脆弱拟杆菌上调ILC3/IL-22/pSTAT3通路促进炎症性肠病黏膜愈合的机制研究

The interaction between enteric commensal microbiota and mucosal epithelium not only influences the progression of inflammatory bowel disease, but also has a key role in mucosal healing. ILC3/IL-22/pSTAT3 axis has been considered as an important signal pathway for mucosal repair mediated by commensal bacteria, but the specific bacteria which make a regulation on it have not been clearly demonstrated. In the previous work, we found that long-time use of antibiotic led to the delay of mucosal healing in experimental colitis, while the bacteroides fragilis we isolated could reverse this process and increased gene expression of IL-22. We speculate that B.fragilis may activate the molecular pathway of ILC3/IL-22 directly or indirectly by regulating the intestinal flora, and then exert its therapeutic function in IBD mucosal healing by promoting the epithelial regeneration. In this project, we plan to construct the mouse model of IBD combined with clinical specimens, transgenic animals and in vitro experiments, to unveil the the regulation pattern and mechanism by which the process of intestinal mucosal healing is affected by the interaction between symbiotic bacteria and bacteria, as well as symbiotic bacteria and Intestinal epithelium. This research will provide a fresh theoretical basis for symbiotic bacteria-mediated IBD mucosal healing, and lay a groundwork for the clinical application of B.fragilis in IBD.

肠道共生菌与黏膜上皮的相互作用不仅影响着炎症性肠病（IBD）的发生发展，还在IBD黏膜愈合中发挥重要作用。ILC3/IL-22/pSTAT3轴被认为是肠道共生菌介导黏膜修复的重要通路，但具体哪种细菌对其进行调控仍未阐明。前期工作中，我们发现长时间抗生素的应用会导致实验性肠炎黏膜修复延迟，而我们自主分离得到的脆弱拟杆菌却能逆转这一过程，且增加了肠道IL-22的表达。我们推测脆弱拟杆菌可能通过调控肠道菌群，直接或间接影响了ILC3/IL-22通路，进而介导肠上皮细胞的增殖迁移，促进IBD黏膜愈合。本项目拟构建小鼠肠炎模型，结合临床标本、转基因动物和体外实验，深入探讨共生菌与共生菌、共生菌与肠上皮互作影响肠黏膜愈合的调控模式和作用机制。本项目将为共生菌促进IBD黏膜愈合提供新的理论依据，也为脆弱拟杆菌防治IBD的临床应用奠定基础。

益生菌在炎症性肠病中的治疗机制研究已成为近年来的研究热点，脆弱拟杆菌ZY-312是一株新型益生菌,我们课题组前期发现脆弱拟杆菌ZY-312在抗生素相关性腹泻模型、致病菌诱导的感染性肠炎模型中具有缓解作用，促进肠黏膜增殖修复，但具体调控机制不明。.基于此该团队构建DSS（葡聚糖硫酸钠盐）诱导的实验性结肠炎小鼠模型，并给予脆弱拟杆菌ZY-312治疗，通过体重下降分数、疾病活动度指数以及结肠组织病理评估疗效，蛋白芯片、免疫印迹等分子生物学检测方式筛选通路，最后结合基因敲除小鼠以及结肠来源的类器官模型，验证脆弱拟杆菌ZY-312在实验性结肠炎中的调控机制。实验结果显示，脆弱拟杆菌ZY-312在DSS诱导的实验性结肠炎中可减轻体重下降分数、疾病活动度指数以及结肠缩短程度，并且降低了结肠上皮脱落以及炎性细胞浸润程度。相应地，免疫组化结果显示脆弱拟杆菌ZY-312上调结肠上皮增殖指标KI-67的表达，这提示脆弱拟杆菌ZY-312可调控结肠黏膜增殖修复进而缓解小鼠实验性结肠炎。结合蛋白芯片及免疫印迹结果发现脆弱拟杆菌ZY-312参与调节JAK-STAT典型增殖通路，促进结肠上皮STAT3磷酸化（pSTAT3），因此该团队构建了结肠上皮STAT3的特异性敲除小鼠（stat3△IEC）模型并验证脆弱拟杆菌ZY-312缓解实验性结肠炎依赖于结肠上皮STAT3。结合在模拟小鼠肠道微环境的体外类器官模型中发现脆弱拟杆菌ZY-312介导的pSTAT3上调依赖于结肠固有层免疫细胞。依据文献报道IL-22、IL-6、IL-10等炎性因子可参与激活STAT3磷酸化，因此该团队将结肠组织来源的上清进行酶联免疫吸附实验检测发现脆弱拟杆菌ZY-312促进结肠组织IL-22的表达，并在IL-22全身性敲除小鼠（IL-22-/-）中验证了脆弱拟杆菌ZY-312介导IL-22缓解实验性结肠炎。最后通过流式细胞术初步检测了IL-22的上游免疫细胞来源并提示脆弱拟杆菌ZY-312可能促进结肠固有层的ILC3分泌IL-22。综合以上说明脆弱拟杆菌可能通过调控固有层免疫细胞ILC3分泌IL-22，进而激活结肠上皮STAT3磷酸化发挥结肠黏膜增殖修复功能，最终缓解实验性结肠炎。这为益生菌在炎症性肠病中的临床转化与应用提供新的潜在前景。

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