A型塞内卡病毒激活炎症因子表达及诱导细胞焦亡

Senecavirus A activates the expression of inflammatory factors and induces pyroptosis

初步探索A型塞内卡病毒(SVA)感染3D4/21细胞后引起细胞因子的变化,以及诱导细胞焦亡的分子机制。用SVA感染3D4/21细胞,不同时间点分别检测IL-1β、IL-6、TNF-α、IL-12、GM-CSF、IL-10、IFN-γ、CXCL10细胞因子mRNA的表达水平和相关炎症因子分泌水平;采用CCK-8检测细胞活力;用乳酸脱氢酶释放试验检测细胞膜的损伤程度;用Western-blot检测目的基因在蛋白水平的表达程度。结果显示:SVA感染细胞后,IL-1β、IL-6、TNF-α、IL-12、GM-CSF、IL-10、IFN-γ、CXCL10细胞因子mRNA表达水平均升高,与转录水平相似,SVA能够诱导IL-1β、IL-6、TNF-α以及IL-12分泌水平上调;SVA感染3D4/21细胞后,焦亡相关基因在mRNA水平表达增加,焦亡相关蛋白GSDMD、IL-1β以及Caspase-1被激活;SVA感染细胞后,导致细胞活力下降,细胞膜完整性丧失;转染sh-NLRP3或加入NLRP3抑制剂MCC950,Caspase-1抑制剂VX765,然后感染SVA,发现焦亡相关活性蛋白表达降低。结论:SVA感染3D4/21细胞后引起上述细胞因子表达增加,激活Caspase-1引起的细胞焦亡,且与NLRP3炎症小体途径相关。

To explore the changes of cytokines with Senecavirus A(SVA)infection and the molecular mechanism of cell pyroptosis induced by SVA,3D4/21 cells were infected with SVA,the level of cytokines mRNA and secretion,including IL-1β,IL-6,TNF-α,IL-12,GM-CSF,IL-10,IFN-γ,and CXCL10 at different time points,were detected respectively by qPCR and ELISA.Cell viability was assayed by CCK-8 detection.The cell membrane integrity was assessed by LDH release test.The expression of target proteins was detected by Western-blot.After SVA infected cells,the mRNA levels of IL-1β,IL-6,TNF-α,IL-12,GM-CSF,IL-10,IFN-γ,CXCL10 cytokines were increased.After SVA infection with 3D4/21 cells,the expression of pyroptosis related genes of NLRP3,Caspase-1 and IL-1βwere increased at the mRNA level.The expressions of active formed pyroptosis related proteins of GSDMD(35 ku),Caspase-1(20 ku)and IL-1β(17 ku)were increased after SVA infection.Moreover,cell viability decreased and cell membrane integrity lost during the SVA infection.After NLRP3 expression interfering with sh-NLRP3 transfection or NLRP3 inhibitor MCC950 addition,and Caspase-1 inhibitor VX765 treatment with 3D4/21,then the cells were infected with SVA,the expressions of active formed pyroptosis related proteins were decreased.In conclusion,the expressions of inflammatory cytokines were increased after SVA infection with pig macrophage of 3D4/21cells.SVA induced Caspase-1-mediated cell pyroptosis was dependent on NLRP3 inflammasome activation.