益生菌E.faeciumL3联合溃结安治疗溃疡性结肠炎的相关分子机制研究

Ulcerative colitis (UC) is a non-specific inflammation of intestine. Although its etiology and pathogenesis is not yet fully clear,We found in the previous study, colon tissue of UC rats: ① appear metabolic disorders ,butyric acid, acetic acid, propionic acid and other short-chain fatty acids increased, indicating short-chain fatty acid oxidation defects appear intestinal flora disorders and metabolic disorders ,② IL-1β, TGF-β1 , TRL2, STAT3, JAK2 and other gene expression increased, indicating that cytokine balance disorders and TLR / JAK-STAT signaling pathway abnormalities.Therefore, we suggest that intestinal flora imbalance may affect the cytokine balance through the TLR / JAK-STAT pathway, leading to metabolic disorders and inflammation. Therefore, we use probiotics E.faecium L3 combined with ulceration (KJA) treatment of rat UC, in terms of symptoms and pathology has achieved significant effect, but the mechanism is not clear. On the basis of previous research, this project will explore the intrinsic relationship between intestinal flora, metabolism, T cell subsets and TLR / JAK-STAT inflammatory signaling pathway, and analyze the pathogenesis of UC and the mechanism of drug combination. Provide provide a theoretical basis for the clinical treatment of UC.

溃疡性结肠炎(UC)是一种病因及发病机制不明确的肠道慢性非特异性炎症。我们在前期研究中发现， UC大鼠结肠组织中：①丁酸、乙酸、丙酸等短链脂肪酸的含量增加，提示短链脂肪酸氧化缺陷，存在肠道菌群失衡及代谢紊乱现象；②IL-1β, TGF-β1、TRL2、STAT3、JAK2等多种基因表达水平上调，提示细胞因子平衡及TLR/ JAK- STAT炎症信号通路发生紊乱；根据本课题组的研究结果结合国内外文献报道我们提出：肠道菌群失衡可能通过TLR/JAK- STAT炎症信号通路影响细胞因子平衡，导致代谢的紊乱及炎症的发生。基于此我们用益生菌E.faecium L3联合溃结安（KJA）治疗大鼠UC，发现联合药物的疗效更显著，但机制不明确。因此本项目通过肠道菌群、代谢、T细胞亚群、TLR／JAK-STAT炎症信号通路的内在联系,进一步分析UC的发病及联合药物的作用机制，为UC的诊治提供实验及科学依据。

通过嗜酸乳杆菌和复方缓溃乐混悬剂联合干预2、4、6-三硝基苯磺酸（trinitrobenzene sulfonic acid，TNBS）诱导的大鼠溃疡性结肠炎（Ulcerative colitis，UC），分析联合干预对UC大鼠肠道菌群、免疫和血清代谢物的影响，探讨UC发病以及联合干预的分子机制。UC大鼠结肠组织中促炎因子表达量上调而抗炎因子表达量下调，从而促炎因子和抗炎因子间的平衡关系被打破，促进UC的发生发展。联合干预使UC大鼠结肠组织中IL-4、IL-13、TGF-β和IL-10的mRNA和蛋白表达水平明显上调，说明联合干预的抗炎效果比较明显，同时联合干预抑制UC大鼠结肠组织中TLR4、TLR9、jak2、stat1蛋白的表达。UC组大鼠肠道菌群丰度下降，但变形菌门中的大肠埃希菌.志贺菌属、Klebsiella等条件致病菌丰度升高。各干预措施均会影响UC大鼠肠道中厚壁菌门、拟杆菌门和变形菌门的分布，其中联合干预组优势菌门的分布最接近于正常组，说明联合干预的调控效果优于单用药物。UC组大鼠体重下降、体质虚弱、腹泻等症状可能与厚壁菌门中的Romboutsia菌属的降低有关；厚壁菌门中的粪球菌属在UC组中显著富集；放线菌门中的双歧杆菌在LH组中显著富集且双歧杆菌与IL-13和TGF-β呈正相关，而与IFN-γ呈负相关，提示联合干预通过双歧杆菌影响TGF-β和IL-13的表达而发挥抗炎作用。联合干预可以通过增加Ruminococcaceae_UCG_014、Ruminococcaceae_ UCG_010、Romboutsia等短链脂肪酸产生菌来缓解UC组大鼠结肠上皮所出现的能量不足状态；UC组大鼠存在能量代谢障碍，具体体现在乳酸、甘氨酸和肌酸含量的升高和葡萄糖、丙氨酸、牛磺酸含量的下降，其与肠黏膜中Romboursia等短链脂肪酸产生菌的丰度下降相关。UC组大鼠出现能量代谢、氨基酸代谢和脂质代谢紊乱。Klebsiella丰度的升高是UC大鼠代谢紊乱的一种重要信号，Klebsiella与乳酸、肌酸、甘氨酸水平呈负相关，而与丙酮水平呈正相关，联合干预通过抑制Klebsiella菌属的生长来影响乳酸、肌酸和甘氨酸代谢，对UC大鼠肠道菌群紊乱所引起的代谢变化有一定的调控作用；联合干预通过促进Th17/Treg细胞间的平衡调节免疫。