RIPK3通过抑制自噬转录因子TFEB在肠上皮细胞非坏死性损伤中的作用及机制

Intestinal epithelial cells (IEC) non-necrotic injury caused by autophagy disorder is an important pathogenesis of inflammatory bowel disease (IBD), but it is not clear if there is correlation between receptor-interacting protein kinase (RIPK)3 and autophagy disorder. It’s known that RIPK3 can phosphorylate serine. Transcription factor EB (TFEB) is a key molecule to maintain autophagy and its inactivation is regulated by phosphorylation on serine. We explored the relation between RIPK3 and TFEB and found that RIPK3 activation and TFEB deactivation both exist in intestinal non-necrotic area of IBD. Silencing RIPK3 in vitro can enhance TFEB activity and autophagy of IEC. RIPK3 and TFEB were found to bind each other during IEC injury with co-immunoprecipitation. Therefore, we put forward the hypothesis that RIPK3 may inhibit IEC autophagy through TFEB phosphorylation. We plan to investigate if RIPK3 can phosphorylate TFEB and its genetic locus with in vitro phosphorylation and mass spectrometry. We also plan to investigate the effect of RIPK3 inhibitor on autophagy and intestinal mucosal barrier. These efforts may elucidate mechanisms of RIP3 mediated IEC autophagy disorder and provide new targets for the treatment of IBD.

“自噬障碍导致肠上皮细胞（IEC）非坏死性损伤”是炎症性肠病（IBD）的重要发病机制。受体相互作用蛋白激酶（RIPK）3与自噬障碍的关系还不清楚。已知RIPK3具有调控丝氨酸磷酸化的能力，且维持自噬的关键分子转录因子EB（TFEB）的失活由丝氨酸磷酸化调控。前期研究发现：在IBD肠道非坏死区存在RIPK3活化和TFEB失活；体外沉默RIPK3可增强IEC的TFEB活性和自噬；IEC损伤时RIPK3和TFEB可相互结合。因此，我们提出“RIPK3可能通过磷酸化修饰TFEB抑制IEC自噬”的假说。我们拟进一步研究：①RIPK3能否磷酸化TFEB以及磷酸化TFEB的位点，②在肠炎模型中研究RIPK3对自噬和肠粘膜屏障的影响。基于以上实验，明确RIPK3在IEC自噬障碍中的作用机制，为IBD治疗提供新靶点。

TNF-α/RIPK3介导的necroptosis是一种新提出来的细胞坏死方式。既往研究提示炎症性肠病可能存在这种细胞死亡方式。本项目首次在小鼠TNBS肠炎模型中观察到TUNEL(+)caspase-3(-)的肠上皮细胞，以及肠炎组织中RIPK3信号活化，为necroptosis参与肠炎发病提供了证据。进一步，我们模拟了caspase-3抑制的炎症条件，用TNF-α+ Z-VAD-fmk刺激caco-2肠上皮细胞，构建了体外研究模型。处理后的细胞胞核碎裂、融合，胞浆出现自噬体；经流式细胞术证实，这种细胞死亡方式为坏死；通路分析表明RIPK3-MLKL通路显著活化。RIPK3抑制剂NSA可抑制上述改变，符合necroptosis定义。为了阐明RIPK3的生理作用，我们利用morpholino技术敲减胚胎期斑马鱼RIPK3分子，结果观察到鱼形态变为弓形，出现心包和肾脏高度水肿，肠蠕动性几乎消失；敲减还导致代表肠道损伤和炎症的巨噬细胞浸润。Real-Time PCR检测Hedgehog信号通路，发现敲减RIPK3鱼的shha和smo显著升高，而Gli3显著下降，表明敲减RIPK3引起的Hedgehog信号通路异常参与了上述发育畸形。因此，过度抑制RIPK3信号通路可能会引起潜在的毒副作用。为了阐明TNF/RIPK3信号通路和炎症性肠病的关系。我们检测了Crohn’s病和Crohn’s病肛瘘患者TNFSF单核苷酸多态性，发现rs6478106位点是Crohn’s病易感位点；IRGM基因的rs72553867，NKX2-3 基因的rs4409764和AOX1基因的rs3731772是Crohn’s病肛瘘的易感位点。上述研究结果初步阐明了肠上皮necroptosis在肠道炎症中的作用和遗传背景，为深入研究肠上皮necroptosis提供了工具。