摘要
目的 探讨人参皂苷Rb1对小鼠脑缺血再灌注诱导的血脑屏障(BBB)损伤的作用及机制.方法 将C57BL/6小鼠随机分为假手术组、模型组和人参皂苷Rb1低、中、高剂量(5、10、20mg·kg-1)组,采用线栓法栓塞颈内动脉1h后复灌建立脑缺血再灌注损伤模型,假手术组不栓塞,其余操作同模型小鼠.缺血1h后ip相应药物,于再灌注24h后处死取材.采用伊文思蓝染色法检测各组小鼠BBB损伤程度;采用实时荧光定量PCR(qRT-PCR)法检测各组小鼠脑组织中炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)以及紧密连接蛋白1(ZO-1)、闭合蛋白(Occludin)的mRNA表达水平;同时采用Western blotting检测各组小鼠脑组织中ZO-1、Occludin蛋白,金属基质蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)以及MAPK通路相关蛋白磷酸化的表达水平.结果 与模型组相比,人参皂苷Rb,可显著减少脑缺血再灌注小鼠脑组织中伊文思蓝的渗漏量(P＜0.05),显著降低脑组织中IL-1β、IL-6和TNF-α的mRNA转录水平(P＜0.05、0.01);显著上调ZO-1和Occludin的mRNA转录和蛋白表达水平(P＜0.05、0.01);显著降低MMP-2、MMP-9的蛋白表达水平(P＜0.05、0.01);显著抑制MAPK通路p38、JNK及ERK磷酸化蛋白的表达(P＜0.05、0.01).结论 人参皂苷Rb1对小鼠脑缺血再灌注诱导的BBB损伤具有一定的改善作用,其作用机制可能与抑制MAPK信号通路激活,减少MMP-2、MMP-9蛋白的表达,进而减轻对ZO-1、Occludin等紧密连接蛋白的降解有关.
Abstract
Objective To explore the effects and underlying mechanisms of ginsenoside Rb,(Rb1)on the blood-brain barrier(BBB)injury induced by cerebral ischemia reperfusion in mice.Methods C57BL/6J mice were randomly divided into sham operated group,model group,low,medium,high dose group of Rb,(5,10,and 20 mg·kg-1).This research used the middle cerebral artery occlusion and reperfusion(MCAO/R)animal model.After 1 h of ischemia and 24 h reperfusion,brain tissues of mice were extracted from executed mice.The evaluation of BBB damage in each group of mice was measured using Evans blue staining.The mRNA expression levels of the inflammatory factors—interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),as well as the expression of Zonula occludens-1(ZO-1)and Occludin were examined in the brain tissues of mice by the qRT-PCR.Western blotting method was uesd to detect the expression levels of ZO-1,Occludin,Matrix metalloproteinase-2/9(MMP-2/9),and MAPK related proteins in the brain tissue of each group of mice.Results Compared with the model group,Rb,significantly reduced the leakage of Evans blue in brain tissue of mice with cerebral ischemia-reperfusion(P＜0.05),and significantly reduced the mRNA transcription level of IL-1β,IL-6 and TNF-α in brain tissue(P＜0.05,0.01),significantly upregulated the mRNA transcription and protein expression levels of ZO-1 and Occludin(P＜0.05,0.01),significantly reduced the protein expression levels of MMP-2 and MMP-9(P＜0.05,0.01),significantly inhibited the expression of phosphorylated proteins p38,JNK,and ERK in the MAPK pathway (P ＜ 0.05, 0.01). Conclusion Rb, can ameliorate BBB damage via inhibiting the activation of MAPK signal pathway, down-regulate the expression of MMP-2 and MMP-9, attenuate the degradation of ZO-1 and Occludin in MCAO/R-induced mice.
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