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中国科技核心期刊

中国科学引文数据库(CSCD)来源期刊

中华重症医学电子杂志 ›› 2021, Vol. 07 ›› Issue (03): 233 -240. doi: 10.3877/cma.j.issn.2096-1537.2021.03.007

基础研究 上一篇    下一篇

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
姜硕 1, 王梦楠 1, 赵慧颖 1, 郭晓夏 1, 王慧霞 1, 安友仲 1 , ( )   
  1. 1. 100044,北京,北京大学人民医院重症医学科
  • 收稿日期:2021-05-27 出版日期:2021-08-28
  • 通信作者: 安友仲

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

Shuo Jiang 1, Mengnan Wang 1, Huiying Zhao 1, Xiaoxia Guo 1, Huixia Wang 1, Youzhong An 1 , ( )   

  1. 1. Department of Critical Care Medicine, Peking University People's Hospital, Beijing 100044, China
  • Received:2021-05-27 Published:2021-08-28
  • Corresponding author: Youzhong An
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/ml LPS组、100 ng/ml LPS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Western Blot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Western Blot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Western Blot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:与空白对照组比较,LPS刺激HPMVECs,siRNA+LPS处理组NLRP3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NLRP3显著降低了NLRP3下游因子白细胞IL-1β和IL-18的表达,差异有统计学意义(P<0.05),而cGAS和STING的表达水平无显著降低;差异无统计学意义(P>0.05)。

结论

(1)LPS刺激下,在HPMVECs中cGAS、STING、NLRP3、IL-1β和IL-18的表达水平均显著升高,参与调控炎症反应;(2)cGAS/STING/NLRP3信号通路顺序参与调控PMVECs炎症作用。

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-1β and IL-18 were significantly reduced in siRNA+LPS stimulation group. (3) STING inhibition intervention experiment: LPS stimulated HPMVECs, and the expression level of STING increased significantly. Compared with the LPS group, when STING was inhibited, the expression levels of NLRP3 and its downstream factors IL-1β and IL-18 were significantly reduced, but the expression level of cGAS did not decrease significantly. (4) NLRP3 inhibition intervention experiment: LPS stimulated HPMVECs, and the expression level of NLRP3 increased significantly in siRNA+LPS stimulation group. Compared with the LPS group, inhibiting NLRP3 significantly reduced the expression of IL-1β and IL-18 in leukocytes, the downstream factors of NLRP3. The expression levels of cGAS and STING did not decrease significantly.

Conclusions

(1) Under the stimulation of LPS injury, the expression levels of cGAS, STING, NLRP3, IL-1β and IL-18 in human lung microvascular endothelial cells are significantly increased, and they participate in the regulation of inflammation. (2) The cGAS/STING/NLRP3 signaling pathway is involved in the regulation of ALI/ARDS in sequence.

图1 不同剂量LPS刺激下HPMVECs中cGAS mRNA和蛋白表达水平的变化
图2 不同剂量LPS刺激下HPMVECs中STING mRNA和蛋白表达水平的变化
图3 不同剂量LPS刺激下HPMVECs中NLRP3 mRNA和蛋白表达水平的变化
表1 不同剂量LPS刺激下HPMVECs中cGAS、STING、NLRP3 mRNA和蛋白表达水平的变化(
x ˉ
±s)
图4 抑制cGAS对HPMVECs中STING、NLRP3、IL-1β与IL-18表达的影响
表2 LPS刺激下抑制cGAS、STING、NLRP3对HPMVECs中对应因子蛋白水平表达的影响(
x ˉ
±s
图5 抑制STING对HPMVECs中cGAS、NLRP3、IL-1β与IL-18表达的影响
图6 抑制NLRP3对HPMVECs中cGAS、STING、IL-1β与IL-18表达的影响
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