卒中后高血糖通过自噬介导脑血管内皮细胞紧密连接蛋白降低的机制研究

Hyperglycemia induced by ischemic stroke increase the incidence of edema and haemorrhagic transformation, which can cause poor outcomes and death of patients. However, it is not clear about the mechanism of stroke aggravation caused by hyperglycemia and specific treatments are still needed. The decrease of tight-junction protein (TJP) in vascular endothelial cell directly increase the permeability of blood-brain barrier, causing cerebral edema and haemorrhage. Our previous study have showed that hyperglycemia in the early period of reperfusion obviously decreased TJP expression and aggravated ischemic stroke injury. However, it is still not clear about the mechanism of TJP reduction caused by post-stroke hyperglycemia. It is reported that TJP can translocate into cytoplasm from cell membrane by vesicle trafficking and autophagy is involved in TJP decrease, which are the major reason of TJP degradation. So the hypothesis of this study is that hyperglycemia is involved in the process of vesicle trafficking and autophagy. We will study the effects of hyperglycemia on vesicle trafficking, autophagy and lysosomal degradation of TJP, and find the key regulatory molecules. We hope that the current research about the specific mechanism of TJP regulation can provide new targets and methods for stroke treatment.

脑卒中后发生的应激性高血糖可加重脑水肿、增加缺血向出血转化的发生率，导致脑卒中预后不良甚至死亡，其发生机制尚不清楚，也缺少针对性治疗措施。脑血管内皮细胞间紧密连接蛋白（TJP）降低可增加血脑屏障通透性，加重脑水肿和脑出血。本申请人前期发现脑缺血再灌注早期高血糖可显著降低TJP表达，加重脑损伤，但高血糖引起TJP降低的分子机制尚待阐明。TJP可经囊泡转运方式由细胞表面进入细胞质，细胞自噬过程也可促进其降解，成为TJP总体及细胞表面表达水平降低的重要原因。据此，本课题假设高血糖可影响脑血管内皮细胞TJP的囊泡转运和／或自噬过程，提出应用脑缺血再灌注动物及细胞模型，通过荧光共定位、电镜等技术，研究高血糖分别对TJP囊泡转运、细胞自噬以及溶酶体降解等分子细胞过程的效应，探索和阐释各个环节的关键调控因子及其作用。本课题有望特异地针对脑血管内皮细胞特定TJP及其调控机制，为脑卒中治疗提供新靶点和新技术

缺血性脑卒中后高血糖增加脑水肿、加重脑损伤，导致脑卒中病人预后不良甚至死亡。在前期研究的基础上，本研究进一步发现脑缺血再灌注早期高血糖可显著降低紧密连接蛋白（TJP）的水平，包括Occludin、Claudin-5及ZO-1，加重脑损伤，体外研究发现氧糖剥夺再灌注早期，高葡萄糖水平（16mM）显著促进脑微血管内皮细胞中ZO-1由细胞膜向细胞质转移，而对Occludin及Claudin-5的胞内分布无影响。本项研究利用大鼠脑缺血再灌注模型进一步发现，卒中后高血糖促进MMP-2/-9的分泌及其对ZO-1的降解。另一方面，卒中后高血糖促进氧化应激和氮化应激，增强自噬，促进caveolin-1介导ZO-1由细胞膜转运到细胞内，使ZO-1通过自噬-溶酶体通路降解。使用雷帕霉素增强自噬会进一步增加ZO-1的降解，而给予3-MA抑制自噬，可以减少ZO-1由细胞膜向细胞质转移，减轻血脑屏障的损伤。所以，本研究初步明确卒中后高血糖增加血脑屏障通透性的分子机理，尤其是对ZO-1降解的调控机制，为脑卒中治疗提供新靶点。