肠上皮中的PKM2在炎症性肠病的作用及机制研究

Inflammatory bowel disease (IBD) is involved in the disorders of mucosal epithelial cell metabolism and immune regulation, but the detailed mechanism remains unknown. In recent years, pyruvate kinase M2 (PKM2) has been found to be a key regulator of cellular metabolism and inflammation. The relationship between IBD and PKM2 is largely unclear. Based on our previous research work of this project, we found that PKM2 is highly expressed in the intestinal epithelial cells and can promote the proliferation of epithelial cells and the repair of the colitis process. RNA-Seq analysis revealed that PKM2 is involved in the regulation of Wnt/β-catenin signaling pathway and defensins family genes during enteritis, but the specific mode of action is still unclear. This project will be based on the existing work basis, using PKM2 conditional knockout mice as animal models, and techniques such as co-immunoprecipitation, CRISPR-Cas9, gene sequencing, and protein profiling to highlight the pathways and molecular mechanisms involved in the regulation of PKM2 in the colitis. Finally, we will establish an effective PKM2 rectum administration method. This project will help elucidate the mechanism of action of PKM2 in colitis and provide new targets for the development of IBD drugs.

炎症性肠病（inflammatory bowel disease，IBD）与肠道粘膜上皮细胞代谢和免疫调节紊乱有关，具体发病机制还不清楚。M2型丙酮酸激酶（pyruvate kinase M2，PKM2）近年来被发现是细胞代谢与炎症反应的关键调节因子，但其与肠炎的作用关系未知。本项目前期研究发现，PKM2在肠上皮细胞中高表达并能促进上皮细胞的增殖和肠炎过程的损伤修复。RNA-Seq分析发现PKM2在肠炎过程中参与调节Wnt/β-catenin信号通路和防御素（defensins）家族基因，但是具体的作用模式还不清楚。本项目将基于现有的工作基础，以PKM2条件敲除鼠为动物模型，利用免疫共沉淀、CRISPR-Cas9、基因测序、蛋白质谱等技术重点阐述PKM2在肠炎进程参与调节的通路和分子机制，并建立有效PKM2肠道给药方法。本项目有助于阐明PKM2在肠炎作用机制，并为研发IBD药物提供新靶标。

炎症性肠病（Inflammatory Bowel Disease，IBD）是最常见的肠道疾病，近年来发病率持续升高。IBD诊断和治疗复杂，预后不佳，严重影响患者生活质量，目前针对IBD 的机制探索以及治疗一直是当今医学研究的热点之一。IBD与肠道粘膜上皮细胞代谢和免疫调节紊乱有关，具体发病机制还不清楚。丙酮酸激酶（Pyruvate kinase，PK）是催化糖酵解反应的关键限速酶之一，主要包含M1型（PKM1）和M2型（PKM2）两种同工酶。PKM2是PK重要的异构酶，主要在胚胎和肿瘤等增殖旺盛的细胞中表达，具有经典的丙酮酸激酶活性和非经典的蛋白激酶活性。PKM2除了参与肿瘤细胞的能量代谢重编程调控外，在免疫炎症通路的激活和效应阶段也起到重要的功能，是细胞代谢、增殖与炎症反应中的关键调节因子，但其与肠道炎症的关系未知。本项目研究发现，PKM2在肠道上皮细胞中高表达，而小鼠肠炎模型和IBD病人肠道组织检测发现PKM2表达水平显著降低。进一步地功能实验证实PKM2能够促进肠上皮细胞的增殖和肠炎的损伤修复。RNA-Seq、荧光定量PCR、免疫荧光和Western Blot等技术分析发现PKM2在肠炎过程中参与调节Wnt/β-catenin信号通路激活，并且促进增殖基因Cyclin D1和肠道α-防御素（α-defensins）家族基因的表达。机制上，我们利用肠道PKM2特异性敲除鼠和肠道类器官为研究对象，采用免疫印记、免疫共沉淀、免疫荧光、转录组测序等技术手段阐述了PKM2通过Wnt/β-catenin-TCF-4信号通路维持肠道稳态的具体分子机制。同时我们表达并建立了有效PKM2肠道给药方法，体外PKM2给药能够有效缓解了小鼠肠炎模型的进展和维持肠道的稳态。本项目的发现有助于阐明PKM2在肠炎作用机制，并为研发IBD药物提供新靶标。