基于自噬调控NLRP3炎症小体活化探讨机械通气致脑功能障碍的作用机制

The protection of vital organs after mechanical ventilation （MV）in intensive care unit is one of the clinical hot topic. Lung-brain interaction is the important mechanism of acute cognitive impairment induced by MV. Autophagy at neurons was closely associated with inflammatory response in cognitive impairment and autophagy regulated the activation of NLRP3 inflammasome, which upregulate the expression of IL-1β, were reported. Our previous studies have found that MV resulted in the autophagy at hippocampal neurons and the increase of NLRP3 inflammasome in hippocampus. However, the specific regulatory mechanism remains unclear. Therefore, we hypothesized that regulation of NLRP3 inflammasome activation by autophagy is one of the key mechanisms about cerebral dysfunction triggered by MV. To test this hypothesis, this proposal choose selective NLRP3 gene knockout mice, co-culture of microglia and neuron, after autophagy agonists and inhibitors are applied, to disclose the mechanism of autophagy-regulated NLRP3 inflammasome activation in cerebral dysfunction induced by MV through animal ethology, molecular biology, immunofluorescence, confocal laser, electron microscope and flow cytometry instrument technology. It will provide new targets and strategies for the protection of central nervous system of mechaincally ventilated patiants in intensive care unit.

重症监护病房机械通气（MV）后重要脏器的保护是临床关注的热点问题之一，MV可通过肺脑交互作用诱发中枢急性认知损害。文献报道，神经元细胞自噬与认知损害炎症反应密切相关，自噬调控NLRP3炎症小体的激活上调IL-1β表达。前期研究发现，MV可导致海马神经元自噬发生，海马组织NLRP3炎症小体表达增加，但具体调控机制未明。因此我们假设自噬调控NLRP3炎症小体活化是MV致脑功能障碍的关键机制之一。本项目将拟通过选择性NLRP3基因敲除小鼠、神经小胶质细胞和神经元细胞的共培养为研究对象，应用自噬激动剂和抑制剂，采用动物行为学、分子生物学、免疫荧光法、激光共聚焦、电镜及流式细胞仪等技术探讨自噬调控NLRP3炎症小体活化在机械通气致脑功能障碍中的作用机制，为ICU机械通气患者的中枢神经保护提供新靶点和新策略。

机械通气可诱发谵妄和认知损害，住院时间延长以及并发症增加，已成为麻醉和危重症领域亟待解决的问题。前期研究提示中枢炎症反应的启动在机械通气致脑损伤中的重要作用，然而具体的调控机制仍不清楚。因此，本项目研究自噬调控NLRP3炎症小体在机械通气致脑损伤的作用机制。本研究首先观察长时间机械通气诱发小鼠急性脑损伤后学习记忆能力等行为学的变化，检测NLRP3炎症小体和自噬的变化，证实了机械通气后1d小鼠海马LC-3I、LC-3II、NLRP3炎症小体（NLRP3、ASC、Caspase-1）表达水平增加，海马神经元凋亡增加，揭示了长时间机械通气诱导中枢NLRP3炎症小体活化致肺脑交互损伤。其次，观察自噬激动剂和抑制剂对机械通气小鼠NLRP3炎症小体的影响，我们发现调控自噬后，机械通气小鼠NLRP3炎症小体活化被抑制，阐明了自噬与NLRP3炎症小体的关系。本项目从“机械通气后急性脑功能障碍”这一临床问题出发，揭示NLRP3炎症小体在机械通气致急性认知损害中的作用，阐明了阻断机械通气中肺脑交互过程，为机械通气后重要脏器肺、脑保护提供特异性干预靶点，为机械通气患者急性脑损害的防治提供新策略。