基于“肺与大肠相表里”理论探讨通腑理肺汤通过PD-1/PD-L1信号通路干预脓毒症肠屏障损伤的作用及机制

Patients with sepsis are prone to intestinal barrier injury, which is the initiating factor of MODS, Targeting intestinal barrier injury is a key point to prevent the progression from sepsis to MODS,however,it needs further in-depth study.Based on the theory of " the lung associated with the large intestine ", the Tongfu Li Fei Decoction was established by the principle of combined treatment of lung and intestine,whitch can reduce the levels of TNF-α,IL-6 and HMGB-1 in sepsis, and show a good effect in regulating gastrointestinalfunction.The study confirmed that PD-1/PD-L1 signaling pathway can be expressed in intestinal epithelial cells, which is related to intestinal mucosal immune tolerance and cause to the decrease of intestinal barrier function by sepsis. It is presumed that Tongfu Li Fei decoction may be the protective effect of protecting septic intestinal barrier by reducing the expression of PD-1/PD-L1 signal pathway.The aim of this study is to observe the intervention effect of Tongfu Li Fei decoction on the rat model of intestinal barrier injury by the intestinal mucosal histopathology,ultrastructure, intestinal permeability and the expression of tight junction protein.Detection the level of PD-1/PD-L1 signaling pathway gene, protein expression and cytokine in intestinal epithelial cells.Part of the possible mechanisms was clarified by the rat model of intestinal barrier injury in the intervention of sepsis through Tongfu Li Fei Decoction, it has important guiding significance for the treatment of intestinal barrier injury in sepsis with traditional Chinese medicine.

脓毒症患者易发生肠屏障损伤,是导致MODS的始动因素,以脓毒症肠屏障损伤为靶点是防止脓毒症进展为MODS的关键环节，但有待深入研究。基于“肺与大肠相表里”理论，应用“肺肠合治”创立的通腑理肺汤，能降低脓毒症TNF-α、IL-6 、HMGB-1细胞因子水平，具有调节脓毒症胃肠功能作用，但具体机制尚待阐明。研究证实PD-1/PD-L1信号通路可在肠上皮细胞表达，与肠道免疫耐受及脓毒症导致肠黏膜屏障功能下降密切相关，推测通腑理肺汤可能是通过降低PD-1/PD-L1信号通路表达，来达到改善脓毒症肠黏膜屏障损伤的作用。本课题通过肠黏膜组织病理及超微结构、肠黏膜通透性及紧密连接蛋白表达，观察通腑理肺汤对脓毒症大鼠模型肠屏障损伤的干预作用；检测肠上皮细胞PD-1/PD-L1信号通路基因、蛋白表达及细胞因子水平，部分阐明通腑理肺汤干预脓毒症肠屏障损伤可能机制。对脓毒症肠屏障损伤中医药治疗具有重要的指导意义

脓毒症肠屏障损伤为靶点是防止脓毒症进展为MODS的关键点，PD-1/PD-L1信号通路可在肠上皮细胞表达，基于“肺与大肠相表里”理论，应用“肺肠合治”创立的通腑理肺汤（TFL），能降低脓毒症炎性细胞因子水平、调节其胃肠功能作用，推测TFL可能是通过降低PD-1/PD-L1信号通路表达，来达到改善脓毒症肠黏膜屏障损伤的作用，但干预作用及机制有待深入研究。实验分3部分：1.TFL对CLP诱导脓毒症肠屏障损伤模型大鼠的肠屏障的干预作用、对大鼠血清和肠组织细胞因子水平及PD-1/PD-L1信号通路的关键蛋白表达的影响。通过观察各组血浆FD4浓度、肠上皮细胞紧密连接蛋白、肠黏膜组织病理改变、肠组织和细胞炎性因子、PD-1/PD-L1信号通路表达。结论：TFL能够提高CLP诱导的脓毒症大鼠的存活率，上调脓毒症模型大鼠肠组织紧密连接蛋白ZO-1、Occludin-1、Claudin-1mRNA及蛋白的表达，修复脓毒症造成的肠屏障损伤；TFL能够减少CLP诱导的脓毒症大鼠的TNF-α、IL-1β和IL-6因子表达，增加IL-10因子表达，减轻炎症反应。TFL能够显著抑制CLP诱导的脓毒症大鼠肠组织中PD-1和PD-L1表达水平升高。2.TFL通过阻断PD-1/PD-L1信号通路对炎症因子的影响。大鼠外周血分离出DC细胞、CD8+T细胞和NK细胞，流式细胞仪分选后予炎性介质干预。结论：TFL能够逆转CLP诱导的SHP-2、PI3K和p-AKT上调作用，使DC细胞和CD8+T细胞的共培养系统中IL-6和TNF-α的浓度升高，SHP-2、PI3K和p-AKT的表达受到抑制，通过阻断PD-1/PD-L1信号通路来抑制炎症因子的释放。3.TFL对LPS诱导的THP1细胞保护作用与机制研究。通过LPS与THP1细胞构建体外炎症模型、检测TFL对THP1细胞的细胞毒性作用、细胞分泌炎性因子TNF-α和IL-6表达、细胞裂解液中PD-1/PD-L1信号通路的表达、细胞中miR-146a，miR-146b、miR-155的表达以及分别加入抑制剂作用后各细胞裂解液中PD-1/PD-L1信号通路的表达情况。结论:TFL能通过调控miR-146a，调节PD-1/PD-L1信号通路，保护LPS诱导的THP1细胞。本研究结果部分阐明TFL干预脓毒症肠屏障损伤机制，对脓毒症肠屏障损伤中医药治疗具有重要意义。