基于PI3K/AKT/mTOR途径桔梗皂苷D对脑缺血再灌注诱导的血脑屏障的保护作用及机制研究

Protective Effect and Mechanismof Platycodonopsis Saponin D on Cerebral Ischemia/Reperfusion-Induced Blood-Brain Barrier on PI3K/AKT/mTOR Pathway Based

本研究旨在探讨桔梗皂苷D (Platycodin D,PLD)通过PI3K/AKT/mTOR途径对脑缺血再灌注诱导的血脑屏障的保护作用及机制研究。试验采用糖尿病Sprague-Dawley大鼠构建大脑中动脉闭塞模型。动物试验分组:sham组、HIR组、PLD组、3-MA组(3-甲基腺嘌呤,3-methyladenine)和PLD+3-MA组。分析各组大鼠神经功能缺损评分、脑含水量、脑梗塞体积和血脑屏障渗透性。透射电子显微镜观察血脑屏障超微结构变化和自噬。免疫印迹法分析紧密连接(Tight junction,TJ)蛋白(ZO-1、claudin-5和occludin)免疫荧光染色和蛋白质免疫印迹(Western blot,WB)法,分析自噬相关蛋白(Beclin-1、LC3B和p62)和PI3K/AKT/mTOR通路相关蛋白的表达。结果表明,PLD和自噬抑制剂3-MA治疗显著降低了脑梗死体积、脑含水量和伊文思蓝渗漏,同时增加了TJ蛋白的表达,降低了自噬相关蛋白的表达(P<0.05)。此外,PLD增加了PI3K、AKT和mTOR的磷酸化水平。PLD通过抑制自噬预防高血糖条件下,脑缺血再灌注损伤后的血脑屏障损伤,可能通过调节PI3K/AKT/mTOR信号通路来实现。

To investigate the mechanism and the protective effect of Platycodin D(PLD)on cerebral ischemia/reperfusion-induced bloodbrain barrier via PI3K/AKT/mTOR pathway in this study.Middle cerebral artery occlusion model was constructed in Sprague-Dawley diabetic rats.Animal experiments were divided into shamgroup,HIRgroup,PLDgroup,3-MAgroup(3-methyladenine,3-methyladenine)and PLD+3-MA group.Nerve function deficit score,cerebral water content,cerebral infarction volume and blood-brain barrier permeability were analyzed in each group.Ultrastructural changes and autophagy of blood-brain barrier were observed by transmission electron microscopy.Western blot analysis of tight junction(tight junction,the expression of autophagy associated proteins(Beclin-1,LC3B and p62)and PI3K/AKT/mTOR pathway related proteins were analyzed by immunofluorescence staining of TJ proteins(ZO-1,claudin-5 and occludin)and western blot.Treatment with PLDand autophagy inhibitor 3-MAsignificantly decreased infarct volume,brain water content and evans blue leakage,increased the expression of TJ protein and decreased the expression of autophagy related proteins(P<0.05).In addition,PLDincreased the phosphorylation levels of PI3K,AKT and mTOR.PLDprevents blood-brain barrier injury after cerebral ischemia-reperfusion injury in hyperglycemia by inhibiting autophagy,which may be achieved by regulating PI3K/AKT/mTOR signaling pathway.