脑清喷鼻微乳对急性脑梗死大鼠 血脑屏障通透性影响及其机制

Influence and Mechanism of Naoqing Penbi Weiru on the Permeability of Blood-brain Barrier in Rats after Acute Ischemic Stroke

摘要:

目的: 观察脑清喷鼻微乳对急性脑梗死大鼠血脑屏障(blood-brain barrier,BBB)超通透性影响及其相关分子机制。方法: 采用自身栓子法制备大鼠大脑中动脉栓塞模型(MCAO),筛选后随机分为对照组、模型组、脑清喷鼻微乳组,并给予药物干预。脑清喷鼻微乳组予脑清喷鼻微乳0.19 mL·kg^-1(相当于含麝香酮0.011 mg·kg^-1)滴鼻给药,每日3次,对照组与模型组给予相同时间点等量生理盐水滴鼻。每组于1,3,7 d 3个时相对大鼠进行神经功能缺损评分后,取材观察各时相大鼠大脑皮质缺血区神经元及内皮细胞形态,检测伊文思蓝(EB)、紧密连接蛋白-1(zonula occludins protins-1,ZO-1)、层黏连蛋白(laminin,LN)及水通道蛋白-4(aquaporin protein-4,AQP-4)含量。结果: 与同一时相正常组相比,模型组Bederson's评分、EB及AQP-4蛋白含量明显升高(P<0.05),ZO-1、Laminin蛋白含量显著降低(P<0.05),神经元坏死较多及内皮细胞损害严重。与同一时相模型组相比,脑清喷鼻微乳组Bederson's评分、EB及AQP-4蛋白含量明显降低(P<0.05),ZO-1,Laminin蛋白含量显著升高(P<0.05),神经元存活率较高及内皮细胞损害较轻。同组内不同时相相比较,脑清喷鼻微乳组Bederson's评分、EB及AQP-4蛋白含量逐渐降低(P<0.05),ZO-1蛋白含量逐渐升高(P<0.05),Laminin蛋白含量3 d后逐渐升高(P<0.05),1 d时神经元及内皮细胞水肿最严重,7 d时神经元变性坏死最多,内皮细胞基膜及细胞器缺损最为严重。结论: 脑清喷鼻微乳通过调控血脑屏障(BBB)3层结构中的主要蛋白ZO-1,Laminin及AQP-4蛋白的含量,调节BBB通透性,保护BBB结构及功能的完整性,减少神经元凋亡,减轻可逆性神经元损伤,促进神经功能的恢复。

Abstract:

Objective: To observe the effects of Naoqing Penbi Weiru on the permeability of blood-brain barrier(BBB) and ultrastructure of cortex endothelial cells after acute ischemic stroke. Method: The model of middle cerebral artery occlusion (MCAO) was established using self-embolus method. After screening,the male SD rats were randomly divided into the control group,the model group,the Naoqing Penbi Weiru group and gived drug intervention. Naoqing Penbi Weiru group nasal drip with Naoqing Penbi Weiru 0.19 mL·kg^-1(amount to contain muscone 0.011 mg·kg^-1), three times one day. Control group and model group nasal drip with equivalent normal saline at the same time. Every group detected the permeability of nerve function by the Bederson's score, the permeability of blood-brain barrier(BBB) by the Evans blue dye (EB dye), watch the ultrastructure of Cortex endothelial cells in ischemic region by electron microscopy, Zonula occludins protins-1(ZO-1),laminin(LN) and Aquaporin Protein-4(AQP-4)by western-blot. Result: Compared with the same phase control group, in the model group, the Bederson's score, the EB dye and the protein of AQP-4 significantly improved(P<0.05). The protein of ZO-1 and LN were significantly lower(P<0.05). Neurons and endothelial cell morphology were worse than the control group.Compared with the same phase model group, in the Naoqing Penbi Weiru group, the Bederson's score, the EB dye and the protein of AQP-4 significantly decreased(P<0.05). The protein of ZO-1 and LN were significantly higher(P<0.05). Neurons and endothelial cell morphology were better than the modol group. With the same group but at different time points compared, in the Naoqing Penbi Weiru group, Bederson's score, the Evans Blue dye (EB dye) and the protein of AQP-4 gradually decreased(P<0.05),the protein of ZO-1 were gradually higher(P<0.05) and the protein of Laminin were gradually higher(P<0.05)at 3 day. Neurons and endothelial edema were the most serious at 1 day. At 7 day, degeneration and necrosis of neurons was the most and basement membrane and organelle of endothelial cell defects most serious. Conclusion: Naoqing Penbi Weiru group can adjust BBB permeability, protect the integrity of BBB structure and function, reduce the apoptosis of neurons,reduce the damage of reversible neurons and promote the recovery of neurological function by regulating and controling the main protein AQP-4, LN and ZO-1 in the three layer structure of BBB.