基于CD4+T细胞探讨新金汁（粪菌移植）干预湿热型溃疡性结肠炎大鼠的机制

Studies indicated that the structure of intestinal flora of patients with IBD changed significantly and involved in CD4+T cell-mediated immune response. The latest evidence-based medical research and our previous animal experiment pointed out that Fecal microbiota transplantation (FMT) for the treatment of UC has a certain clinical efficacy, but the mechanism is unknown. It was reported that FMT is inherited and innovated from traditional Chinese medicine (TCM) gold juice and can be regarded as a new gold juice and should be affirmed as "bitter and cold" attribute. This study is to analyze FMT from the perspective of traditional Chinese medicine for the first time, the feces of healthy rats were used as the donor source, Damp-heat syndrome and common-type model of rats with UC were established respectively by means of DSS-induced + high fat and sucrose feeding + hot-humid environment and DSS-induced method, to study the efficacy of FMT on UC by disease activity index and histopathological score in rats, To compare the curative effect index of FMT on the two model rats, the changes of the differentiation characteristics of CD4+T lymphocyte subsets (Th1, Th2, Th17 and Treg) and the related transcription factors and cytokine expression profiles in rats intestinal mucosa after FMT intervention were analyzed by flow cytometry and ELISA respectively, to confirm the herb attribute (bitter cold) of FMT and its possible immunological mechanism of intervention UC, in order to provide TCM and immunological theoretical support for the clinical use of FMT.

研究指出溃疡性结肠炎（UC）患者肠道菌群发生显著结构改变，参与了CD4+T细胞介导的免疫反应。最新循证数据和我们前期动物实验表明粪菌移植（FMT）治疗UC具有一定的疗效，但机制不明。FMT是对中药金汁继承和创新而来，可视为一种新金汁，理当具有“苦寒”性味，但有待证实。本研究首次从中药角度立意分析FMT，拟以健康大鼠粪便为供体，通过葡聚糖硫酸钠（DSS）诱导+高脂高糖喂养+湿热环境的方式和单纯DSS诱导的方式分别建立湿热型和普通型UC大鼠模型，研究FMT对大鼠疾病活动指数、组织病理积分的影响；比较FMT对两种模型大鼠的疗效指数；通过流式细胞和ELISA分析结肠组织内CD4+T细胞亚群（Thl,Th2,Thl7和Treg）分化及其转录因子和细胞因子表达的变化，旨在证实FMT的性味（苦寒）和其干预UC可能的免疫学机制，以期为FMT的临床运用提供中医和免疫学理支持。

项目背景：研究指出，IBD 患者肠道菌群结构发生显著改变，有益菌减少，有害菌增多，从而抑制 Treg 细胞，激活 Thl 和 Th17 介导的免疫反应，进而失去免疫耐受功能。另有实验表明肠道菌群的改变可能激活 IL-33 和 Th2，激活肠道免疫反应，诱导炎症产生。国内动物实验也指出，FMT 对小鼠实验性结肠炎具有一定的治疗效果，可能通过重建肠道菌群，调节肠道 T 细胞免疫稳态来发挥治疗作用。然而，CD4+T 细胞主要亚群（Thl,Th2, Thl7 和 Treg）及其转录因子和细胞因子如何参与 FMT 干预 UC，是单独还是协同发挥作用，具体机制目前仍不清楚，有待进一步研究。重要结果：根据HE染色病理学、透射电镜超微结构分析，FMT对CUCM与DUCM两种UC模型小鼠均有显著的疗效，且对DUCM组效果优于CUCM组。流式检测结果表明FMT可以改善T细胞比例，使DUCM模型组中Th1细胞显著减少，Th2细胞显著增多。血常规表明FMT可以使UC模型中WBC、PLT下调，RBC、HGB上调，并且DUCM组的各项指标改善均优于CUCM组。疗效指数分析FMT可以使UC模型组中的疾病活动指数均较治疗前降低，且DUCM组的疗效指数优于CUCM组。同时16s菌群多样性结果也显示FMT可以使UC模型中的菌群趋于正常。FMT治疗UC模型小鼠效果明显，并且湿热型UC模型组（DUCM）疗效优于普通型UC模型组（CUCM）。总而言之，FMT对DUCM和CUCM组模型小鼠均具有不错的疗效，对DUCM组模型小鼠疗效更好。本研究为FMT治疗UC提供了理论依据，也首次采用新金汁中药属性苦寒针对性的治疗湿热型UC的研究，为新金汁的中药性味苦寒提供了理论依据，但还需更进一步深入的研究。