PKR/STAT3介导线粒体自噬清除异常磷酸化tau蛋白在POCD中的信号传导机制

Postoperative cognitive dysfunction (POCD) is a complication characteristic as a decline in cognitive performance after major surgery and general anesthesia in all ages. Profound systemic inflammation or neuroinflammation mediated by overwhelming inflammation, which might contribute to the overall severity of neurological injury as the causative factor for cognitive decline after peripheral surgery, however, the mechanisms for POCD remain unclear. In our previous study, we found the significant hyperphospohrylation of tau and mislocalization in the neuronal neucleus after laparotomy, associated with chronic neuroinflammation, which finally caused neuronal apoptosis. The inhibiton of STAT3 activity may contribute the critical role in the progress of POCD. For the further pilot study, we found the activation of PKR, which inhibits the transcriptional activity of STAT3 by direct interaction. And the mitophagy is suppressed simutaneously. In this project, laparotomy will be performed to wild type and PKR-/- mice to establish POCD in vivo. For the in vitro part, mix culture of primary neurons and glia will be used to determine the effect of mitophagy on tau clearance via the signal transduction of PKR/STAT3.The expression or activity of tau, PKR/STAT3 in different cellular compartments will be determined by series of biochemical assays, including western blotting analysis, multiple immunofluorescence staining with or without specific gene silencing or overexpression. And mitochondria function, oxidative stress, apoptosis and mitophagy will be examined as well. So that our proposed studies will strengthen our understanding on the molecular mechanism underlying POCD, and further facilitate the optimal therapy strategy for POCD.

术后认知功能障碍（POCD）是重要脏器手术和麻醉后的一种严重并发症，可影响患者的长期预后，但机制尚不完全明确，多认为与神经系统炎症有关。本课题组前期研究发现，海马组织中的tau蛋白过度磷酸化和核内错位聚积参与了POCD的进展，伴有持续性的神经系统炎症反应，引起神经元凋亡，STAT3在细胞核内的转录活化抑制可能是一个关键。研究证实，细胞自噬是清除异常磷酸化tau蛋白的主要途径，STAT3可以在转录后调控癌细胞中的自噬相关基因表达。我们的预实验发现，POCD中PKR活性增强，负性调控STAT3，线粒体自噬受到抑制。本项目拟在野生型和PKR-/-小鼠，原代神经元、胶质细胞混合培养两个模型上，利用分子生物学方法，多重免疫荧光显微成像、基因过表达/沉默、双光子激光共聚焦活细胞显微成像技术深入探究PKR/STAT3在剖腹探查术引起的POCD中的信号传导机制，为临床防治POCD提供新思路。

越来越多的证据表明，术后认知功能障碍（POCD）极大的增加了术后死亡率、住院率和社会成本。长期认知能力下降与高龄、先前存在的轻度认知障碍、术后谵妄或术后感染显著相关。本项目在前期研究基础上，利用剖腹探查术诱导的POCD模型，针对不同年龄阶段的野生型和痴呆模型小鼠的空间学习和记忆能力测试发现：①剖腹探查术显著降低各个年龄阶段的小鼠的认知能力，并同时存在海马神经元的氧化应激损伤，表现为持续的中枢神经系统炎症反应（包括促炎因子水平的增加和胶质细胞的激活），抗炎治疗或激动胆碱能受体均能显著改善行为学和分子生物学的改变；②tau蛋白的异常磷酸化不仅仅聚集在细胞质，还大量沉积在细胞核中，并受线粒体自噬的影响，PKR和STAT3相互作用后调控细胞核和线粒体内的异位的磷酸化tau蛋白的清除。而这些改变在特异性基因敲除的PKR-/-小鼠中显著减轻甚至消失，表明PKR在POCD发生发展中的重要作用。拓展研究线粒体自噬在认知功能障碍中的作用发现，脑缺血再灌注损伤（CIRI）的小鼠与POCD有着类似的空间学习及记忆水平的降低，在野生型小鼠海马组织、HT22小鼠海马神经元和原代大鼠海马神经元中，均发现CIRI引起的广泛的氧化应激反应、线粒体功能障碍和神经元凋亡，均可被SIRT1抑制剂显著逆转，表明SIRT1在线粒体自噬中起着显著的调控作用。本项目的一系列研究提示，中枢神经系统炎症和神经递质系统损害是POCD的主要病理机制。