异氟烷基于TLR4/RISK/NF-κB调控糖尿病缺血性脑卒中后NLRP3炎症小体形成的机制研究

Stroke is common in diabetes mellitus (DM), and inflammation cascade reaction aggravates brain injury after stroke. It is unknown whether and how NLRP3 inflammasome plays an essential role in cerebral injury after stroke in DM. Our previous study showed that isoflurane alleviated inflammation response after cerebral injury via RISK pathway, as well as reduced the expression of NLRP3 inflammasome. Further study indicated that cerebral infarct volume was decreased in NLRP3 gene knockout mice after stoke. May isoflurane involved in regulating NLRP3 inflammasome formation, hence improve prognosis after stoke in DM? And what’s the mechanism of the regulation? Researches showed that isoflurane could activate NF-κB pathway and inhibit NIRP3 expression via mitochondrial autophagy. Furthermore, mitochondrial autophagy could be regulated by TLR4/NF-κB pathway. In our preliminary experiments, TLR4 was increased in DM mice, and isoflurane could induce the enhancement of autophagy related proteins in a DM stroke model. We therefore hypothesize that isoflurane inhibits NLRP3 inflammasome formation via a RISK/NF-κB pathway, which resulting in TLR regulation for the mitochondrial autophagy. We will establish stroke models in wild-type and NLRP3 or TLR4 genes knock-out DM mice, as well as the neuron-macrophage co-culture system to test our hypothesis via in vitro and in vivo experiments.

脑卒中患者常合并糖尿病（DM），炎症瀑布效应加重卒中后脑损伤。NLRP3炎症小体是否主导DM脑卒中后脑损伤及其调控机制尚未明确。前期研究结合文献表明：异氟烷通过RISK通路减轻炎症反应，降低脑损伤；异氟烷可诱导NLRP3下降；NLRP3敲除后小鼠脑卒中后脑损伤减轻。那么异氟烷是否调控NLRP3而改善脑卒中预后？如何启动？已知异氟烷可激活NF-κB，诱发线粒体自噬，抑制NLRP3炎症小体；而调控TLR4/NF-κB可影响线粒体自噬。预实验发现：DM小鼠TLR4表达增加；异氟烷增加DM小鼠脑卒中后自噬蛋白的表达。据此推测：DM合并脑卒中时，异氟烷通过TLR4调控RISK/NF-κB通路，诱发线粒体自噬，抑制NLRP3炎症小体。本项目拟构建基因敲除小鼠DM脑卒中模型，建立神经元与巨噬细胞共培养体系，通过体内外实验，阐明异氟烷通过TLR4/RISK/NF-κB途径调控NLRP3炎症小体的形成机制。

本课题建立糖尿病与MCAO模型，评估吸入麻醉药异氟醚ISO脑保护效应与NLRP3炎症小体的相关性，明确了NLRP3炎症小体活性及IL-1β生成在ISO预处理脑保护效应中的作用；明确RISK/NF-κB 通路、线粒体自噬在ISO脑保护中的作用及其与NLRP3炎症小体、IL-1β的相关性；明确ISO通过TLR4受体调控RISK/NF-κB 通路而影响线粒体自噬。.结果发现，糖尿病合并缺血性脑卒中时，ISO预处理通过调控巨噬细胞TLR4受体影响RISK/NF-κB通路，诱发线粒体自噬，进一步抑制NLRP3炎症小体的活性，降低IL-1β释放，减轻炎症反应，产生脑保护效应。.研究的科学意义：该研究结果有助于阐明ISO启动糖尿病CIRI中内源性炎症机制相关的脑保护效应，为更好防治此类患者围术期脑卒中及有效实施脑保护提供科学依据；为更好防治此类患者围术期脑卒中及有效实施脑保护提供科学依据；可为围术期此类患者其他重要脏器保护的研究及临床应用提供参考依据。.研究的现实意义：（1）围术期脑卒中事件影响患者安全及转归，合并糖尿病明显降低救治成功率并影响预后，加强围术期重要脏器尤其是脑功能的保护，对提高围术期安全并降低医疗费用具有重要意义。（2）心脏、颈动脉等手术中需临时阻断或降低脑血流，『人为』造成脑缺血，本课题有助于为此类患者选择何种方式的麻醉方法（如静脉麻醉或吸入麻醉）乃至围术期管理提供指导。（3）围术期脑卒中不良事件的主要原因是脑遭受不同程度的CIRI和/或缺氧损伤，故寻求简便可行、切实有效的防治方法，对降低围术期并发症和死亡率具有重要的科学、社会和经济意义。