七叶皂苷钠对低氧暴露下大鼠血-脑脊液屏障通透性变化的影响及其抗渗漏机制研究

目的探讨七叶皂苷钠对急性低氧暴露下大鼠血-脑脊液屏障(BBB)的保护作用及抗渗漏机制。方法取健康成年雄性SD大鼠75只,随机分为常氧对照组(NC组)、单纯缺氧组(SH组)和药物治疗组(DT组),每组25只。NC组大鼠不做减压处理。SH组和DT组大鼠放入模拟海拔4000m的高原环境模拟舱内,力竭运动2d,再将模拟海拔高度调整至7000m,继续减压3d。光暗周期24h,舱内温度18℃,自由饮食水。减压至第3天,DT组大鼠给予七叶皂苷钠5mg/kg,腹腔注射,1次/d。其余两组在相应时间点给予等量生理盐水同法处理。减压结束后,NC组在平原环境下取材,其余两组在模拟海拔5000m环境中迅速取材。采用干湿重法测定脑含水量,伊文思蓝(EB)示踪法检测BBB通透性,HE染色法观察脑组织病理学改变,硝酸镧示踪法观察BBB紧密连接(TJ)和脑组织超微结构变化,免疫组织化学法和Western blotting检测咬合蛋白(Occludin)的表达,实时荧光定量PCR法检测Occludin、胶质附着蛋白(Zo-1)和闭锁蛋白-5(Claudin-5)的基因表达。结果与NC组及DT组相比,急性缺氧5d后SH组脑含水量和EB含量显著增加(P<0.01),而NC组与DT组比较无显著差异(P>0.05)。光镜下可见NC组海马神经元脱失严重,水肿明显,DT组神经元脱失减少,水肿减轻;透射电镜示NC组硝酸镧颗粒通过开放的紧密连接弥散性沉积至脑间质,毛细血管周围水肿明显,DT组镧颗粒渗漏减少、水肿程度减轻。Occludin基因和蛋白表达水平在NC组最低(P<0.01),在DT组最高(P<0.05)。与SH组比较,Zo-1和Claudin-5 mRNA在NC组和DT组均显著升高(P<0.05),后两组比较无显著差异。结论急性低氧暴露可使大鼠脑Occludin蛋白和Occludin、Zo-1、Claudin-5基因表达显著降低,BBB通透性增加,七叶皂苷钠能上调Occludin蛋白和Occludin、Zo-1、Claudin-5基因表达,降低BBB通透性,从而发挥抗渗漏和保护BBB的作用。

Anti-leakage mechanism and effect of sodium aescinate on the permeability of rat blood-brain barrier exposed to hypoxia

Objective To study the anti-leakage mechanism and protective effect of sodium aescinate on the blood-brain barrier of rat acutely exposed to hypoxia.Methods Seventy-five healthy SD rats were randomly divided into 3 groups(25 each):normoxic control(NC),simple hypoxic(SH) and drug treated(DT) group.Acute hypoxia brain edema rat model was established by a simulation of plateau acute hypoxia for 5 days.Dry-wet method to determine cerebral water content,evans blue(EB) method to evaluate the permeability of blood-brain barrier(BBB).HE staining to detect pathological changes of brain.Lanthanum nitrate tracer method with transmission electron microscopy(TEM) to observe the state of BBB tight junction(TJ) and ultrastructure of brain tissue.Protein and mRNA expression of Occludin,Zo-1 and Claudin-5 were investigated by immunohistochemistry,western-blotting and real-time PCR respectively.Results After exposure to acute hypoxia for 5 days,compared with NC group,the water content of brain in SH group increased obviously(P<0.01) while that in DT group decreased significantly(P<0.05),and EB content analysis revealed a similar result.It was observed by microscopy that hippocampus neurons were lost and edemas occurred heavily in SH group while slightly in DT group.It was detected by TEM that lanthanum nitrate infiltrated and deposited into cerebral interstitium through widened TJ gap of the brain microvascular endothelial cells(BMVECs),and the perivascular edema was obvious in SH group,while these changes were lessened in DT group.The lowest expression levels of Occludin mRNA and protein were found in SH group(P<0.01),and the highest levels were in DT group(P<0.05).Meanwhile,to compare with SH group,the expression of zo-1 and Claudin-5 mRNA increased remarkably in both NC and DT group(P<0.05),while no significant difference existed between the latter two groups.Conclusion Acute hypoxia exposure may lead to a remarkable decline of the expressions of rat’s brain Occludin protein and the Occludin,Zo-1 and Claudin-5 mRNA,and an obvious increase of BBB permeability.Sodium aescinate can up-regulate the expression level of these molecules and decrease BBB permeability,thus play a profitable role of anti-leakage and BBB protection.