPS刺激HPMVECs,cGAS表达水平显著升高,差异有统计学意义(P<0.05);与LPS刺激组比较,抑制cGAS时,siRNA+LPS处理组STING、NLRP3及其下游因子IL-1β、IL-18的表达水平均显著降低,差异有统计学意义(P<0.05)。(3)STING抑制干预实验:与空白对照组比较,LPS刺激HPMVECs,STING表达水平显著升高,差异有统计学意义(P<0.05);与LPS刺激组比较,抑制STING时,NLRP3及其下游因子IL-1β、IL-18的表达水平均显著降低,差异有统计学意义(P<0.05),而cGAS的表达水平无显著降低(P>0.05)。(4)NLRP3抑制干预实验:与空�Objective To explore the role and mechanism of cyclic guanine adenine synthase(cGAS)/stimulator of interferon genes(STING)in regulating the inflammation of human pulmonary microvascular endothelial cells(HPMVECs)through NOD-like receptor protein 3(NLRP3)inflammasome.Methods Primary culture of HPMVECs,lipopolysaccharide(LPS)dose-effect experiment and cGAS,STING and NLRP3 inhibition intervention experiments.(1)Dose-effect experiment:After 24 hours of LPS exposure at 50,100,1000 ng/ml(50,100,1000 ng/ml group,respectively),real-time fluorescent reverse transcription-polymerase chain reaction(qRT-PCR)and western blotting(Western Blot)were used to detect cGAS,STING and NLRP3 expression.(2)cGAS inhibition intervention experiment:transfect with cGAS small interfering RNA(siRNA),and then add 100ng/ml LPS for 24 h;at the same time set up a blank control group,LPS stimulation group,and siRNA single treatment group,and Western Blot was used to detect STING,NLRP3,and The expression of NLRP3 downstream factors interleukin-1β(IL-1β)and interleukin-18(IL-18).(3)STING inhibitory intervention experiment:Transfect with STING small interfering RNA(siRNA),and then add 100ng/ml LPS for 24 h;Set up multiple control groups at the same time to detect the protein expression of other factors.(4)NLRP3 inhibition intervention experiment:pretreated with NLRP3 inflammatory body inhibitor MCC950 for 30 minutes,and then added 100ng/ml LPS for 24 h;Set up multiple control groups at the same time to detect the protein expression of other factors.Results(1)Dose-effect experiment:the mRNA level and protein expression of cGAS,STING and NLRP3 in HPMVECs were significantly increased.(2)cGAS inhibition intervention experiment:LPS stimulated HPMVECs,and the expression level of cGAS increased significantly.Compared with the LPS group,when cGAS was inhibited,the expression levels of STING,NLRP3 and its downstream factors IL-1βand IL-18 were significantly reduced in siRNA+LPS stimulation group.(3)STING inhibition intervention experiment:LPS stimulated HP

关 键 词：急性呼吸窘迫综合征 人肺微血管内皮细胞炎性损伤 鸟嘌呤核苷酸腺嘌呤核苷酸合成酶 干扰素激活蛋白 NOD样受体蛋白3 

分 类 号：R563.8[医药卫生—呼吸系统]