肠-淋巴-肺轴通过TLR4信号通路对SAP-ALI中性粒细胞活化的影响机制

Severe acute pancreatitis associated acute lung injury (SAP-ALI) is a common but critical illness with a considerable mortality. Underlying mechanisms are complex and poorly understood. Polymorphonuclear neutrophils (PMNs) activation is the key process in the pathogenesis of SAP-ALI. Recent studies indicated that the mesenteric lymph and the gut-lymphatic-lung axis may plays an important role in the development of SAP-ALI. We first establish an animal model of SAP-ALI in rats by 4% sodium taurocholate infusion into the pancreatic duct. The mesenteric lymph duct was ligated, or drainaged, and the mesenteric lymph samples collected from SAP rats was injected intravenously into normal recipient rats, or incubated in vitro with normal PMNs, to clarify the pathogenicity of SAP mesenteric lymph to ALI and PMNs activation. Then, lipidomics, differential proteomics, and bioinformatics analysis were performed to identify the biologically active mediators in the SAP mesenteric lymph. As an inflammatory lipid mediator, the free fatty acid（FFA）acted as the ligands of the Toll-like Receptor-4（TLR4）expressed on the PMNs, and potentially play an important part in the gut-lymphatic-lung axis. The in vivo and ex vivo PMN priming effect ( intracellular adhesion, respiratory burst, delayed apoptosis, and cytokine cascade) of cytotoxic lymph from SAP rats was observed in the TLR4-/- mice, TRIF-/- mice, Myd88-/- mice, and widetype mice, to investigate the role of FFAs, TLR4, and the downstream signal pathway in SAP-ALI. Finally, we try to block the FFA mediated lipotoxicity by different ways, such as lipase inhibitor orlistat, improved FFA-to-protein ratio, mesenteric lymph ligation or drainage. We look forward to providing a new strategy for the management of SAP-ALI.

重症急性胰腺炎相关性急性肺损伤（SAP-ALI）是临床常见危重症，死亡率高，发病机制亟待阐明。中性粒细胞（PMNs）活化是SAP-ALI的研究重点。国外研究表明，“肠-淋巴-肺轴”可能是SAP-ALI的致病通路。我们建立SAP-ALI动物模型，观察肠淋巴液（ML）阻断或引流对ALI的影响，观察SAP-ML输注对正常大鼠体内和体外PMNs的损伤效应。针对不同时段的SAP-ML，进行脂质代谢组学、差异蛋白质组学和生物信息学分析，鉴定SAP-ML中能诱发ALI的活性介质。针对前期研究发现的脂类活性介质：游离脂肪酸（FFA），和PMNs活化的关键受体-TLR4，利用TLR4、Myd88、TRIF突变小鼠，从动物水平和细胞水平，深入研究FFA-TLR4及下游通路诱导SAP肺PMNs活化（细胞粘附，呼吸爆发，凋亡延迟）的分子机制，和不同手段阻断FFA脂毒性的治疗前景，为SAP-ALI治疗提供新的策略。

本课题通过研究肠、淋巴、肺之间致病性活性介质释放的级联反应，探讨“肠-淋巴-肺轴”对 SAP 急性肺损伤和肺 PMNs活化的调节机制，从一个新的角度阐述 SAP-ALI 的发病机制，为 SAP-ALI 的诊断和治疗提供了新的靶点。遵循“科赫法则”，通过三方面的研究：①研究 ML 阻断、ML 引流对 SAP 大鼠急性肺损伤和肺 PMNs活化的影响，②研究 SAP-ML 诱导体外正常 PMNs活化、PMVECs 损伤的情况，③研究供体 SAP-ML 经中心静脉注射对正常受体大鼠的肺损伤效应，证实SAP-ML诱发了SAP急性肺损伤和PMNs活化。针对不同时段的 SAP-ML，进行脂质代谢组学、差异蛋白质组学及生物信息学分析，鉴定出 SAP-ML中能诱发 PMNs活化、加重肺损伤的致病性活性介质。探索这些活性介质在 SAP-ALI 发病机制中的可能作用。通过 PMNs 剔除实验，证实了PMNs活化在 SAP-ALI 发病机制中的作用。利用 TLR4-/-、Myd88-/-、TRIF-/-小鼠，发现TLR4及其下游 Myd88 和 TRIF通路在 SAP 肺损伤的作用，阐明 TLR4-TRIF通过介导 PMNs活化而导致SAP-ALI。通过不同手段阻断 FFA 脂毒性：脂肪酶抑制剂（Orlistat），调整FFA/albumin.ratio，ML 阻断或ML 引流，从而抑制 FFA-TLR4通路对PMNs活化的诱导，阻断致病性“肠-淋巴-肺轴”，达到改善 SAP-ALI的治疗目的。通过上述多个层面的研究，阐明“肠-淋巴-肺轴”通过FFA-TLR4-TRIF信号通路诱导 SAP-ALI 中性粒细胞活化的致病机制，为 SAP 治疗提供新的靶点和思路。