TRPM2离子通道在H9N2流感病毒感染小鼠肺微血管内皮细胞线粒体损伤中的作用

doi:10.11843/j.issn.0366-6964.2019.10.011

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (10): 2053-2060.doi: 10.11843/j.issn.0366-6964.2019.10.011

TRPM2离子通道在H9N2流感病毒感染小鼠肺微血管内皮细胞线粒体损伤中的作用

李军^1,2, 高晶萍¹, 张雅琪³, 罗强², 梁亭², 李珮瑶¹, 王少华¹, 张瑞华¹, 徐明举¹, 徐彤^1,2*

1. 河北北方学院预防兽医学重点实验室, 张家口 075000;
2. 河北北方学院生命科学研究中心, 张家口 075000;
3. 河北北方学院基础医学院, 张家口 075000

收稿日期:2019-05-07 出版日期:2019-10-23 发布日期:2019-10-23
通讯作者: 徐彤,主要从事动物传染病的教学和科研工作,Tel:0313-4029358,E-mail:xutong1969@sohu.com
作者简介:李军(1983-),男,河北阳原人,助理研究员,硕士,主要从事畜禽基础药理、病理的研究,E-mail:lijun_167@163.com
基金资助:
国家自然科学基金（31672522；31602030）；河北省教育厅自然科学研究重点课题（ZD2019078）；河北省第二期农业现代化产业体系蛋肉鸡创新团队专项经费（HBCT2018150207）；河北自然科学基金（C2019405060）

The Effect of TRPM2 Ion Channel on Mitochondrial Damage of Pulmonary Microvascular Endothelial Cells in Mice Infected with H9N2 Influenza Virus

LI Jun^1,2, GAO Jingping¹, ZHANG Yaqi³, LUO Qiang², LIANG Ting², LI Peiyao¹, WANG Shaohua¹, ZHANG Ruihua¹, XU Mingju¹, XU Tong^1,2*

1. Key Laboratory of Preventive Veterinary Medicine, Department of Veterinary Medicine, Animal Science College, Hebei North University, Zhangjiakou 075000, China;
2. Life Science Research Center, Hebei North University, Zhangjiakou 075000, China;
3. Basic Medical College, Hebei North University, Zhangjiakou 075000, China

Received:2019-05-07 Online:2019-10-23 Published:2019-10-23

摘要/Abstract

摘要： 旨在探讨瞬时电位受体M2离子通道（TRPM2）在H9N2流感病毒感染小鼠肺微血管内皮细胞（PMVEC）导致线粒体损伤过程中的作用。在已建立TRPM2 shRNA PMVEC的基础上，采用5MOI H9N2猪流感病毒感染细胞，在病毒作用后24和48 h提取各组细胞的线粒体进行蛋白定量，检测各组细胞线粒体超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、一氧化氮合成酶（NOS）、线粒体呼吸链复合物Ⅳ活性和ATP酶水平；利用激光共聚焦显微镜观察线粒体膜电位变化（JC-1染色）及细胞凋亡（Annexin V-FITC/PI双染色法）情况。结果表明：H9N2-SIV感染后TRPM2 shRNA PMVEC内SOD活性、GSH水平和Na⁺-K⁺-ATP、线粒体呼吸链复合物Ⅳ活性显著高于对照shRNA PMVEC（P<0.05或P<0.01）；mtNOS活性则极显著低于shRNA PMVEC（P<0.01）。JC-1染色显示TRPM2-siRNA PMVEC线粒体膜电位水平高于对照shRNA PMVEC，但是AnnexinV-FITC/PI染色显示细胞凋亡则低于H9N2感染对照shRNA PMVEC。结果提示：TRPM2基因沉默有效减缓H9N2感染导致NOS产生，显著降低SOD、GSH、线粒体呼吸链复合物Ⅳ、Na⁺-K⁺-ATP的消耗以及线粒体膜电位的下降程度，进而有效缓解PMVEC线粒体损伤及细胞凋亡。

Abstract: This study aimed to investigate the effect of transient receptor potential melastatin 2 (TRPM2) ion channel on pulmonary microvascular endothelial cell (PMVEC) mitochondrial damage induced by H9N2 influenza virus infection. Basing on the established TRPM2 shRNA PMVEC, in this study, TRPM2 shRNA PMVEC and shRNA PMVEC (control) were infected by the dose of five multiplicity of infection (MOI) H9N2 influenza virus. At 24 and 48 h post-infection, the mitochondria of PMVECs mentioned above was extracted and the protein level was measured by BCA protein assay kit; The SOD activity, GSH level, NOS activity, Na⁺-K⁺-ATPase and mitochondrial complex Ⅳ were measured according to the manufacturer's instructions;The mitochondrial membrane potential change (using mitochondrial membrane potential assay kit with JC-1) and apoptosis (using annexin V-FITC/PI apoptosis detection kit) of PMVEC were observed by laser scanning confocal microscope. The results showed that the activity of SOD, Na⁺-K⁺-ATPase and mitochondria complex Ⅳ and the level of GSH in TRPM2-shRNA PMVEC were significantly higher than those of shRNA PMVEC (control) after H9N2 influenza virus infection (P<0.05 or P<0.01); However, the NOS activity was dramatically lowered than that of control PMVEC (P<0.01). Moreover, the observation of electron microscope showed that the damage of organelles of TRPM2-shRNA PMVEC was not as serious as that of control shRNA PMVEC; Furthermore, the mitochondrial membrane potential level also was higher than that of control shRNA PMVEC. The apoptosis of TRPM2 shRNA PMVEC detected by annexin V-FITC/PI staining showed that there were less PMVECs stained by green fluorescence in cytomembrane and red fluorescence in nucleus than that of control shRNA PMVEC. The results demonstrated that gene silencing of TRPM2 by shRNA alleviated effectively SOD, GSH, Na⁺-K⁺-ATPase, mitochondrial complex Ⅳ in PMVEC infected by H9N2 virus, and further to prevent the mitochondrial damage and apoptosis of PMVEC.

中图分类号:

李军, 高晶萍, 张雅琪, 罗强, 梁亭, 李珮瑶, 王少华, 张瑞华, 徐明举, 徐彤. TRPM2离子通道在H9N2流感病毒感染小鼠肺微血管内皮细胞线粒体损伤中的作用[J]. 畜牧兽医学报, 2019, 50(10): 2053-2060.

LI Jun, GAO Jingping, ZHANG Yaqi, LUO Qiang, LIANG Ting, LI Peiyao, WANG Shaohua, ZHANG Ruihua, XU Mingju, XU Tong. The Effect of TRPM2 Ion Channel on Mitochondrial Damage of Pulmonary Microvascular Endothelial Cells in Mice Infected with H9N2 Influenza Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2053-2060.

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参考文献 28

[1]	POOVORAWAN Y, PYUNGPORN S, PRACHAYANGPRECHA S, et al. Global alert to avian influenza virus infection:from H5N1 to H7N9[J]. Pathog Glob Health, 2013, 107(5):217-223.
[2]	GAO R B, CAO B, HU Y W, et al. Human infection with a novel avian-origin influenza A (H7N9) virus[J]. N Engl J Med, 2013, 368(20):1888-1897.
[3]	何军,刘丽萍,侯赛,等. 安徽省2株人感染H9N2流感病毒基因特征[J]. 中华流行病学杂志, 2016, 37(5):708-713.HE J, LIU L P, HOU S, et al. Genomic characteristics of 2 strains of influenza A (H9N2) virus isolated from human infection cases in Anhui province[J]. Chinese Journal of Epidemiology, 2016, 37(5):708-713. (in Chinese)
[4]	游胜,冯秀,刘晓青,等. 江西省首例人感染H9N2禽流感病例流行病学调查[J]. 现代预防医学, 2017, 44(5):782-784.YOU S, FENG X, LIU X Q, et al. Epidemiological investigation on the first case of human infection with H9N2 avian influenza in Jiangxi[J]. Modern Preventive Medicine, 2017, 44(5):782-784. (in Chinese)
[5]	王玥莲, 鲁丽莉,冯琛,等. 2016年四川省首例人感染禽流感H9N2危重症临床救治转运路径[J]. 世界最新医学信息文摘, 2016, 16(88):152-153.WANG Y L, LU L L, FENG C, et al. Discussion on clinical pathway of the first human avian influenza H9N2 critical disease in Sichuan province, 2016[J]. World Latest Medicine Information (Electronic Version), 2016, 16(88):152-153. (in Chinese)
[6]	World Health Organization. Influenza at the human-animal interface Summary and assessment, 13 February to 9 April 2019[EB/OL]. (2019-04-09). https://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_09_04_2019.pdf?ua=1.
[7]	WAN H Q, PEREZ D R. Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells[J]. J Virol, 2007, 81(10):5181-5191.
[8]	张增峰, 樊晓晖,陈晓燕,等. 禽流感病毒H9N2在人肺组织的复制[J]. 病毒学报, 2013, 29(2):206-210.ZHANG Z F, FAN X H, CHEN X Y, et al. Avian influenza virus subtype H9N2 replicates in human lung tissues[J]. Chinese Journal of Virology, 2013, 29(2):206-210. (in Chinese)
[9]	ZENG H, PAPPAS C, BELSER J A, et al. Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses:possible involvement in the pathogenesis of human H5N1 virus infection[J]. J Virol, 2012, 86(2):667-678.
[10]	IMAI Y, KUBA K, NEELY G G, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury[J]. Cell, 2008, 133(2):235-249.
[11]	XU T, WANG C L, ZHANG R H, et al. Carnosine markedly ameliorates H9N2 swine influenza virus-induced acute lung injury[J]. J Gen Virol, 2015, 96(10):2939-2950.
[12]	RU X C, YAO X Q. TRPM2:a multifunctional ion channel for oxidative stress sensing[J]. Acta Physiol Sin, 2014, 66(1):7-15.
[13]	HECQUET C M, MALIK A B. Role of H2O2-activated TRPM2 calcium channel in oxidant-induced endothelial injury[J]. Thromb Haemost, 2009, 101(4):619-625.
[14]	HECQUET C M, AHMMED G U, VOGEL S M, et al. Role of TRPM2 channel in mediating H2O2-induced Ca2+ entry and endothelial hyperpermeability[J]. Circ Res, 2008, 102(3):347-355.
[15]	HANSFORD R G. Physiological role of mitochondrial Ca2+ transport[J]. J Bioenerg Biomembr, 1994, 26(5):495-508.
[16]	梁亭,李珮瑶,罗强,等. 稳定抑制TRPM2基因表达肺微血管内皮细胞系的构建与鉴定[J]. 中国细胞生物学学报, 2019, 41(3):439-445.LIANG T, LI P Y, LUO Q, et al. Construction and verification of a mouse pulmonary microvascular endothelial cell lines to inhabit TRPM2 gene expression with shRNA lentivirus system[J]. Chinese Journal of Cell Biology, 2019, 41(3):439-445. (in Chinese)
[17]	HE G M, DONG C G, LUAN Z H, et al. Oxygen free radical involvement in acute lung injury induced by H5N1 virus in mice[J]. Influenza Other Respir Viruses, 2013, 7(6):945-953.
[18]	XU M J, LIU B J, WANG C L, et al. Epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor and effectively alleviates acute lung injury induced by H9N2 swine influenza virus[J]. Int Immunopharmacol, 2017, 52:24-33.
[19]	WANG Q W, SU Y, SHENG J T, et al. Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways[J]. Plos One, 2018,13(1):e0191793.
[20]	LU S L, XIANG L S, CLEMMER J S, et al. Oxidative stress increases pulmonary vascular permeability in diabetic rats through activation of transient receptor potential melastatin 2(TRPM2) channels[J]. Microcirculation, 2014, 21(8):754-760.
[21]	AVERY S V. Molecular targets of oxidative stress[J]. Biochem J, 2011, 434(2):201-210.
[22]	KOWALTOWSKI A J, DE SOUZA-PINTO N C, CASTILHO R F, et al. Mitochondria and reactive oxygen species[J]. Free Radical Biol Med, 2009, 47(4) 333-343.
[23]	ARMSTRONG J S, STEINAUER K K, HORNUNG B, et al. Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line[J]. Cell Death Differ, 2002, 9(3):252-263.
[24]	GRAZZINI E, PUMA C, ROY M O, et al. Sensory neuron-specific receptor activation elicits central and peripheral nociceptive effects in rats[J]. Proc Natl Acad Sci U S A, 2004, 101(18):7175-7180.
[25]	杨慧玲, 潘景轩,吴伟康. 高级病理生理学[M]. 北京:科学出版社, 1998:10.YANG H L, PAN J X, WU W K. Advanced pathophysiology[M]. Beijing:Science Press, 1998:10. (in Chinese)
[26]	ÇAKIR M, TEKIN S, TA?LIDERE A, et al. Protective effect of N-(p-amylcinnamoyl) anthranilic acid, phospholipase A2 enzyme inhibitor, and transient receptor potential melastatin-2 channel blocker against renal ischemia-reperfusion injury[J]. J Cell Biochem, 2019, 120(3):3822-3832.
[27]	LI F F, MUNSEY T S, SIVAPRASADARAO A. TRPM2-mediated rise in mitochondrial Zn2+ promotes palmitate-induced mitochondrial fission and pancreatic β-cell death in rodents[J]. Cell Death Differ, 2017, 24(12):1999-2012.
[28]	LI F F, ABUARAB N, SIVAPRASADARAO A. TRPM2:shredding the mitochondrial network[J]. Channels, 2017, 11(6):507-509.

TRPM2离子通道在H9N2流感病毒感染小鼠肺微血管内皮细胞线粒体损伤中的作用

The Effect of TRPM2 Ion Channel on Mitochondrial Damage of Pulmonary Microvascular Endothelial Cells in Mice Infected with H9N2 Influenza Virus

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