Abstract：Objective To explore the killing mechanism induced by Coxsackievirus A16（CV-A16）in primary muscle cells of gerbils，and to lay the foundations for elucidation the pathogenesis of CV-A16 and the further application of gerbil model.Methods The primary muscle cell model was established by digestion of trypsase/collagenase double enzyme hydrolysis. Primary muscle cells were infected by different dose of CV-A16 and the cell viability was detected by CCK-8 assays. Chromatin condensation and break were measured by Hoechst 33258 staining. The early and last stage of apoptosis cells were measured by AnnexinV/PI double staining. Expression changes of Caspase-3，Caspase-8，JNK and NF-κB pathway proteins were detected by Western Blot.Results The cell viability were 88.95% and 64.05% at groups of different multiplicity of infection（MOI=0.50 and 1.00），which was significantly different from those of the negative control group. The cell viability and multiplicity of infection were negative correlation（rs=-0.857，P=0.014）. The apoptosis rates were 7.2%，21.8% and 50.7% at MOI=0.01,0.10 and 1.00 groups，respectively. The apoptosis rate and MOI were positive correlation（rs=1.000，P<0.001）. When the primary cells were infected by CV-A16，cleavage of Caspase-3 and Caspase-8 were detected. Western Blot assays showed that the expression of NF-κB pathway proteins IκBα，p65 and p-p65 were reduced，which was different in enterovirus 71-infected cells. The JNK kinase was actived.Conclusion CV-A16 could induce apoptosis in primary muscle cells from gerbils.
 LIU S L，PAN H，LIU P，et al. Comparative epidemiology and virology of fatal and nonfatal cases of hand，foot and mouth disease in mainland China from 2008 to 2014[J] . Rev Med Virol，2015，25（2）：115-128.  SUN Y S，LI Y J，XIA Y，et al. Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation[J] . Sci Rep，2016，6：34299.  ZHU F，XU W，XIA J，et al. Efficacy，safety，and immunogenicity of an enterovirus 71 vaccine in China[J] . N Engl J Med，2014，370（9）：818-828.  TAKAHASHI S，LIAO Q，VAN BOECKEL T P，et al. Hand，foot，and mouth disease in China：Modeling epidemic dynamics of enterovirus serotypes and implications for vaccination[J] . PLoS Med，2016，13（2）：e1001958.  ZHU G，ZHENG Y，ZHANG L，et al. Coxsackievirus A16 infection triggers apoptosis in RD cells by inducing ER stress[J] . Biochem Biophys Res Commun，2013，441（4）：856-861.  SHI Y，HE X，ZHU G，et al. Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways[J] . PLoS ONE，2015，10（4）：e0122109.  周彬，孙一晟，姚苹苹，等. 肠道病毒71型诱导沙鼠肌肉细胞凋亡及其机理研究[J] . 预防医学，2017，29（2）：109-112，116.  徐黎明，石巧娟，徐磊，等. 蒙古沙鼠非酒精性脂肪性肝病模型的建立与评价[J] . 浙江预防医学，2007，19（11）：1-2，7.  NOTO J M，ROMERO-GALLO J，PIAZUELO M B，et al. The mongolian gerbil：a robust model of helicobacter pylori-induced gastric inflammation and cancer[J] . Methods in Molecular Biology，2016：263-280.  XU F，YAO P P，XIA Y，et al. Enterovirus 71 infection causes severe pulmonary lesions in gerbils，meriones unguiculatus，which can be prevented by passive immunization with specific antisera[J] . PLoS ONE，2015，10（3）：e0119173.  YAO P P，QIAN L，XIA Y，et al. Enterovirus 71-induced neurological disorders in young gerbils，Meriones unguiculatus：development and application of a neurological disease model[J] . PLoS ONE，2012，7（12）：e51996.  SONG S，TAN J，MIAO Y，et al. Crosstalk of autophagy and apoptosis：Involvement of the dual role of autophagy under ER stress[J] . Journal of Cellular Physiology，2017，232（11）：2977- 2984.  RAJMANI R S，GUPTA S K，SINGH P K，et al. HN protein of Newcastle disease virus sensitizes HeLa cells to TNF-α-induced apoptosis by downregulating NF-κB expression[J] . Arch Virol，2016，161（9）：2395-2405.  YE J，CHEN Z，LI Y，et al. Japanese encephalitis virus NS5 inhibits the type I interferon production by blocking the nuclear translocation of IRF3 and NF-kappaB[J] . Journal of Virology，2017，91（8）：e00039-17.  XU H，SU C，PEARSON A，et al. Herpes simplex virus 1 UL24 abrogates the DNA sensing signal pathway by inhibiting NF-κB activation[J] . Journal of Virology，2017，91（7）：e00025-17.  余冬梅，安输，杨洋，等. JNK 激酶在细胞凋亡中的作用及其与癌症的关系[J] . 中国药理学通报，2015，31（12）：1641- 1645.  ZHANG C，WANG L M. Inhibition of autophagy attenuated curcumol-induced apoptosis in MG-63 human osteosarcoma cells via Janus kinase signaling pathway[J] . Oncology Letters，2017，14（6）：6387-6394.  ZHANG H，LI F，PAN Z，et al. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection[J] . Virus Res，2014，192：74-84.