Caspase11非经典炎症小体在视网膜缺血再灌注损伤中的作用及分子机制研究

Inflammation is one of the important cause of retinal ischemia reperfusion (RIR) injury.However, the mechanism of how inflammatory cytokines are regulated is unclear. Caspase11 non-canonical inflammasomes can regulate the synthesis and secretion of inflammatory cytokines. But its role in RIR injury is unclear. Our previous study found that the number of retinal ganglion cells in Caspase11 knockout mice increased significantly compared with that in wild type mice after RIR injury. Based on the above assumption, this project is aimed to: ① To clarify the role of Caspase11 in RIR, retinal function and morphological changes will be compared between Caspase11 knockout mice and wild type mice after RIR injury. ② The molecular mechanism of Caspase11 in rat RIR will be revealed by RNA and protein detection techniques.③ The mechanism of Caspase11 will be further verified in vitro experiments. This project is expected to lay a theoretical foundation for the mechanism of inflammatory injury induced by RIR, and to provide new direction in the targeted therapy for RIR.

炎症是造成视网膜缺血再灌注（Retinal ischemia reperfusion, RIR）损伤的重要原因之一，但是炎症因子是如何被调控，其机制并不清楚。Caspase11非经典炎症小体可以调控炎症因子的合成与分泌，但是在RIR损伤中能否发挥作用并不清楚。本项目组前期研究发现RIR后，Caspase11基因敲除鼠视网膜神经节细胞数目较野生型鼠显著增加。由此我们推测Caspase11可能通过调控炎症因子介导RIR视网膜神经节细胞损伤。基于上述设想，本项目拟：①对比Caspase11基因敲除鼠和野生型鼠RIR后视网膜功能学和形态学改变，明确Caspase11在RIR中的作用。②利用RNA及蛋白检测技术揭示Caspase11在RIR中作用的分子机制。③进一步在体外实验中验证Caspase11作用机制。本项目的完成将为探究RIR中炎性损伤机制奠定新的理论基础，亦为RIR靶向治疗提供新的思路。

有关caspase11非经典炎症小体的研究目前主要集中在由外源的细菌诱导免疫细胞活化的领域，而体内的非外源微生物的损伤是否能活化caspase11非经典炎症小体，目前并不清楚。因此，我们通过小鼠视网膜缺血再灌注（ischemia/reperfusion，IR）模型，研究caspase11非经典炎症小体能否活化及可能的信号通路。本项目通过将 C57BL/6小鼠的眼内压升高到 110 mmHg，维持 60 分钟，从而诱导形成视网膜IR损伤模型。Western blot法分析IL-1β、NLRP3炎症小体相关蛋白、caspase11、IL-1α及HMGB1的蛋白表达。Caspase11基因敲除鼠视网膜IR损伤后，再次Western blot法分析IL-1β、caspase1、IL-1α及HMGB1的蛋白表达。研究发现，小鼠视网膜IR损伤后16h内，视网膜内caspase11、caspase1、IL-1β、IL-1α、HMGB1蛋白表达均由显著升高。而NLRP3和ASC蛋白表达无显著变化。Caspase11基因敲除后，IR损伤所致的这些蛋白的表达均下降。小鼠视网膜IR损伤可以诱导caspase11非经典炎症小体的活化。Caspase11活化后可以进一步活化caspase-1及其下游的IL-1β，caspase11的活化还同时可以诱导IL-1a和HMGB1蛋白表达的增加。