胰高血糖素样肽1调控烧伤脓毒症免疫反应的信号转导机制

Burn-induced sepsis is characterized by metabolic disorder and immune dysfunction, resulting organ failure and even death. The novel diabetic drug, glucagon-like peptide 1 (GLP-1) and its analog Exendin-4 have been in the trial in critical care. Unexpectedly, we found that although Exendin-4 could reduce the level of blood glucose, it could markedly increase the mortality and affect the immune function as demonstrated with early augmented proinflammatory response, and later immune paralysis. Firstly, we perform the study in the mouse model of burn-injury to observe if the modulation of monocyte by Exendin-4 could be changed in acute insults, and if it is associated with the abnormality in its signaling pathway, especially the imbalance of monocyte Gsα/Gαi. Then we will investigate if Exendin-4 could affect the monocyte differentiation to result in the late immunosuppression. Finally, our hypothesis is further verified in peripheral monocytes isolated from patients with major burns. The purpose of the project is to elucidate the signaling pathway underlying the mechanism for Exendin-4 to work on the immune response secondary to major burns, and to provide warranty for clinical pratice.

烧伤脓毒症以代谢紊乱和免疫障碍为主要特征，导致器官衰竭甚至死亡。新型糖尿病药物胰高血糖素样肽-1（GLP-1）及其类似物Exendin-4已进入危重症临床试验。我们意外发现Exendin-4虽然能降低烧伤小鼠血糖，但却促进其死亡，同时影响免疫功能，动物在早期出现过度炎症反应，而在后期则产生免疫麻痹。本课题拟采用小鼠烧伤模型，进一步观察在严重烧伤中Exendin-4对单核细胞的调控作用是否发生改变？是否与GLP-1信号转导通路异常，尤其是关键分子Gsα/Gαi失衡相关？针对后期出现的免疫抑制状态，探究Exendin-4是否通过影响单核细胞分化而产生免疫抑制作用；最后，采用烧伤患者单核细胞加以验证。本课题旨在阐明Exendin-4影响严重烧伤后免疫反应的信号转导机制，为其临床应用的潜在风险提供预警和可能对策。

本课题采用小鼠15%体表面积Ⅲ°烫伤模型，首先比较胰高血糖素样肽-1（GLP-1）长效类似物Exendin-4（Ex-4）对烫伤和假伤小鼠单核细胞免疫功能和信号转导的不同影响，再通过miRNA芯片检测烫伤小鼠脾脏单核细胞差异表达miRNA。其次通过信号转导阻断剂探讨Ex-4对烫伤小鼠单核细胞产生不同免疫调控作用的信号转导途径，阐明Ex-4促炎作用的机制。最后通过β肾上腺素能受体阻断剂普萘诺尔探讨Ex-4调控小鼠T细胞功能和炎症反应的交感神经机制。.研究发现烫伤小鼠单核细胞对Ex-4的免疫抑制作用产生抵抗，表现为促炎因子TNF-α水平升高，抑炎因子IL-10升高延迟。相应地，体外Ex-4刺激烫伤单核细胞cAMP水平并不增加，而1h时PKA活性却意外增强，提示烫伤单核细胞有其它信号通路被激活。进一步研究发现，烫伤小鼠脾脏单核细胞GLP-1R和GRK2表达减少，Gαi/Gαs失衡，造成烫伤单核细胞Ex-4的信号传导由Gαs向Giɑ偏移，激活促炎的MAPK通路，使TNF-α水平升高。.Ex-4在体内和体外对T淋巴细胞功能和分化产生不同作用：在体外能够促进假伤和烫伤小鼠脾脏T淋巴细胞向Th2漂移，而对T细胞功能没有影响，表明Ex-4是通过体内机制影响T细胞功能。体内预先注射普萘诺尔，发现可以阻断Ex-4对脾细胞核内NF-κB的激活作用；而NF-κB调控的下游分子，如肺和血清TNF-α、MCP-1水平在Ex-4处理的烫伤小鼠升高，但并不被普萘诺尔阻断。体内Ex-4对烫伤小鼠T细胞功能，如脾细胞计数、T细胞增殖和IL-2分泌的抑制作用可被普萘诺尔阻断；体内Ex-4对Th1/Th2的分化作用不受交感调控，而是通过GLP-1R-cAMP途径抑制T细胞的核因子（NFAT），促进T细胞向NFAT的下游调控因子Th2漂移，如IL-10和IL-4的分泌增加。.本研究阐明了GLP-1对烫伤小鼠产生促炎作用的神经内分泌和信号转导机制，即GLP-1通过交感途径抑制烫伤小鼠T细胞功能，而通过细胞内信号传导途径影响其分化；由于烫伤小鼠单核细胞Gαi/Gαs失衡，Ex-4从抑炎的GLP-1R→Gsα→cAMP→ PKA→↓TNF-α向促炎的GLP-1R→Giα→MAPK→ ↑TNF-α偏移，从而引起全身的炎症反应。我们的研究为GLP-1类胰泌素在危重症的临床应用提供预警。