神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路

doi:10.11843/j.issn.0366-6964.2021.011.023

摘要/Abstract

摘要： NMB/NMBR通过调节A型流感病毒（IAV/H1N1/PR8）感染诱导的细胞因子表达而参与抗IAV的先天性免疫反应。为探究其发挥抗IAV/H1N1感染的信号通路，本文用PR8和WSN毒株分别感染MLE-12细胞和小鼠，用NF-κB抑制剂BAY11-7028单独或联合NMB处理MLE-12细胞，小鼠后腿肌内注射NMB和NMBRA，采用RT-PCR和qRT-PCR分析NMB、NMBR、IL-6、IFN-α和NP基因表达变化，采用Western blot分析NMB、NMBR、P65/p-P65、IκBα和NP蛋白表达的变化。结果显示，BAY11-7028可促使PR8和WSN感染的MLE-12细胞中NMB、NMBR、IL-6和IFN-α基因表达水平均下降和NP基因表达水平上升，并降低NMB、NMBR和p-P65蛋白表达水平和提升IκBα和NP蛋白表达水平。然而，NMB联合BAY 11-7028诱导PR8或WSN感染后的细胞中IL-6和NP表达出现极显著下降和IFN-α显著上升。此外，NMB抑制PR8和WSN感染的小鼠肺组织内p-P65和NP蛋白表达水平和促进IκBα蛋白表达水平；NMBRA联合NMB抵消NMB对PR8或WSN感染后的这些蛋白表达水平的调节作用。综上表明，NMB/NMBR通过调节PR8和WSN感染的MLE-12细胞和小鼠体内的NF-κB信号通路上P65蛋白磷酸化和IκBα的表达，进而影响下游细胞因子IL-6和IFN-α基因的表达，从而发挥抗IAV/H1N1感染的先天性免疫应答反应。

关键词: 神经介素B, 神经介素B受体, A型流感病毒H1N1, NF-κB

Abstract: NMB/NMBR had been involved in anti-influenza A virus (IAV/H1N1/PR8) infection by regulating the expression of cytokines. To explore the signaling pathway of NMB/NMBR against IAV/H1N1 infection, the MLE-12 cells and mice were infected with IAV/H1N1 subtype PR8 or WSN strain respectively, and the cells were treated with NF-κB inhibitor BAY11-7028 alone or in combination with NMB, meanwhile, the mice were injected with NMB or NMBRA. The expression profiles of NMB, NMBR, IL-6, IFN-α and NP genes in transcript levels were analyzed by the methods of RT-PCR and qRT-PCR, and the expression profiles of NMB, NMBR, P65/p-P65, IκBα and NP in protein levels were analyzed by Western blot. The results showed that the decreased expression levels of NMB, NMBR, IL-6 and IFN-α, and increased NP levels in MLE-12 cells after PR8 or WSN infection were induced by BAY11-7028 treatment. Meanwhile, the significantly decreased expression levels of NMB, NMBR and p-P65 proteins, and increased expression levels of IκBα and NP proteins were also induced by BAY11-7028. However, NMB combined with BAY11-7028 could induce a significant decrease in IL-6 and NP, and a significant increase in IFN-α in MLE-12 cells after PR8 or WSN infection. NMB could inhibit the expression of p-P65 and NP proteins, and promoted the expression level of IκBα protein in lung tissues of PR8 and WSN infected mice. NMBR inhibitor NMBRA combined with NMB could counteract the regulatory effects of NMB on the expression of these proteins after PR8 or WSN infection. These results suggest that NMB could regulate the phosphorylation of P65 protein and the expression of IκBα involved in NF-κB signaling pathway in PR8 or WSN infected MLE-12 cells and mice, and affect the expressions of downstream cytokines, IL-6 and IFN-α, thereby play the innate immune response against IAV/H1N1 infection.

Key words: neuromedin B(NMB), neuromedin B receptor(NMBR), influenza A virus H1N1, NF-κB

中图分类号:

S852.6

唐梦瑶, 马逸杰, 田世茂, 万乾晖, 杨桂红. 神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路[J]. 畜牧兽医学报, 2021, 52(11): 3215-3223.

TANG Mengyao, MA Yijie, TIAN Shimao, WAN Qianhui, YANG Guihong. The Signaling Pathway of Neuromedin B and Its Receptor NMBR Involvement in Anti-influenza A Virus H1N1 Subtype Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3215-3223.

参考文献 39

[1]	MEDINA R A, GARCÍA-SASTRE A. Influenza A viruses:new research developments[J]. Nat Revi Microbiol, 2011, 9(8):590-603.
[2]	DAI M L, DU W J, MARTÍNEZ-ROMERO C, et al. Analysis of the evolution of pandemic influenza A(H1N1) virus neuraminidase reveals entanglement of different phenotypic characteristics[J]. mBio, 2021, 12(3):e00287-21.
[3]	JIMENEZ-BLUHM P, SEPULVEDA A, BAUMB-ERGER C, et al. Evidence of influenza infection in dogs and cats in central Chile[J]. Prev Vet Med, 2021, 191:105349.
[4]	ZHANG X H, LI Y G, JIN S, et al. PB1 S524G mutation of wild bird-origin H3N8 influenza A virus enhances virulence and fitness for transmission in mammals[J]. Emerg Microbes Infect, 2021, 10(1):1038-1051.
[5]	SALVESEN H A, WHITELAW C B A. Current and prospective control strategies of influenza A virus in swine[J]. Porcine Health Manag, 2021, 7(1):23.
[6]	ASANO Y, MIZUMOTO K, MARUYAMA T, et al. Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP[J]. J Biochem, 1995, 117(3):677-682.
[7]	LI X L, GU M, ZHENG Q M, et al. Packaging signal of influenza A virus[J]. Virol J, 2021, 18(1):36.
[8]	VASIN A V, TEMKINA O A, EGOROV V V, et al. Molecular mechanisms enhancing the proteome of influenza A viruses:an overview of recently discovered proteins[J]. Virus Res, 2014, 185:53-63.
[9]	PÉREZ-RUBIO G, PONCE-GALLEGOS M A, DOMÍNGUEZ-MAZZOCCO B A, et al. Role of the host genetic susceptibility to 2009 pandemic influenza A H1N1[J]. Viruses, 2021, 13(2):344.
[10]	GANTI K, BAGGA A, DASILVA J, et al. Avian influenza A viruses reassort and diversify differently in mallards and mammals[J]. Viruses, 2021, 13(3):509.
[11]	YIN X, DENG G H, ZENG X Y, et al. Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China[J]. PLoS Pathog, 2021, 17(4):e1009561.
[12]	CHANG P X, SEALY J E, SADEYEN J R, et al. Immune escape adaptive mutations in the H7N9 avian influenza hemagglutinin protein increase virus replication fitness and decrease pandemic potential[J]. J Virol, 2020, 94(19):e00216-20.
[13]	HUANG S W, WANG S F. The effects of genetic variation on H7N9 avian influenza virus pathogenicity[J]. Viruses, 2020, 12(11):1220.
[14]	LANZ C, SCHOTSAERT M, MAGNUS C, et al. IFITM3 incorporation sensitizes influenza A virus to antibody-mediated neutralization[J]. J Exp Med, 2021, 218(6):e20200303.
[15]	NENASHEVA V V, NIKITENKO N A, STEPANENKO E A, et al. Human TRIM14 protects transgenic mice from influenza A viral infection without activation of other innate immunity pathways[J]. Genes Immun, 2021, 22(1):56-63.
[16]	WANG Y C, ZHANG X J, BI K F, et al. Critical role of microRNAs in host and influenza A (H1N1) virus interactions[J]. Life Sci, 2021, 277:119484.
[17]	WU J, GU J Q, SHEN L, et al. The role of host cell Rab GTPases in influenza A virus infections[J]. Future Microbiol, 2021, 16(6):445-452.
[18]	KAMEDA H, MIYOSHI H, SHIMIZU C, et al. Expression and regulation of neuromedin B in pituitary corticotrophs of male melanocortin 2 receptor-deficient mice[J]. Endocrinology, 2014, 155(7):2492-2499.
[19]	DEMAS G E, ADAMO S A, FRENCH S S. Neuroendocrine-immune crosstalk in vertebrates and invertebrates:Implications for host defence[J]. Funct Ecol, 2011, 25(1):29-39.
[20]	DELGADO M, VARELA N, GONZALEZ-REY E. Vasoactive intestinal peptide protects against β-amyloid-induced neurodegeneration by inhibiting microglia activation at multiple levels[J]. GLIA, 2008, 56(10):1091-1103.
[21]	GONZALEZ-REY E, DELGADO M. Anti-inflammatory neuropeptide receptors:new therapeutic targets for immune disorders?[J]. Trends Pharmacol Sci, 2007, 28(9):482-491.
[22]	JENSEN R T, BATTEY J F, SPINDEL E R, et al. International union of pharmacology. LXVⅢ. Mammalian bombesin receptors:nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states[J]. Pharmacol Rev, 2008, 60(1):1-42.
[23]	MINAMINO N, KANGAWA K, MATSUO H. Neuromedin B:a novel bombesin-like peptide identified in porcine spinal cord[J]. Biochem Biophys Res Commun, 1983, 114(2):541-548.
[24]	MINAMINO N, SUDOH T, KANGAWA K, et al. Neuromedin B-32 and B-30:two"big"neuromedin B identified in porcine brain and spinal cord[J]. Biochem Biophys Res Commun, 1985, 130(2):685-691.
[25]	SAYEGH A I. The role of bombesin and bombesin-related peptides in the short-term control of food intake[J]. Prog Mol Biol Trans Sci, 2013, 114:343-370.
[26]	SU P Y, KO M C. The role of central gastrin-releasing peptide and neuromedin B receptors in the modulation of scratching behavior in rats[J]. J Pharmacol Exp Ther, 2011, 337(3):822-829.
[27]	MISHRA S K, HOLZMAN S, HOON M A. A nociceptive signaling role for neuromedin B[J]. J Neurosci, 2012, 32(25):8686-8695.
[28]	BOUGHTON C K, PATEL S A, THOMPSON E L, et al. Neuromedin B stimulates the hypothalamic-pituitary-gonadal axis in male rats[J]. Regul Pept, 2013, 187:6-11.
[29]	GAJJAR S, PATEL B M. Neuromedin:An insight into its types, receptors and therapeutic opportunities[J]. Pharmacol Rep, 2017, 69(3):438-447.
[30]	MA Z Y, ZHANG Y, SU J, et al. Effects of neuromedin B on steroidogenesis, cell proliferation and apoptosis in porcine Leydig cells[J]. J Mol Endocrinol, 2018, 61(1):13-23.
[31]	YANG G H, HUANG H P, TANG M Y, et al. Role of neuromedin B and its receptor in the innate immune responses against influenza A virus infection in vitro and in vivo[J]. Vet Res, 2019, 50(1):80.
[32]	MULERO M C, HUXFORD T, GHOSH G. NF-κB, IκB, and IKK:Integral components of immune system signaling[J]. Adv Exp Med Biol 2019, 1172:207-226.
[33]	MITCHELL J P, CARMODY R J. NF-κB and the transcriptional control of inflammation[J]. Int Rev Cell Mol Biol, 2018, 335:41-84.
[34]	CHEN J F, SHI Y, HUANG J R, et al. Neuromedin B receptor mediates neuromedin B-induced COX-2 and IL-6 expression in human primary myometrial cells[J]. J Investig Med, 2020, 68(6):1171-1178.
[35]	JAYAKUMAR T, HOU S M, CHANG C C, et al. Columbianadin dampens in vitro inflammatory actions and inhibits liver injury via inhibition of NF-κB/MAPKs:Impacts on ·OH Radicals and HO-1 Expression[J]. Antioxidants (Basel), 2021, 10(4):553.
[36]	DE CASTRO BARBOSA M L, DA CONCEICAO R A, FRAGA A G M, et al. NF-κB signaling pathway inhibitors as anticancer drug candidates[J]. Anticancer Agents Med Chem, 2017, 17(4):483-490.
[37]	DURAND J K, BALDWIN A S. Targeting IKK and NF-κB for therapy[J]. Adv Protein Chem Struct Biol, 2017, 107:77-115.
[38]	ESTARAS M, GONZALEZ-PORTILLO M R, MARTINEZ R, et al. Melatonin modulates the antioxidant defenses and the expression of proinflammatory mediators in pancreatic stellate cells subjected to hypoxia[J]. Antioxidants (Basel), 2021, 10(4):577.
[39]	DENG S, GU B, YU Z, et al. MIR210HG aggravates sepsis-induced inflammatory response of proximal tubular epithelial cell via the NF-κB signaling pathway[J]. Yonsei Med J, 2021, 62(5):461-469.