老龄小鼠肠黏膜上皮老化致T合酶表达降低的调控机制研究

As the age increases, intestinal mucosa shows signs of aging, and meanwhile the incidence of chronic colitis and tumors is increased significantly, which affects health and has a major impact on quality of life among elderly individuals. The alteration in mucus properties, i.e., decline in expression of mature mucins, resulted from aging on intestinal epithelium, is often accompanied by gut microbiota dysbiosis. Mucins secreted from intestinal epithelial goblet cells compose the structure of biological barriers after glycosylation modification and T-synthase is the rate-limiting enzyme in mucin-type O-linked glycosylation. Our previous research found that comparing with which in young mice, opportunistic pathogen was markedly increased while T-synthase expression in the intestinal mucosa was decreased in aging mice. Low expression of T-synthase is considered critical for obstruction of O-glycan modification and property changes of mucus. Therefore, this project intends to regard the mechanism of reduction in T-synthase expression in the aged intestinal epithelium and figure out the regulatory role of a set of microRNAs, which were up-regulated in aged mucosa using transcriptome analyses during the pre-experiment, on T-synthase mRNA in vivo and in vitro. Furthermore, the cellular and animal models of oxidative stress as well as microRNA agomirs/antagomirs are applied for clarifying the relationship between elevated expression of microRNAs and low expression of T-synthase which caused by aging-induced oxidative stress and the impairment of intestinal mucus barrier. This study will provide a new research perspective and theoretical basis for further study on colorectal inflammation and cancer in the geriatric population.

随年龄增加，肠黏膜上皮明显老化，慢性肠炎和肿瘤的发生率显著增加，严重威胁老年人健康。肠上皮老化主要表现为表面黏液性质改变，即黏液中成熟黏蛋白减少，并伴有肠道菌群失调。已知肠上皮杯状细胞分泌的黏蛋白需经O-型糖基化修饰后发挥生物屏障功能，T合酶是该糖基化修饰的限速酶。我们预实验发现：与青壮年小鼠相比，老龄小鼠肠上皮T合酶表达下降，肠道条件致病菌比例升高；而T合酶减少被认为是导致黏蛋白糖基化受阻和黏液性质改变的关键因素。因此，本项目重点关注老化肠上皮T合酶表达降低的机制，针对前期转录组学检测发现的一组老化时增多的miRNAs，从离体和在体层面研究其沉默T合酶表达的作用；进而构建氧化应激的细胞和动物模型，结合促进或抑制相关miRNA的表达，探讨肠上皮老化出现氧化应激引起相应miRNA升高和T合酶减少，最终导致黏液屏障受损的调控机制，为深入理解老年人群易罹患肠炎和肿瘤提供新的研究视角和理论依据。

人口老龄化已成为世界性的严峻公共卫生问题。随年龄增加，机体组织器官的结构和功能均呈现退行性改变，消化系统尤为明显，极易发生结直肠炎症和肿瘤等，严重危害老年人健康和生命。正常生理状态下，肠黏膜表面的黏液层是维持肠道菌群稳态、抵御细菌入侵的第一道防线；而随年龄增加，肠黏膜表面黏液中的成熟黏蛋白逐渐减少，并伴随肠道细菌易位和菌群失调，可能是肠黏膜炎性病变的直接诱因。已知肠上皮杯状细胞合成的黏蛋白需经O-型糖基化修饰后发挥生物屏障功能，T合酶是糖基化修饰的限速酶。本研究显示，老化肠上皮T合酶表达下降，肠黏液屏障受损，并发现网站预测靶向调控T合酶的一组microRNAs，miR-124-3p和miR-1-3p，在老年人和老龄小鼠的肠黏膜组织表达增多；进一步应用老年人正常肠黏膜组织、自然衰老小鼠模型、microRNA agomir/mimic处理小鼠/细胞、DSS诱导炎症小鼠模型，证明老化肠上皮细胞miR-124-3p、miR-1-3p增多可靶向沉默T合酶表达，可能是导致黏蛋白糖基化受阻和黏液屏障受损、菌群失调和易位、肠炎易感的重要因素。结合老化肠黏膜组织缺血缺氧状态，推测氧化应激可能是老年人肠黏膜上皮microRNAs高表达的主要诱因。因此，给予小鼠抗氧化剂褪黑素进行干预，结果表明，与对照组相比，褪黑素干预组的老龄小鼠结肠氧化应激程度减轻，肠上皮miR-124-3p、miR-1-3p表达下降，而T合酶表达增加，提示抗氧化物质能够通过影响microRNAs和T合酶表达，延缓老化肠黏液屏障受损。上述研究结果证实了“氧化应激/microRNAs/T合酶/黏蛋白O-型糖基化”调控轴在老化肠黏液屏障受损、肠道菌群稳态失衡中的作用，本研究结果不仅为深入理解老年人易罹患肠炎和肿瘤提供了新的研究视角和理论依据，更为临床延缓衰老、健康老龄化、预防老年慢性肠病开辟了新的思路和方法。