Effect of chloroquine phosphate on vascular endothelial cell injury induced by double-stranded RNA

doi:10.3969/j.issn.1674-8115.2022.10.005

Abstract

Abstract:

Objective ·To investigate the inhibitory effect of chloroquine phosphate (CQ) on vascular endothelial cell injury induced by double-stranded RNA (dsRNA) and its mechanism. Methods ·An in vitro model of vascular endothelial cell injury induced by dsRNA in viral sepsis was established via stimulating human umbilical vein endothelial cells (HUVECs) with polyinosinic-polycytidylic acid (poly I:C). HUVECs were randomly divided into normal control group (NC group), poly I:C group with different concentrations and CQ+poly I:C group. The cell viability of HUVECs before and after poly I:C treatment was detected by using Cell Counting Kit-8 (CCK-8). The morphological changes of HUVECs were observed by using optical microscope. The integrity of cell membrane was assessed by calculating the rate of propidium iodide (PI) positive cells by using flow cytometry. The expression level of mature proinflammatory cytokine interleukin 1β (IL-1β), and key proteins of pyroptosis were detected by Western blotting. Results ·Compared with the NC group, HUVECs treated with poly I:C showed a decreased cell viability, more expanded "Vesicles" and more ruptured cells. An increased level of IL-1β expression and positive rate of PI⁺ cells were also observed. The expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), and cleaved caspase-1 and gasdermin D N-terminal (GSDMD-N) were increased significantly (all P<0.05) under the treatment of poly I:C and the changes were concentration-dependent. Interestingly, compared with poly I:C group, the cell viability of HUVEC pretreated by CQ was significantly increased, and a decreased IL-1β level were observed. The phenomena of cell swelling and rupture was improved, and a lower positive rate of PI⁺ cells was detected. The expression levels of cleaved caspase-1 and GSDMD-N of cell pyrosis were also decreased (all P<0.05). CQ pretreatment did not reduce NLRP3 expression induced by dsRNA, but it did inhibit the expression of Toll-like receptor 3 (TLR3). Conclusion ·Pyroptosis is involved in vascular endothelial cells injury induced by dsRNA, and CQ inhibits dsRNA-induced pyroptosis, which could be related to TLR3 regulation.

Key words: chloroquine phosphate (CQ), pyroptosis, double-stranded RNA (dsRNA), vascular endothelial cell

CLC Number:

ZHANG Jiaojiao, SUN Junnan, WANG Hairong. Effect of chloroquine phosphate on vascular endothelial cell injury induced by double-stranded RNA[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(10): 1404-1412.

Figures/Tables 5

Fig 1 Pyroptosis induced by dsRNA in HUVECs

Fig 2 Expression of pyroptosis-associated proteins in HUVECs in response to dsRNA

Fig 3 Inhibition effect of CQ on pyroptosis induced by dsRNA

Fig 4 Expression of key proteins of cell pyrosis with different treatment

Fig 5 Potential mechanisms of CQ inhibition of dsRNA-induced pyroptosis in HUVECs

TrendMD

References 24

1	ACKERMANN M, VERLEDEN S E, KUEHNEL M, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19[J]. N Engl J Med, 2020, 383(2): 120-128.
2	VARGA Z, FLAMMER A J, STEIGER P, et al. Endothelial cell infection and endotheliitis in COVID-19[J]. Lancet, 2020, 395(10234): 1417-1418.
3	JANEWAY C A Jr, MEDZHITOV R. Innate immune recognition[J]. Annu Rev Immunol, 2002, 20: 197-216.
4	WANG T, TOWN T, ALEXOPOULOU L, et al. Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis[J]. Nat Med, 2004, 10(12): 1366-1373.
5	GITLIN L, BARCHET W, GILFILLAN S, et al. Essential role of mda-5 in type Ⅰ IFN responses to polyriboinosinic: polyribocytidylic acid and encephalomyocarditis picornavirus[J]. Proc Natl Acad Sci USA, 2006, 103(22): 8459-8464.
6	TRAYNOR T R, MAJDE J A, BOHNET S G, et al. Intratracheal double-stranded RNA plus interferon-gamma: a model for analysis of the acute phase response to respiratory viral infections[J]. Life Sci, 2004, 74(20): 2563-2576.
7	YU P, ZHANG X, LIU N, et al. Pyroptosis: mechanisms and diseases[J]. Signal Transduct Target Ther, 2021, 6(1): 128.
8	KOVACS S B, MIAO E A. Gasdermins: effectors of pyroptosis[J]. Trends Cell Biol, 2017, 27(9): 673-684.
9	ZHAO N, DI B, XU L L. The NLRP3 inflammasome and COVID-19: activation, pathogenesis and therapeutic strategies[J]. Cytokine Growth Factor Rev, 2021, 61: 2-15.
10	BAI B C, YANG Y Y, WANG Q, et al. NLRP3 inflammasome in endothelial dysfunction[J]. Cell Death Dis, 2020, 11(9): 776.
11	BAUERNFRIED S, SCHERR M J, PICHLMAIR A, et al. Human NLRP1 is a sensor for double-stranded RNA[J]. Science, 2021, 371(6528): eabd0811.
12	SCHREZENMEIER E, DÖRNER T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology[J]. Nat Rev Rheumatol, 2020, 16(3): 155-166.
13	HUANG L Y, STUART C, TAKEDA K, et al. Poly(I: c) induces human lung endothelial barrier dysfunction by disrupting tight junction expression of claudin-5[J]. PLoS One, 2016, 11(8): e0160875.
14	LUNDBERG A M, DREXLER S K, MONACO C, et al. Key differences in TLR3/poly I: c signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems[J]. Blood, 2007, 110(9): 3245-3252.
15	朱莹莹, 张姣姣, 孙俊楠, 等. 氯喹对脂多糖诱导的Ⅱ型肺泡细胞损伤的影响[J]. 内科理论与实践, 2021, 16(3): 197-201.
	ZHU Y Y, ZHANG J J, SUN J N, et al. Effect of chloroquine on type Ⅱ alveolar cell injury caused by lipopolysaccharide[J]. J Int Med Concepts Pract, 2021,16: 197-201.
16	王宁, 朱莹莹, 张姣姣, 等. 氯喹对脂多糖诱导的人脐静脉内皮细胞损伤的影响[J]. 内科理论与实践, 2020, 15(2): 111-115.
	WANG N, ZHU Y Y, ZHANG J J, et al. The dose dependent bidirectional effect of chloroquine on lipopolysaccharide-induced injury to human umbilical vein endothelial cells[J]. J Int Med Concepts Pract, 2020, 15(2): 111-115.
17	国家卫生健康委员会, 国家中医药管理局. 新型冠状病毒肺炎诊疗方案(试行第八版)[J]. 中国病毒病杂志, 2020, 10(5): 321-328.
	National Health Commission, National Administration of Traditional Chinese Medicine. Guidelines for the diagnosis and treatment of coronavirus disease 2019 (trial version eighth)[J]. Chin J Viral Dis, 2020,10(5): 321-328.
18	WU L Q, DAI J P, ZHAO X F, et al. Chloroquine enhances replication of influenza A virus A/WSN/33 (H1N1) in dose-, time-, and MOI-dependent manners in human lung epithelial cells A549[J]. J Med Virol, 2015, 87(7): 1096-1103.
19	SATO R, IMAIZUMI T, AIZAWA T, et al. Inhibitory effect of anti-malarial agents on the expression of proinflammatory chemokines via Toll-like receptor 3 signaling in human glomerular endothelial cells[J]. Ren Fail, 2021, 43(1): 643-650.
20	CARTER W A, DE CLERCQ E. Viral infection and host defense[J]. Science, 1974, 186(4170): 1172-1178.
21	MAJDE J A. Viral double-stranded RNA, cytokines, and the flu[J]. J Interferon Cytokine Res, 2000, 20(3): 259-272.
22	SHIBAMIYA A, HERSEMEYER K, SCHMIDT WÖLL T, et al. A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells[J]. Blood, 2009, 113(3): 714-722.
23	MIAN M F, AHMED A N, RAD M, et al. Length of dsRNA (poly I: C) drives distinct innate immune responses, depending on the cell type[J]. J Leukoc Biol, 2013, 94(5): 1025-1036.
24	HÄCKER H, MISCHAK H, MIETHKE T, et al. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation[J]. EMBO J, 1998, 17(21): 6230-6240.

[1]	Chi-hsiang CHUANG, Jia-chen DONG, Rong SHU. Biological and angiogenic effects of enamel matrix derivative on periodontal regeneration-related cells [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1099-1102.
[2]	PENG Hai-yan, LI Hua-ping . Regulation of miR-137 overexpressed trophoblast cells on biological functions of vascular endothelial cells#br# [J]. , 2017, 37(10): 1350-.
[3]	ZHANG Yi-wei, TIAN Kun, WANG Yan, et al. Regulation of aquaporin 1 expression in rat pulmonary microvascular endothelial cells by free fatty acid mixture and action mechanism [J]. , 2016, 36(3): 322-.
[4]	JIANG Zhen, CAO Yong-mei, ZENG Zhen, et al. Regulation effect of hypoxia response element-mediated Bcl-2-shRNA on hypoxia-induced anti-apoptotic pulmonary vascular endothelial cells [J]. , 2015, 35(5): 674-.
[5]	LI Jing, SHANG Jia-wei, LIU Xi, et al. Effects of free fatty acid mixture on toll-like receptor-4 of pulmonary microvascular endothelial cells of rats and mechanism of FFA-induced inflammation response [J]. , 2014, 34(5): 590-.
[6]	LI Ya-chun, LI Ying-chuan, ZHOU Ming, et al. Effects of lipolysaccharide on expression of ACE and ACE2 in rat pulmonary microvascular endothelial cells and intervention effects of angiotensin-converting enzyme inhibitor [J]. , 2012, 32(4): 452-.
[7]	ZHANG Xia, JIN Hui-min. Research progress of vascular endothelial function in chronic kidney disease [J]. , 2012, 32(4): 528-.
[8]	LI Cong, HAO Xu, ZHOU Qiao, et al. In vitro injury effects of sera of patients with uremia on vascular endothelial cells [J]. , 2011, 31(7): 900-.
[9]	GUO Ying, LI Yan-qin, LIU Bin, et al. Relationship between vascular endothelial function and chronic obstructive pulmonary disease [J]. , 2010, 30(9): 1152-.
[10]	WANG Li-hua, GU Le-yi, LIANG Xin-yue, et al. Effects of Rapamycin on nephropathy and expression of VEGF and VEGF receptors in PAN nephritic mice [J]. , 2010, 30(4): 375-.
[11]	LI Ying-chuan, JIANG Wei, ZHOU Ming, et al. Improvement of method for primary cultivation and identification of rat pulmonary microvascular endothelial cells [J]. , 2010, 30(11): 1440-.
[12]	XIAO Li-fang, GU Le-yi, LIANG Xin-yue, et al. Effects of rapamycin on nephropathy of diabetic rats and expressions of vascular endothelial growth factor and its receptor [J]. , 2009, 29(9): 1040-.