ATP酶抑制因子1对脂多糖诱导的小鼠肺泡巨噬细胞炎症反应及线粒体自噬的影响

Effects of ATPase inhibitory factor 1 on lipopolysaccharide-induced mouse alveolar macrophage activation and mitophagy

目的:本研究旨在探索ATP酶抑制因子1(ATPase inhibitory factor 1,IF1)在脂多糖(lipopolysaccha⁃ride,LPS)诱导的肺泡巨噬细胞炎症模型中的作用。方法:用LPS刺激小鼠肺泡巨噬细胞系MH-S作为体外细胞炎症模型。利用CRISRP activation质粒构建过表达IF1的MH-S细胞系,采用Western blot及RT⁃qPCR检测IF1的表达;ELISA法检测细胞炎症因子白细胞介素6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和IL-1β水平;JC-1和MitoSOX™Red分别检测细胞线粒体膜电位(mitochondrial membrane potential,MMP)和活性氧(reactive oxygen species,ROS)水平;Western blot检测自噬相关蛋白LC3、线粒体膜蛋白TOM20和线粒体自噬蛋白parkin水平;荧光共定位检测线粒体的标记探针MitoTracker Red与自噬相关蛋白LC3的共定位情况。结果:LPS刺激肺泡巨噬细胞后IF1表达水平降低,细胞炎症因子分泌增加(P<0.01),MMP下降,ROS水平升高、LC3-II/LC3-I比值与parkin蛋白水平升高,TOM20蛋白水平下降(P<0.01),Mito-Tracker Red与LC3蛋白共定位增加。上调IF1后,过表达组中IF1表达水平升高,细胞炎症因子分泌也相应减少,MMP和ROS水平恢复,LC3-II/LC3-I比值与parkin蛋白水平下降,TOM20蛋白水平升高,MitoTracker Red与LC3蛋白共定位减少。结论:IF1可能通过抑制肺泡巨噬细胞线粒体自噬并改善线粒体功能,从而减轻巨噬细胞炎症因子的分泌。

AIM:To explore the role of ATPase inhibitory factor 1(IF1)in lipopolysaccharide(LPS)-in⁃duced alveolar macrophage activation and subsequent inflammatory response.METHODS:Murine alveolar macrophages(MH-S cells)were stimulated by LPS in vitro.Overexpression of IF1 in MH-S cell line was established with the CRISPR activation system,and the expression of IF1 was detected by Western blot and RT-qPCR.The levels of inflammatory fac⁃tors such as interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and IL-1βwere detected by ELISA.Mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were detected by JC-1 and MitoSOX™Red,respectively.The expression of autophagy protein LC3,mitochondrial protein TOM20,and mitophagy protein parkin was detected by Western blot.Immunofluorescence was used to detect the colocalization of mitochondrial marker probe MitoTracker Red and autophagy protein LC3.RESULTS:LPS decreased the expression of IF1,MMP,and TOM20,while increased the release of inflammatory factors and ROS,increased LC3-II/LC3-I ratio and parkin expression(P<0.01),and enhanced the colocalization of Mito-Tracker Red and LC3 in alveolar macrophages.In the overexpression group,the expression of IF1 and TOM20 was upregulated,the level of MMP was upregulated,ROS and inflammatory factors were downregulated,the expression of parkin and LC3-II/LC3-I ratio were decreased,and the colocalization of Mito-Tracker Red with LC3 was re⁃duced.CONCLUSION:IF1 may downregulate macrophage-derived inflammatory cytokines by inhibiting mitochondrial autophagy and improving mitochondrial function.