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  • 徐铮昊,唐海琳,任瑞文,赵平,戚中田,赵兰娟.西尼罗病毒对人神经母细胞瘤细胞p38丝裂原活化蛋白激酶途径的调控[J].第二军医大学学报,2019,40(11):1169-1175    [点击复制]
  • XU Zheng-hao,TANG Hai-lin,REN Rui-wen,ZHAO Ping,QI Zhong-tian,ZHAO Lan-juan.Regulation of p38 mitogen-activated protein kinase pathway by West Nile virus in human neuroblastoma cells[J].Acad J Sec Mil Med Univ,2019,40(11):1169-1175   [点击复制]
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西尼罗病毒对人神经母细胞瘤细胞p38丝裂原活化蛋白激酶途径的调控
徐铮昊1,唐海琳1,任瑞文2,赵平1,戚中田1,赵兰娟1*
0
(1. 海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海 200433;
2. 南部战区疾病预防控制中心, 广州 510507
*通信作者)
摘要:
目的 探讨西尼罗病毒(WNV)感染人神经母细胞瘤细胞SH-SY5Y后对p38丝裂原活化蛋白激酶(MAPK)途径的影响,以及该途径在WNV复制及应激应答、炎性应答相关分子表达调控中的作用。方法 WNV分别短时孵育(5、15、30、60 min)和感染(12、24、48、60 h)SH-SY5Y细胞,用蛋白质印迹法检测p38 MAPK和磷酸化p38 MAPK表达,用qRT-PCR检测WNV感染细胞内C/EBP同源蛋白(CHOP)、白细胞介素6(IL-6)、活化转录因子6α(ATF6α)和干扰素刺激基因15(ISG15)mRNA表达水平的动态变化。用WNV感染p38 MAPK siRNA转染的SH-SY5Y细胞,用qRT-PCR检测WNV RNA水平及CHOPIL-6ATF6αISG15 mRNA水平。结果 与未孵育的细胞相比,WNV短时孵育细胞内p38 MAPK的磷酸化水平升高。WNV感染SH-SY5Y细胞12 h、24 h时激活了p38 MAPK途径,而感染48 h、60 h时明显抑制了p38 MAPK途径。WNV感染促进了CHOPIL-6ISG15 mRNA表达而抑制了ATF6α mRNA表达。与对照siRNA转染的细胞相比,p38 MAPK siRNA转染的细胞内WNV RNA(P<0.05)水平和ATF6α mRNA(P<0.01)水平升高,而CHOP mRNA水平降低(P<0.05)。结论 WNV感染早期激活了p38 MAPK途径,该途径的激活可能负反馈调控WNV的复制。WNV经p38 MAPK途径调控与应激应答相关的CHOP、ATF6α表达及与炎性应答相关的IL-6表达。
关键词:  西尼罗病毒  人神经母细胞瘤细胞  p38丝裂原活化蛋白激酶  病毒复制
DOI:10.16781/j.0258-879x.2019.11.1169
投稿时间:2019-06-17修订日期:2019-09-23
基金项目:国家重点研发计划(2016YFC1202903),上海市公共卫生三年行动计划重点学科建设项目(15GWZK0103).
Regulation of p38 mitogen-activated protein kinase pathway by West Nile virus in human neuroblastoma cells
XU Zheng-hao1,TANG Hai-lin1,REN Rui-wen2,ZHAO Ping1,QI Zhong-tian1,ZHAO Lan-juan1*
(1. Department of Biodefense, Faculty of Naval Medicine, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Center for Disease Control and Prevention of Southern Theater Command of PLA, Guangzhou 510507, Guangdong, China
*Corresponding author)
Abstract:
Objective To explore the regulation of p38 mitogen-activated protein kinase (MAPK) pathway by West Nile virus (WNV) in human neuroblastoma SH-SY5Y cells and the contributions of p38 MAPK to WNV replication as well as stress and inflammatory response related molecule expression. Methods Total and phosphorylated p38 MAPK levels were analyzed in SH-SY5Y cells incubated with WNV for short (5, 15, 30 and 60 min) and long (12, 24, 48 and 60 h) durations by Western blotting. Dynamic changes of CCAAT/enhancer-binding protein homologous protein (CHOP), interleukin 6 (IL-6), activating transcription factor 6α (ATF6α) and interferon-stimulated gene 15 (ISG15) mRNA expression in WNV infected cells were detected by qRT-PCR. In response to WNV infection, WNV RNA level and CHOP, IL-6, ATF6α and ISG15 mRNA levels were assessed in SH-SY5Y cells transfected with p38 MAPK siRNA. Results Incubation with WNV for short durations enhanced p38 MAPK phosphorylation compared to the untreated control. The p38 MAPK signaling pathway was activated at 12 h and 24 h in WNV-infected SH-SY5Y cells, but down-regulated at 48 h and 60 h. WNV infection led to increased mRNA expression of CHOP, IL-6 and ISG15 and reduced ATF6α mRNA. In comparison with control siRNA transfection, the levels of WNV RNA (P<0.05) and ATF6α mRNA (P<0.01) were increased and CHOP mRNA level was decreased (P<0.05) in WNV-infected SH-SY5Y cells with the p38 MAPK siRNA transfection. Conclusion The p38 MAPK pathway is activated during early stage of WNV infection and such activation may negatively regulate WNV replication. WNV-induced stress response molecules CHOP and ATF6α and proinflammatory cytokine IL-6 production by SH-SY5Y cells are coupled with the regulation of p38 MAPK pathway.
Key words:  West Nile virus  human neuroblastoma cells  p38 mitogen-activated protein kinase  virus replication