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

doi:10.3877/cma.j.issn.2096-1537.2021.03.007

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。

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

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.

Key words: Acute respiratory distress syndrome, Inflammatory injury of pulmonary microvascular endothelial cells, Cyclic guanine adenine synthase, Stimulator of interferon genes, NOD-like receptor protein 3

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）

信号物质	LPS剂量（ng/ml）	mRNA	蛋白水平
cGAS	50	1.18±0.16	1.08±0.09
	100	1.69±0.07^b	1.44±0.05^b
	1000	1.10±0.09	1.34±0.07^b
STING	50	1.24±0.14	1.11±0.06
	100	1.67±0.07^a	1.60±0.08^b
	1000	1.47±0.47	0.94±0.13
NLRP3	50	1.41±0.13^b	2.05±0.10^b
	100	1.30±0.12^a	1.86±0.07^b
	1000	1.23±0.21	1.26±0.11

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）

信号物质	LPS剂量（ng/ml）	mRNA	蛋白水平
cGAS	50	1.18±0.16	1.08±0.09
	100	1.69±0.07^b	1.44±0.05^b
	1000	1.10±0.09	1.34±0.07^b
STING	50	1.24±0.14	1.11±0.06
	100	1.67±0.07^a	1.60±0.08^b
	1000	1.47±0.47	0.94±0.13
NLRP3	50	1.41±0.13^b	2.05±0.10^b
	100	1.30±0.12^a	1.86±0.07^b
	1000	1.23±0.21	1.26±0.11

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）

信号物质	蛋白水平
信号物质	抑制cGAS	抑制STING	抑制NLRP3
cGAS	—	0.86±0.05	1.00±0.24
STING	0.37±0.06^b	—	0.98±0.25
NLRP3	0.61±0.03^b	0.63±0.05^b	—
IL-1β	0.44±0.06^b	0.39±0.09^b	0.57±0.17^b
IL-18	0.46±0.02^b	0.67±0.12^a	0.54±0.14^b

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）

信号物质	蛋白水平
信号物质	抑制cGAS	抑制STING	抑制NLRP3
cGAS	—	0.86±0.05	1.00±0.24
STING	0.37±0.06^b	—	0.98±0.25
NLRP3	0.61±0.03^b	0.63±0.05^b	—
IL-1β	0.44±0.06^b	0.39±0.09^b	0.57±0.17^b
IL-18	0.46±0.02^b	0.67±0.12^a	0.54±0.14^b

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

1	Fan E, Brodie D, Slutsky AS. Acute respiratory distress syndrome: advances in diagnosis and treatment [J]. JAMA, 2018, 319(7): 698-710.
2	Kumar P, Shen Q, Pivetti CD, et al. Molecular mechanisms of endothelial hyperpermeability: implications in inflammation [J]. Expert Rev Mol Med, 2009, 11: e19.
3	Wang J, Dai M, Cui Y, et al. Association of Abnormal Elevations in IFIT3 with overactive cyclic GMP-AMP synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes [J]. Arthritis Rheumatol, 2018, 70(12): 2036-2045.
4	Anghelina D, Lam E, Falck-Pedersen E. Diminished innate antiviral response to adenovirus vectors in cGAS/STING-deficient mice minimally impacts adaptive immunity [J]. J Virol, 2016, 90(13): 5915-5927.
5	An X, Zhu Y, Zheng T,et al. An analysis of the expression and association with immune cell infiltration of the cGAS/STING pathway in Pan-Cancer [J]. Mol Ther Nucleic Acids, 2019, 14: 80-89.
6	Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling [J]. Nature, 2008, 455(7213): 674-678.
7	Ozaki E, Campbell M, Doyle SL. Targeting the NLRP3 inflammasome in chronic inflammatory diseases: current perspectives [J]. J Inflamm Res, 2015, 8: 15-27.
8	Bauernfeind FG, Horvath G, Stutz A, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression [J]. J Immunol, 2009, 183(2): 787-791.
9	Tang T, Lang X, Xu C, et al. CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation [J]. Nat Commun, 2017, 8(1): 202.
10	Grailer JJ, Canning BA, Kalbitz M, et al. Critical role for the NLRP3 inflammasome during acute lung injury [J]. J Immunol, 2014, 192(12): 5974-5983.
11	郭晓夏, 安友仲. A2b腺苷受体活化降低脂多糖诱导的肺微血管内皮通透性 [J]. 中华危重病急救医学, 2018, 30(6): 588-593.
12	王慧霞, 郭晓夏, 赵慧颖, 等. 腺苷A2B受体活化对TNF-α致人肺微血管内皮细胞炎性损伤的影响 [J]. 中国中西医结合急救杂志, 2018, 25(4): 337-341.
13	Sharma S, tenOever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway [J]. Science, 2003, 300(5622): 1148-1151.
14	Tanaka Y, Chen ZJ. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway [J]. Sci Signal, 2012, 5(214): ra20.
15	Fitzgerald KA, McWhirter SM, Faia KL, et al. IKK and TBK1 are essential components of the IRF3 signaling pathway [J]. Nat Immunol, 2003, 4(5): 491-496.
16	Yuan L, Mao Y, Luo W, et al. Palmitic acid dysregulates the Hippo-YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS-STING-IRF3 signaling mechanism [J]. J Biol Chem, 2017, 292(36): 15002-15015.
17	Li W, Li W, Zang L, et al. Fraxin ameliorates lipopolysaccharide-induced acute lung injury in mice by inhibiting the NF-κB and NLRP3 signalling pathways [J]. Int Immunopharmacol, 2019, 67: 1-12.
18	Kolliputi N, Shaik RS, Waxman AB. The inflammasome mediates hyperoxia-induced alveolar cell permeability [J]. J Immunol, 2010, 184(10): 5819-5826.
19	Yan Y, Lu K, Ye T, et al. MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB [J]. Int J Mol Med, 2019, 43(3): 1467-1477.
20	Li D, Ren W, Jiang Z, Zhu L. Regulation of the NLRP3 inflammasome and macrophage pyroptosis by the p38 MAPK signaling pathway in a mouse model of acute lung injury [J]. Mol Med Rep, 2018, 18(5): 4399-4409.
21	Zhang H, Chen S, Zeng M, et al. Apelin-13 administration protects against LPS-induced acute lung injury by inhibiting NF-κB pathway and NLRP3 inflammasome activation [J]. Cell Physiol Biochem, 2018, 49(5): 1918-1932.

[1]	江雅婷, 刘林峰, 沈辰曦, 陈奔, 刘婷, 龚裕强. 组织相关巨噬素3 保护肺血管内皮糖萼治疗急性呼吸窘迫综合征的机制研究[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(05): 353-362.
[2]	李振翮, 魏长青, 甄国栋, 李振富. 脓毒症并发急性呼吸窘迫综合征患者血清S1P、Wnt5a变化及其临床意义[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 293-300.
[3]	杨茂宪, 沈鹏, 王倩倩, 吴旺, 沈永帅, 蒋禛, 徐龙生, 朱建刚, 刘倍倍. 吡啶甲酸镁联合地塞米松对急性呼吸窘迫综合征大鼠的治疗作用研究[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(03): 196-203.
[4]	李智, 冯芸. NF-κB 与MAPK 信号通路及其潜在治疗靶点在急性呼吸窘迫综合征中的研究进展[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 840-843.
[5]	魏丁, 乔艳艳, 顾兴, 张燕, 李艳燕, 钱卫生, 潘蕾, 高永恒, 金发光. 体外膜肺氧合救治急性呼吸窘迫综合征不良预后危险因素分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(03): 363-367.
[6]	杨东星, 沈鹏, 赵慧颖. 免疫球蛋白联合依库珠单抗治疗GBS 并发重度ARDS 患者一例[J/OL]. 中华重症医学电子杂志, 2024, 10(04): 404-408.
[7]	王晓霞, 乌丹, 张江英, 乌雅罕, 郝颖楠, 斯日古楞. 《2023 年美国胸科学会关于成人急性呼吸窘迫综合征患者管理的临床实践指南更新》解读[J/OL]. 中华重症医学电子杂志, 2024, 10(04): 338-343.
[8]	田学, 谢晖, 王瑞兰. 急性呼吸窘迫综合征相关肺纤维化的研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(03): 258-264.
[9]	李松栗, 黄蔚, 巢杰, 杨毅, 邱海波. 单核/巨噬细胞来源的细胞外囊泡在急性呼吸窘迫综合征中的研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(03): 253-257.
[10]	杨永红, 杨莹, 齐红蕾, 刘福瑞, 朱金源. 单细胞测序在急性呼吸窘迫综合征中的应用进展[J/OL]. 中华重症医学电子杂志, 2024, 10(03): 248-252.
[11]	倪韫晖, 杨毅, 袁雪燕, 邱海波. 胸壁加压在急性呼吸窘迫综合征中的应用和临床进展[J/OL]. 中华重症医学电子杂志, 2024, 10(03): 243-247.
[12]	孙藏岚, 黄丽丽, 李小雨, 邱海波. 脂质组学在急性呼吸窘迫综合征中的应用和临床进展[J/OL]. 中华重症医学电子杂志, 2024, 10(02): 127-135.
[13]	史楠, 袁雪燕, 邱海波. 肺复张在急性呼吸窘迫综合征中的应用和临床进展[J/OL]. 中华重症医学电子杂志, 2024, 10(02): 118-126.
[14]	王永广, 朱鹏, 许千金, 甘桂芬, 石钟山, 潘纯. 急性呼吸窘迫综合征诊断标准亟需更新[J/OL]. 中华重症医学电子杂志, 2024, 10(02): 113-117.
[15]	张梅, 陈卉, 李转霞, 王瑞, 李林娟. Metrnl和NLRP3炎症小体：糖尿病肾病的潜在诊断标志物[J/OL]. 中华肥胖与代谢病电子杂志, 2024, 10(03): 193-199.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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