IL-17A活化小胶质细胞在脓毒症脑病中的作用与机制研究

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction that occurs secondary to infection in the body without overt CNS infection. SAE is a rapid decline in cognitive functions, especially memory. Septic patients with SAE showed higher mortality. Microglia activation plays critical role in the mechanism of SAE. IL-17A, a core pro-inflammatory cytokine, activates microglia and their interaction acts as a major mechanism in neuro-inflammation associated diseases, such as multiple sclerosis, Alzheimer disease and brain injury. So far, whether the change of IL-17A and the activation of microglia by IL-17A invlolved in SAE was poorly understood. We have reported that the earlier and higher elevated IL-17A derived from gammadelta T cells in peritoneal fluid was very important during sepsis and blockade of IL-17A decreases proinflammatory cytokine production, neutrophil infiltration, and lung injury, thereby improving septic mice survival. Our preliminary experiment found that IL-17A in sepsis mice cerebrospinal fluid elevated, and there were the similar tendency in TNF-α concentration detection. We speculate that IL-17A also activates microglia which results in the magnified inflammation in SAE. Our research will elucidate role of activation of microglia by IL-17A and the following biological changes in SAE with the experimental sepsis model and mcroglia derived from septic mouse. The research will also use Laser scanning confocal microscope combined with immunohistochemical double labeling, flow cytometry, anti-IL-17A and anti-IL17 receptor antibody administration to elucidate the IL-17A induced microglia activation in SAE.

脓毒症脑病（sepsis-associated encephalopathy，SAE）是脓毒症全身炎症反应所致弥漫性脑功能障碍和意识改变，合并SAE时脓毒症病死率明显升高。以小胶质细胞激活为标志的中枢炎症反应在SAE中起重要作用。IL-17A促炎作用显著，其和小胶质细胞在中枢炎性疾病（多发性硬化、老年痴呆、创伤等）中扮演重要角色。SAE中IL-17A变化与小胶质细胞IL-17受体表达、对细胞活化及认知功能影响和调节机制知之甚少。预实验发现脓毒症小鼠脑脊液IL-17A升高，且TNF-α上调也与之趋势相同，推测IL-17A是SAE中激活小胶质细胞的重要分子，进而影响SAE进展。为此本研究拟通过脓毒症模型及原代小胶质细胞，采用免疫荧光、流式细胞、IL-17A及其抗体、IL-17受体抗体干预等方法，多水平层面探讨IL-17A活化小胶质细胞在SAE中的作用与机制，为SAE防治提供新思路。

脓毒症脑病（sepsis-associated encephalopathy，SAE）是脓毒症全身炎症反应所致弥漫性脑功能障碍和意识改变，合并SAE时脓毒症病死率明显升高。本研究着眼于SAE发病机制中的一个方面，即以小胶质细胞激活为标志的中枢炎症反应，及其进而在SAE中起到的起重要作用，对这方面的可能机制进行了相关的探索。研究设计基于细胞因子IL-17A显著的促炎作用，IL-17A和小胶质细胞在中枢炎性疾病中扮演重要角色这一既往研究结论。结合实验前期的初步探索，最终聚焦于SAE中IL-17A变化与小胶质细胞IL-17受体表达、对细胞活化及认知功能影响和调节机制这一具体生物反应途径，推测IL-17A是SAE中激活小胶质细胞的重要分子，进而影响SAE进展。. 本研究在进行过程中，通过C57小鼠盲肠结扎穿孔致脓毒症模型，以及模型小鼠原代小胶质细胞，采用免疫荧光、流式细胞等方法，对各组小鼠体内、体外IL-17A-IL-17R-小胶质细胞活化这一途径中各分子变化进行了探索，并应用IL-17A及其抗体、IL-17受体抗体干预等方法，多水平层面探讨IL-17A活化小胶质细胞在SAE中的作用与机制，期望为SAE防治提供新思路。. 研究结果发现，接受了CLP手术的小鼠出现了明显的认知功能受损，并且其认知功能受损的情况与小鼠脑组织内IL-17A, IL-1β 及TNF-α水平以及小胶质细胞活化情况相关。CLP手术致脓毒症后，小鼠脑内小胶质细胞明显激活，而在体应用anti-IL17A 或anti-IL-17R抗体后，脓毒症小鼠脑内小胶质细胞活化程度降低，脑内炎症水平降低，同时认知功能也得到改善；体外研究提取假手术和脓毒症小鼠脑内小胶质细胞，并应用IL-17A予以刺激，发现IL-17A确实可以促使脓毒症小胶质细胞活化并分泌更多促炎细胞因子。. 通过本研究的结果，证明脓毒症过程中脑内IL-17A水平变化是小胶质细胞活化微环境中的重要启动因子，是SAE的重要原因之一；实验通过干预IL-17A—IL-17R途径，有效减轻了脓毒症小胶质细胞活化，并一定程度上改善了脓毒症脑病所致认知功能障碍，为脓毒症脑病的机制研究和防治策略提供了一定的研究依据。