Prohibitin 1 Modulates Mitochondrial Stress-Related Autophagy in Human Colonic Epithelial Cells

IntroductionAutophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells. MethodsWe measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. ResultsTNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability. ConclusionsDecreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.引言 细胞自噬（autophagy）是细胞应对胞外与胞内应激的适应性反应，通过降解细胞质组分及细胞器（包括受损线粒体），以促进细胞存活并恢复细胞内稳态。部分参与细胞自噬的基因会增加克罗恩病（Crohn's disease）的易感风险。活性氧（reactive oxygen species, ROS）与促炎细胞因子（如肿瘤坏死因子α（TNFα））在活动性炎症性肠病中水平升高，可通过引发线粒体损伤促进细胞损伤与细胞自噬。抑制素（PHB）在维持线粒体正常呼吸功能中发挥作用，其在活动性炎症性肠病中表达水平下调。恢复结肠上皮细胞中PHB的表达可使小鼠免受实验性结肠炎损伤，并对抗氧化应激。本研究旨在探讨PHB在肠上皮细胞中调控线粒体应激相关细胞自噬的潜在作用。 方法 本研究通过RNA干扰（RNA interference, RNAi）下调Caco2-BBE、HCT116野生型（WT）及p53敲除细胞中PHB的表达，以此检测细胞自噬的激活情况。本研究评估了外源性PHB表达对TNFα及干扰素γ（IFNγ）诱导的细胞自噬的影响。在PHB下调过程中，本研究使用巴佛洛霉素A1（Bafilomycin A1）或siATG16L1抑制细胞自噬，并检测其对胞内氧化应激、线粒体膜电位及细胞活力的影响。本研究使用ROS清除剂N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine），检测胞内ROS在siPHB诱导的细胞自噬中的必要性。 结果 TNFα与IFNγ诱导的细胞自噬水平与PHB蛋白表达呈负相关。外源性PHB表达可降低基础细胞自噬水平及TNFα诱导的细胞自噬水平。肠上皮细胞中PHB基因沉默可通过升高胞内ROS水平诱导线粒体自噬。在PHB下调过程中抑制细胞自噬会加重线粒体去极化，并降低细胞活力。 结论 PHB水平下调伴细胞自噬功能异常，会使肠上皮细胞更易受到线粒体损伤与细胞毒性作用的影响。恢复PHB的表达或可作为治疗炎症性肠病等疾病中氧化应激与细胞因子诱导的线粒体损伤的潜在疗法。