甲型流感病毒H1N1/FM1对小鼠巨噬细胞NLRP3炎症小体信号通路的影响

Effect of influenza A virus H1N1/FM1 on macrophage NLRP3 inflammosome signaling pathway in mouse

目的探讨甲型流感病毒H1N1/FM1感染小鼠巨噬细胞不同时间点NOD样受体P3(NLRP3)炎症小体信号通路的变化。方法以小鼠单核巨噬细胞系RAW264.7为研究对象,甲型流感病毒H1N1/FM1感染1周作为实验组,感染6 h作为对照组,ELISA检测细胞培养上清中IL-1β的浓度,荧光定量PCR检测炎症小体信号通路中NLRP3、caspase-1、IL-1β的mRNA相对表达量,蛋白免疫印迹法检测NLRP3、caspase-1、cleaved-caspase-1的蛋白相对表达量,免疫荧光染色法定性检测NLRP3的蛋白表达。结果细胞培养上清液中IL-1β的浓度实验组高于对照组(P<0.01),其NLRP3 mRNA和蛋白的表达量低于对照组降低(P均<0.05),caspase-1、IL-βmRNA相对表达量以及caspase-1、cleaved-caspase-1蛋白相对表达量均高于对照组(P均<0.05)。免疫荧光染色中实验组NLRP3蛋白荧光强度弱于对照组。结论甲型流感病毒H1N1/FM1感染小鼠巨噬细胞可以激活NLRP3炎症小体信号通路,促进IL-1β的分泌,且在感染后期(约1周)有更强的激活效应。

Objective To investigate the variation of NOD-like receptor P3 （NLRP3） inflammosome signaling pathway at different time points in mouse macrophages infected by influenza A vims H1N1/FM1.Methods Mouse monocyte-macrophage cell line RAW264. 7 was used in this experiment. In the study group, the cells were infected with H1N1/FM1 for 1 week, and those were infected for 6 h in the control group. The expression of IL-1β in the cell supernatant was measured by ELISA. The relative expression of NLRP3, caspase-1 and IL-1β mRNA was investigated by fluorescent quantitative PCR. The expression levels of NLRP3, caspase-1 and cleaved caspase-1 proteins were quantitatively detected by Western blot. The expression of NL- RP3 protein was observed by immunofluorescent staining. Results In the study group, the expression of IL-1β in the cell supernatant was significantly up-regulated （P 〈0. 01） , the expression levels of NLRP3 mRNA and protein were significantly down-regulated （both P 〈0. 05） , the relative expression of caspase-1 and IL-β mR-NA ,and caspase-1 and cleaved-caspase-1 proteins were significantly up-regulated （ all P 〈 0. 05 ） compared with those in the control group. The fluorescent intensity of NLRP3 protein in the study group was lower than that in the control group. Conclusions Infection of macrophage cells with influenza A vims H1N1/FM1 can activate the NLRP3 inflammosome signaling pathway and promote the secretion of IL-1β. Futhermore, the acti-vating effect can be enhanced during the late stage of infection （approximately 1 week）.