基于代谢组学的清肠化湿方上调真杆菌调整肠道菌群影响NLRP6炎症小体治疗溃疡性结肠炎机制研究

The lack of intestinal barrier integrity leads to the increase of bacterial antigen translocation, and stimulate the inflammatory response of intestinal mucosa, which is the core pathological feature of UC. Intestinal flora and its metabolites play an important role in the development of ulcerative colitis by regulating the activation of inflammasome and promoting the expression of inflammatory factors and intestinal mucosal injury. Qingchang Huashi recipe is an effective prescription for treatment of ulcerative colitis. Previous studies have shown that it can reduce the intestinal immune inflammatory response and restore the injured intestinal mucosa; The macrogenomic method was used to detect the intestinal flora of UC patients. The results showed that abundance and quantity of Eubacterium in active phase of UC patients compared with healthy people, and this phenomenon was closely related to the expression of CHBP gene. Based on the previous research results, this project is combined with microbial genomics, genomics and metabolomics around the intestinal flora (Eubacterium), CHBP gene and inflammasome to study mechanism of Qingchang Huashi recipe regulated mucosal immunity, reconstruction the intestinal mucosal barrier to treat the ulcerative colitis from the perspective of intestinal flora and immune angle. Therefore, it provides scientific basis for improving the curative effect of Qingchang Huashi recipe in the treatment of ulcerative colitis.

肠道屏障完整性缺失导致细菌抗原易位增加，刺激肠道黏膜的炎症反应，是溃疡性结肠炎的核心病理特征。肠道菌群及其代谢产物通过调节炎症小体的活化，促进炎症因子表达和肠黏膜损伤，参与了溃疡性结肠炎的发生发展。清肠化湿方是临床治疗溃疡性结肠炎的有效处方，前期研究表明能够减轻肠道免疫炎症反应，修复受损肠黏膜；采用宏基因组方法检测溃疡性结肠炎患者肠道菌群，结果显示活动期患者真杆菌的丰度和数量显著降低，且与CHBP基因的表达量密切相关。本项目基于前期研究结果，采用微生物组学、基因组学联合代谢组学的多组学方法，从肠道-菌群-免疫角度，围绕肠道菌群（菌株）、CHBP基因、炎症小体研究清肠化湿方调节黏膜免疫、重建肠黏膜屏障治疗溃疡性结肠炎的的作用机制，从而为优化处方提高溃疡性结肠炎的临床疗效提供科学依据。

溃疡性结肠炎（ulcerative colitis, UC）发病机制涉及肠道菌群和代谢紊乱、肠道屏障损伤、免疫功能失衡等。清肠化湿方（QCHS）治疗UC临床有效，但其作用机制和及功能靶标仍需进一步探索。本研究围绕肠道菌群及其代谢、NLRP6炎症小体信号通路、免疫细胞平衡等对清肠化湿方的作用机制进行了深入研究，结果如下：（1）证实了NLRP6是清肠化湿方调节UC小鼠肠道免疫的可能靶标：QCHS显著减轻了DSS诱导的小鼠结肠炎并修复结肠黏膜屏障，维持肠系膜淋巴结中的Th17/Treg平衡抑制UC小鼠结肠炎症，且能够激活NLRP6信号通路，调节IL-1β和 IL-18成熟；利用湿热证UC病证结合小鼠模型，结果表明QCHS可能促进Nlrp6的表达，抑制caspase-1、IL-1β的表达；体外研究也表明QCHS含药血清能够抑制LPS诱导的细胞炎症并激活NLRP6通路；（2）证实了清肠化湿方能够调节UC小鼠肠道菌群：DSS诱导后UC小鼠粪便菌群中部分属水平的真杆菌相对丰度相较于对照组显著下调，但清肠化湿方干预之后，真杆菌相对丰度变化不显著。在属水平上，QCHS明显降低了Romboutsia和Turicibacter 相对丰度，显著增加了乳杆菌Lactobacillus相对丰度，尤其是格氏乳杆菌（L.gasseri）相对丰度；（3）代谢组学结果显示清肠化湿方重塑UC小鼠肠道代谢谱并影响铁死亡代谢通路：UHPLC-MS非靶向代谢组学检测QCHS对UC小鼠粪便代谢组的影响，QCHS处理后小鼠粪便中有311种代谢物发生变化，QCHS显著提高了铁死亡核心物质谷胱甘肽GSH的水平；（4）有效成分黄芩苷通过激活NLRP6信号改善DSS诱导的小鼠结肠炎，调节Th17/Treg细胞平衡，减轻免疫炎症反应，调整肠道菌群丰度及结构，促进细菌分解代谢SCFAs；（5）此外，本研究发现了L.gasseri与铁死亡代谢之间的直接相关性，并且L.gasseri可以作为潜在的铁死亡抑制剂来减轻小鼠UC，体外实验还显示L.gasseri活菌可以激活NLRP6信号通路。综上，通过本研究阐明了清肠化湿方治疗溃疡性结肠炎的肠道微生态机制及代谢组学基础，为清肠化湿方及其效应组分治疗UC的临床应用提供了进一步的科学依据。