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

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。

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

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-1β and caspase-1 p20,IL-1β in 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-1β were significantly higher than the control group (P<0.05). Pro-IL-1β was higher in the intervention group than in the activation group (P<0.05). The caspase-1 p20 and IL-1β in the supernatant of the activation group were higher than those in the control group (P<0.05), the caspase-1 p20 and IL-1β in 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.［Key words］cardiac fibroblast; mitochondrial reactive oxygen species； NLRP3 inflammasome; sepsis