Th2介导的JAK-STAT信号通路活化在肠内营养保护肠道粘膜免疫屏障中的作用机制研究

Intestinal barrier dysfunction caused by the damaged intestinal mucosal immunity and the disruption of intestinal microflora balance is a main factor leading to infectious complication and even death of critically ill patients after trauma, infection and major surgery. Initial trophic enteral nutrition can maintain the intestinal mucosal immunity molecules and obviously decrease patients' infection morbidity, however, the exact mechanisms are still unclear. Previous evidence indicated that the decreased levels of Th2 cytokines due to the absence of enteral stimulation suppressed the activation of JAK-STAT pathway, and subsequently led to the dysfunction of intestinal mucosal immunity. We thus hypothesize that the activation of JAK-STAT pathway mediated by Th2 cytokines may play a crucial role in enteral nutrition protecting the intestinal mucosal barrier function and this process will be initiated when the dosage of enteral nutrition cross the threshold value. We investigate the changes and relationship among the levels of Th2 cytokines, the JAK-STAT pathway, intestinal mucosal immunity and intestinal barrier function in a mice model of parenteral and enteral nutrition, using gene knockout, intervention of JAK-1 agonist and inhibitor, immunological fluorescence, high-throughput sequencing and in vitro bacterial culture. We aim to explore the certain threshold dose of enteral nutrition upregulating the intestinal mucosal immunity and further discuss the underlying molecular mechanisms. Our results will provide rationale for the clinical enteral nutrition implementation and infection complication prevention in critical illness.

肠粘膜免疫损害及菌群改变引发的肠屏障功能障碍是创伤及外科大手术等重症病人发生感染甚至导致死亡的重要原因。早期肠内营养有助于上调肠粘膜免疫效应分子，降低感染的发生，但其中涉及的具体机制尚不清楚。前期研究表明缺乏肠内刺激时，Th2类细胞因子分泌减少，JAK-STAT通路活化受抑，导致肠粘膜免疫功能受损。因此我们提出假设：Th2类细胞因子介导的JAK-STAT信号通路活化在肠内营养保护肠屏障功能中发挥重要作用，且肠内营养存在剂量阈值启动肠粘膜免疫效应上调。项目采用小鼠肠外和肠内营养模型，通过基因敲除、JAK蛋白激动和抑制干预、免疫荧光、高通量测序鉴定肠道菌种和体外细菌培养等技术，探讨不同剂量的肠内营养刺激下Th2类细胞因子、JAK-STAT信号通路蛋白、肠粘膜免疫和肠屏障功能变化及其间相关性，明确肠内营养保护肠粘膜免疫的阈值剂量和分子机制，为肠内营养的临床应用和重症病人感染的防治提供理论依据。

肠粘膜免疫损害及菌群改变引发的肠屏障功能障碍是创伤及外科大手术等重症病人发生感染甚至导致死亡的重要原因。早期肠内营养有助于上调肠粘膜免疫效应分子，降低感染的发生，但其中涉及的具体机制尚不清楚。前期研究表明缺乏肠内刺激时，Th2类细胞因子分泌减少，JAK-STAT通路活化受抑，导致肠粘膜免疫功能受损。因此我们提出假设：Th2类细胞因子介导的JAK-STAT信号通路活化在肠内营养保护肠屏障功能中发挥重要作用，且肠内营养存在剂量阈值启动肠粘膜免疫效应上调。本项目采用小鼠肠外和肠内营养模型，通过基因敲除、JAK蛋白激动和抑制干预、免疫荧光、高通量测序鉴定肠道菌种和体外细菌培养等技术，探讨不同剂量的肠内营养刺激下Th2类细胞因子、JAK-STAT信号通路蛋白、肠粘膜免疫和肠屏障功能变化及其间相关性，本研究明确了肠内营养保护肠粘膜免疫的阈值剂量（EN达到40%目标剂量可以显著改善肠屏障）及其分子机制（EN可以活化JAK-STAT信号通路）。此外，由于临床上缺乏有效的措施应对肠外营养引起的肠屏障损伤，因此，我们用动物实验对比了在PN中分别添加不同脂肪乳剂（大豆油（n-6PUFA）和鱼油（n-3PUFA））以及TPN导致肠道菌群紊乱对肠道屏障及潘氏细胞功能的影响。结果表明鱼油组可以减轻肠道损伤且增加溶菌酶，RegIIIγ以及α-cryptdin 5的蛋白和转录水平，且其机制主要为上调IL-22/STAT3信号通路； TPN引起的肠屏障障碍与潘氏细胞功能受损是由菌群介导所产生，菌群改变引起色氨酸代谢异常进而影响环芳香烃受体（aryl hydrocarbonreceptor receptor (AhR)）的活化进而影响IL-22/STAT3信号通路从而影响肠屏障与潘氏细胞的功能。本项目为肠内营养的临床应用和重症病人感染的防治提供了重要的理论依据。