去泛素化酶ABRO1对李斯特菌感染单核巨噬细胞IL-1β释放的影响及其机制

Effect of deubiquitinase ABRO1 on IL-1βrelease of monocyte macrophage induced by listeriolysin O

目的观察去泛素化酶Abraxas兄弟蛋白(ABRO1)对李斯特菌(LM)感染的小鼠单核巨噬细胞J774A.1白细胞介素(IL)-1β释放的影响,并探讨相关机制。方法培养J774A.1细胞,分别感染有李斯特菌溶血素O(LLO)的野生型LM菌株(野生型组)、敲除LLO的hly基因缺失(Δhly)LM菌株(基因缺失组)及Δhly株回补hly基因的LM菌株(回补株组),采用Western blotting法检测ABRO1蛋白,ELISA法检测细胞培养液上清中的IL-1β。将J774A.1细胞分为NI组(未感染LM菌株)、WT组(感染WT LM菌株)与Δhly组(感染Δhly LM菌株),各组分别转染NC si RNA、ABRO1 si RNA,采用ELISA法检测细胞培养液上清中的IL-1β,采用Western blotting法检测炎症小体相关分子Caspase-1、p20(Caspase-1活化形式)、IL-1β、p17。结果随着感染时间延长,野生型组、回补株组ABRO1表达、IL-1β水平逐渐增高,在感染120 min达到最高、均高于基因缺失组(P均<0.05);基因缺失组不同时点ABRO1表达、IL-1β水平无明显变化。WT组转染ABRO1 si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于转染NC si RNA的细胞(P均<0.05);WT组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达高于NI组(P均<0.05);Δhly组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于WT组(P均<0.05)。结论李斯特菌感染的J774A.1细胞中ABRO1表达增高,下调ABRO1表达后,J774A.1细胞中IL-1β释放减少;LLO可能通过激活炎症小体从而促进LM感染诱导的IL-1β释放。

Objective To observe the effect of deubiquitinase Abraxas brother 1(ABRO1)on the release of interleukin(IL)-1βfrom monocyte macrophage J774A.1 in mice induced by listeriolysin O(LLO),and to explore the relevant mechanisms.Methods J774A.1 cells were cultured and infected with wild-type Listeria monocytogenes(LM)strains with LLO(wild-type group),hly gene deficient(Δhly)LM strains with LLO knockout(gene deficient group),andΔhly strains supplemented with hly gene LM strains(replenishment group),respectively.Western blotting was used to detect ABRO1 protein,and ELISA was used to detect IL-1βin the supernatant of cell culture medium.We divided J774A.1 cells into NI group(uninfected LM strain),WT group(infected WT LM strain),andΔhly group(infectedΔhly LM strain),and cells in each group were transfected with NCsiRNA and ABRO1siRNA,respectively.The IL-1βin the supernatant of the cell culture medium was detected by ELISA,and Western blotting was used to detect inflammasome-related molecules Caspase-1,p20,and IL-1β,and p17.Results Over the infection time,the expression of ABRO1 and IL-1βlevels gradually increased in the wild-type group and the replenishment group,reaching their highest levels at 120 minutes of infection,and both were higher than those in the gene deficient group(all P<0.05).There were no significant changes in ABRO1 expression or IL-1βlevels at different time points in the gene deficient group.The level of IL-1βand expression of p20 and p17 in the supernatant of cells transfected with ABRO1 siRNA in the WT group were lower than those in cells transfected with NC siRNA(all P<0.05).The level of IL-1βand expression of p20 and p17 in the supernatant of cells transfected with NC siRNA in the WT group were higher than those in the NI group(all P<0.05).The level of IL-1βand expression of p20 and p17 in the supernatant of cells transfected with NC siRNA in theΔhly group were lower than those in the WT group(all P<0.05).Conclusions Under the induction of LLO,the expression of ABRO1 increased in J774A.1 cells.After down-regulating ABRO1 expression,the release of IL-1βin LLO-induced J774A.1 cells decreased;LLO may promote LLO-mediated IL-1βrelease by activating inflammasomes.