硫酸吲哚酚抑制DRP1介导的线粒体自噬在CKD肠道功能紊乱中的作用及机制研究

Uremic toxin indoxyl sulfate (IS) is a significant injury factor of chronic kidney disease (CKD) and also an important cause of intestinal dysfunction of CKD patients, but the mechanism remains elusive. Recent research has shown IS could lead to mitochondrial dysfunction. Intestinal mucosal injury of CKD mice, inhibition of mitophagy and its regulatory gene DRP1 regulated by IS were observed in our preliminary work. Considering the important role of mitophagy in intestinal epithelial cells, we speculate that IS might inhibit DRP1-mediated mitophagy, which contributes to intestinal dysfunction. Therefore, based on these results, we would detect intestinal injury and mitophagy caused by CKD and analyze the effect of IS on mitophagy and STAT3-DRP1 signaling pathway to illuminate the mechanism. Finally, we elaborate the intervention effect of intestinal adsorbent AST-120 on IS-mediated intestinal dysfunction and mitophagy in intestinal cells and the mouse model, and validate the relevance among IS, STAT3 and DRP1 expression in clinical samples. This research explores the pathogenesis of CKD complicated with intestinal disorders and its prevention strategy, which has clinical significance.

尿毒症毒素硫酸吲哚酚(IS)是慢性肾病(CKD)的重要致伤因素，也是CKD肠道损伤的重要原因，但机制不清。近期研究表明IS能抑制线粒体功能，我们前期研究发现CKD小鼠肠黏膜受损；且IS能抑制肠上皮细胞的线粒体自噬及其调控基因DRP1表达。鉴于线粒体自噬对肠上皮细胞的重要作用，我们推测：IS可能通过DRP1抑制线粒体自噬，进而导致肠道功能紊乱。本项目拟在此基础上，首先在动物模型和临床样本中检测CKD导致的肠道损伤和线粒体自噬变化；然后在细胞实验中分析IS抑制线粒体自噬的效应与机制，阐明STAT3-DRP1信号通路在其中的作用；最后在体外和小鼠模型中解析肠道吸附剂AST-120对IS引起的肠道损伤和线粒体自噬效应的干预效果，并在临床样本中验证肠道损伤程度与IS、STAT3、DRP1的相关性。本项目对探索CKD并发肠功能紊乱的发病机制及其防治策略具有重要的临床指导意义。

项目背景：我国慢性肾脏病（chronic kidney disease，CKD）的患病率高达10.8%，且发病率和死亡率居高不下。肠道功能紊乱是CKD的常见并发症，反过来，肠道功能紊乱又会促进尿毒症毒素的生成，进一步加重肾脏损伤。既往研究多集中于肠道损伤对CKD的影响，但是CKD引发肠道功能紊乱尚未引起足够的重视，并且其机制尚未明确。因此探索CKD合并肠道功能紊乱的发病机制及其防治策略具有重要的临床意义。.研究内容：本研究首先构建CKD小鼠模型，明确肠黏膜屏障破坏的现象。然后收集Sham、CKD小鼠粪便行16s核糖体RNA测序，证实肠道菌群紊乱。跨上皮电阻、qPCR实验发现：菌群代谢的吲哚衍生物硫酸吲哚酚（Indoxyl sulfate，IS）导致肠上皮细胞损伤；腹腔注射IS能破坏紧密连接、促进肠道通透性、诱导肠黏膜损伤。.接下来的机制探讨发现：IS并未影响经典的mTOR信号通路，而是通过抑制线粒体分裂相关蛋白动力相关蛋白1（dynamin-related protein 1，DRP1）表达而诱导线粒体自噬障碍，进而导致肠上皮细胞紧密连接损伤。进一步生的物信息学分析、qPCR、Western blot、基因敲低、重组质粒，双荧光素酶报告基因、点突变和ChIP实验证实：IS通过促进转录因子干扰素调节因子1（interferon regulatory factor 1，IRF1）直接结合于DRP1基因启动子区而抑制DRP1表达及其介导的线粒体自噬，进而诱导肠上皮细胞损伤。此外，CKD小鼠喂食IS清除剂（AST-120）或给予IRF1基因敲除小鼠注射IS，均证实：小鼠肠组织损伤减轻，而且肠道组织中升高的IRF1和降低的DRP1表达均有所恢复，蓄积的线粒体自噬小体也显著减少。最后，HPLC检测发现CKD患者血清IS水平较高，而肠镜所取的组织标本中IRF1表达升高、DRP1表达降低、线粒体自噬障碍、肠黏膜屏障破坏。.研究意义：本研究证实尿毒症毒素IS是诱导CKD并发肠道功能紊乱的关键因素，而转录因子IRF1则是其中的重要环节。机制上，蛋白结合毒素IS通过促进IRF1直接结合于DRP1基因启动子区而抑制DRP1表达及其介导的线粒体自噬，进而诱导肠黏膜屏障损伤。抑制IS蓄积或靶向IRF1-DRP1轴可能是一种缓解CKD并发肠道功能障碍的新型治疗策略。