PepT1对MDP诱导草鱼肠道炎症反应的调控作用及分子机制研究

The oligopeptide transporter 1 (PepT1) plays a crucial role in bacterial-induced intestinal inflammation through transport bacterial peptides muramyl dipeptide (MDP) into intestinal epithelial cells. We previously found that PepT1 was involved in MDP-induced intestinal inflammation of grass carp. However, their related regulation network and mechanism remain mysterious. Therefore, this project will further intervene the expression of PepT1 to clarify the regulation role of PepT1 in MDP-induced intestinal inflammation through the analysis of intestinal morphology, MPO activity and expression level of inflammatory cytokines at molecular levels. By analyzing response relation between the expression of PepT1, intestinal inflammation, upstream transcription factors (NF-κB and STAT3) and downstream signal transduction molecules (NOD2 and RICK), we will further reveal the upstream regulatory mechanism and downstream signaling pathways of PepT1 in MDP-induced intestinal inflammation. The approval and successful implementation of this project will not only contribute to understanding the function and molecular mechanisms of PepT1 in MDP-induced intestinal inflammation, but also provide a new insight into the mechanisms of intestinal inflammation in fish.

小肽转运载体1（PepT1）通过介导肠道细菌代谢产物胞壁酰二肽(MDP)的跨膜运输，对动物肠道细菌性炎症反应发挥着关键的调控作用。我们前期研究表明PepT1参与了MDP诱导的草鱼肠道炎症反应，但对其调控网络和作用机制尚不清楚。基于这一科学问题，本项目拟进一步通过干预草鱼肠道PepT1的功能，分析MDP刺激前后肠道形态、MPO活性和炎症因子水平的变化规律，在分子水平系统阐明PepT1对MDP诱导肠道炎症反应的调控作用；通过研究草鱼PepT1表达、肠道炎症水平、PepT1上游转录因子（NF-κB、STAT3等）及下游信号转导分子（NOD2、RICK等）之间的响应关系，解析草鱼PepT1在MDP诱导肠道炎症反应中的上游调控机制和下游信号转导途径。本研究不仅有助于理解PepT1在草鱼肠道细菌性炎症中的功能及MDP诱导肠道炎症反应的分子机制，同时还将为解析鱼类肠道炎症发生的机制提供理论基础。

研究表明，细菌胞壁酰二肽/小肽转运载体1（MDP/PepT1）途径对哺乳动物细菌性肠道炎症反应发挥着关键的调控作用。然而，在低等脊椎动物鱼类中是否存在MDP/PepT1信号途径，其是否参与调节肠道炎症反应尚不清楚。因此本研究以草鱼为研究对象，1）利用现代分子克隆技术鉴定了草鱼肠道PepT1基因序列，通过同源建模的方法获得了蛋白空间结构，解析了草鱼PepT1对细菌二肽MDP分子结合模式及免疫应答规律，阐明了草鱼肠道PepT1对细菌MDP诱导肠道炎症反应的调控作用；2）克隆了草鱼PepT1基因上游调控序列，生物信息学方法预测了转录因子结合位点，利用双荧光素酶报告基因系统验证了转录因子对肠道PepT1的表达调控作用，研究了谷氨酰胺二肽、丁酸钠和三丁酸甘油酯在活体和离体条件下对草鱼肠道PepT1表达的营养调控规律，从而揭示了草鱼肠道PepT1的上游调控及营养干预机制；3）克隆并鉴定了草鱼肠道MAPK通路关键基因序列，利用细菌MDP诱导的肠炎模型研究了MAPK通路对免疫刺激的应答规律，通过特异性抑制剂干预MAPK信号通路活性，研究MDP刺激后对肠道炎症因子表达的影响，从而解析MAPK信号通路对MDP/PepT1诱导的肠道炎症反应的调控作用。本项目研究结果不仅为深入解析草鱼细菌性肠炎发生的分子机理提供理论基础，同时亦可为鱼类细菌性肠炎病害防控提供潜在的治疗靶点。