线粒体活性氧在心肌成纤维细胞NLRP3炎症小体激活中的作用

Role of mitochondrial reactive oxygen species in activation of NLRP3 inflammasome in cardiac fibroblasts

目的:探讨线粒体活性氧在心肌成纤维细胞NLRP3炎症小体激活中的作用。方法:培养原代小鼠心肌成纤维细胞,细胞分为对照组(正常培养组),使用脂多糖刺激的引发组,使用脂多糖加三磷酸腺苷的激活组,以及再加用mito-TEMPO的干预组。通过MitoSOX染色检测各组细胞线粒体活性氧水平;通过蛋白质印迹法检测细胞内NLRP3、凋亡相关微粒样蛋白(ASC)、Pro-caspase-1、Pro-IL-1β及上清液中caspase-1 p20、IL-1β蛋白水平;用酶联免疫吸附法检测上清液中IL-1β蛋白的含量;使用免疫共沉淀法观察ASC与NLRP3蛋白的结合情况。结果:激活组细胞胞内线粒体活性氧水平较对照组明显增加(P<0.05),而干预组线粒体活性氧水平较激活组明显下降(P<0.05);心肌成纤维细胞经脂多糖刺激后,细胞内NLRP3和Pro-IL-1β蛋白较对照组明显升高(P<0.05),干预组中Pro-IL-1β较激活组明显升高(P<0.05)。激活组上清液中caspase-1 p20和IL-1β较对照组明显升高(P<0.05),干预组caspase-1 p20和IL-1β较激活组明显减少(P<0.05)。激活组NLRP3-ASC连接形成复合体,干预组NLRP3和ASC的结合减少。结论:心肌成纤维细胞中线粒体活性氧通过促进NLRP3蛋白和ASC蛋白的结合,使NLRP3炎症小体激活。

Objective:To investigate the role of mitochondrial reactive oxygen species(mtROS)in NLRP3 inflammasome activation in cardiac fibroblast(CF).Methods:Primary mouse CF was cultured.CFs were divided into control group,initiation group(LPS),activation group(LPS+ATP)and intervention group(LPS+mito-TEMPO+ATP).MtROS in CF was detected with MitoSOX reagent.CF NLPR3 inflammasome activation was assessed with Western blotting for intracellular NLRP3,ASC,Pro-caspase-1,Pro-IL-1βand caspase-1 p20,IL-1βin the supernatant and ELISA was used to detect the level of IL-1β.The interaction between NLRP3 and ASC was examined by co-immunoprecipitation.Results:The expression of intracellular mtROS in the activation group was significant increased compared with the control group(P<0.05),while the mtROS expression in the intervention group was lower than that in the activation group(P<0.05).After challenging by LPS,the CF intracellular NLRP3 and Pro-IL-1βwere significantly higher than the control group(P<0.05).Pro-IL-1βwas higher in the intervention group than in the activation group(P<0.05).The caspase-1 p20 and IL-1βin the supernatant of the activation group were higher than those in the control group(P<0.05),the caspase-1 p20 and IL-1βin the intervention group were significantly lower than those in the activation group(P<0.05).Protein NLRP3 interacted with ASC in activation group,and the binding of NLRP3 and ASC in the intervention group was reduced.Conclusion:MtROS promoted the NLRP3 inflammasome activation in CF via facilitated the binding of NLRP3 and ASC.