Objective To investigate the effect of transcription factor FOXO3a on mitophagy in hepatic ischemia-reperfusion injury in mice. Methods A total of 30 male C57BL/6 mice were randomly divided into Sham operation group (Sham) and ischemia reperfusion (IR) 2 h, 6 h, 12 h and 24 h groups, 6 mice in each group. The mouse model of liver ischemia -reperfusion injury was established. Blood biochemical methods were used to detect changes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). HE staining and TUNEL were used to observe the damages of liver tissue and apoptosis. Western blot assay and qRT-PCR were used to detect the expressions of transcription factor FOXO3a, mitochondrial autophagy-related protein Nix protein and its mRNA expression in each group. Mouse liver AML12 cells were treated with FOXO3a and Nix interfering RNA, and the model was established for 6 h after hypoxia for 1.5 h.These cells were divided into siRNA-NC group, FOXO3a siRNA group and Nix siRNA group. MTT assay was used to detect the viability of cells in each group. The number and distribution of autophagy in each group were observed by confocal microscopy. The expressions of FOXO3a, Nix, microtubule-associated protein LC3, apoptotic protein P62 and Caspase-3 were detected by Western blot assay. Results The levels of ALT and AST in all groups of IR were reduced, and reached the peak value at 6 h (P<0.05). HE and TUNEL results showed that liver injury and apoptosis were the most serious at 6 h after reperfusion. The expression of FOXO3a and Nix was higher in IR group than that in the Sham group, and the expression level of FOXO3a mRNA was the highest at 12 h after reperfusion, the expression of Nix mRNA was the highest at 6 h after reperfusion (P<0.05). Western blot assay showed the highest expression of FOXO3a in the reperfusion of 12 h, and the highest expression levels of Nix, Caspase-3 and LC3Ⅱin reperfusion 6 h. After interfering with the expression of FOXO3a, MTT showed a marked reduction in cell survival (P<0.05), Western blot assay showed that the expression level of FOXO3a was significantly higher in siRNA-NC group than that in FOXO3a siRNA group, and the expression levels of Nix, Caspase-3 and LC3Ⅱwere significantly lower than those of FOXO3a siRNA group. Confocal microscopy showed that the number and distribution of autophagosomes were significantly lower in siRNA-NC group than those in FOXO3a siRNA group. After interfering with the expression of Nix, MTT showed a marked increase in cell survival (P<0.05), Western blot assay showed that the expression levels of Nix, P62 and LC3Ⅱ were significantly higher in siRNA-NC group than those in Nix siRNA group. Conclusion FOXO3a can reduce the hepatic ischemia-reperfusion injury in mice, which may be related to the FOXO3a inhibition for liver cell mitophagy and apoptosis.%目的 探讨转录因子FOXO3a调控线粒体自噬对小鼠肝脏缺血再灌注损伤的影响.方法 雄性C57BL/6小鼠30只,随机分为Sham组(只模拟开腹手术不夹闭)和再灌注(IR)2、6、12、24 h组,每组6只,建立小鼠肝缺血再灌注损伤模型.血生化检测各组丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)变化,HE染色及TUNEL法观察肝组织损伤及细胞凋亡情况,Western blot及qRT-PCR检测各组细胞中转录因子FOXO3a、线粒体自噬相关蛋白Nix蛋白及其mRNA表达水平.培养小鼠肝AML12细胞,用FOXO3a和Nix干扰RNA处理细胞建立缺氧1.5 h复氧6 h的模型,分为siRNA-NC组(加入10μL siRNA空载对照转染细胞)、FOXO3a siRNA组(加入10μL FOXO3a siRNA转染细胞)以及Nix siRNA组(加入10μL Nix siRNA转染细胞).MTT法检测各组细胞活力,共聚焦显微镜观察各组细胞内自噬体的数量与分布,Western blot检测FOXO3a、Nix、微管相关蛋白LC3、凋亡蛋白P62及Caspase-3的表达情况.结果 IR各组ALT、AST均明显升高,且再灌注6 h时达到峰值(P<0.05).HE及TUNEL结果示再灌注6 h时小鼠肝组织损伤以及细胞凋亡最严重.IR各组FOXO3a及Nix的mRNA相对表达量均高于Sham组,且再灌注12 h时FOXO3a的mRNA表达量最高,再灌注6 h时Nix的mRNA表达量最高(P<0.05).Western blot示再灌注12 h时FOXO3a相对表达水平最高,再灌注6 h时Nix、Caspase-3、LC3Ⅱ相对表达水平最高.干扰FOXO3a的表达后,MTT显示FOXO3a siRNA组细胞存活率降低(P<0.05);Western blot检测显示siRNA-NC组FOXO3a表达水平高于FOXO3a siRNA组,Nix、Caspase-3、LC3Ⅱ表达水平明显低于FOXO3a siRNA组(P<0.05).共聚焦显微镜观察示siRNA-NC组细胞内自噬体的数量低于FOXO3a siRNA组.干扰Nix的表达后,MTT显示Nix siRNA组细胞存活率升高(P<0.05);Western blot检测显示siRNA-NC组Nix、P62、LC3Ⅱ表达水平高于Nix siRNA组,而FOXO3a表达水平低于Nix siRNA组(P<0.05).结论 FOXO3a可减轻小鼠肝缺血再灌注损伤,其机制可能与FOXO3a抑制肝细胞线粒体自噬且抑制细胞凋亡有关.
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