川芎嗪通过激活PKCα/Nrf2信号转导抑制七氟烷诱导的氧化损伤和血脑屏障破坏研究

Ligustrazine inhibits sevoflurane-induced oxidative damage and blood-brain barrier disruption by activating PKCα/Nrf2 signaling

目的:研究川芎嗪对七氟烷引起的氧化损伤以及血脑屏障(BBB)生理功能损伤的影响及其机制。方法:将新生幼鼠随机分组,设置对照组,并分别使用七氟烷、七氟烷和溶剂、七氟烷和川芎嗪处理小鼠,暴露于七氟烷中不同时长处死并收集小鼠大脑和海马组织,通过免疫酶联吸附检测ROS,SOD,MDA,GSH和S100β蛋白水平,制作小鼠大脑和海马组织切片并通过免疫化学染色和免疫荧光染色检测小鼠大脑和海马组织中MMP-2、MMP-9、TIMP-1、TIMP-2以及HO-1的表达,并检测Nrf2的核易位,HE染色后通过透射电子显微镜检查海马神经元损伤情况,通过蛋白质印迹分析PKC/Nrf2以及MMP-2、MMP-9表达情况,使用原位酶谱检测明胶酶活性并通过PCR检测ZO-1的表达。结果:与对照组相比,七氟烷组海马神经元损伤明显,而川芎嗪处理可以明显改善七氟烷诱导的海马神经元损伤;与对照组相比,七氟烷处理后MMP-2、MMP-9表达和明胶酶活性明显增加,ZO-1表达明显减少,BBB通透性显著增加,而TIMP-1和TIMP-2表达无显著改变;与七氟烷组相比,川芎嗪可以抑制明胶酶活性,促进ZO-1表达,改善BBB生理状态;与溶剂组和七氟烷组相比,对照组和川芎嗪组ROS和BMD水平明显降低,MDA和GSH活性显著增加,PKCα、Nrf2和HO-1的表达以及Nrf2的核易位显著增加。结论:川芎嗪可以通过PKCα/Nrf2信号通路以及抑制明胶酶活性,改善BBB通透性,保护海马免受氧化应激的伤害,改善海马体组织病理学结构。

Objective:To study the effect and mechanism of ligustrazine on oxidative damage induced by sevoflurane and physiological function of blood-brain barrier(BBB).Methods:The newborn pups were randomly divided into a control group and 3 other groups which were treated with sevoflurane,sevoflurane and solvent,sevoflurane and ligustrazine.There were all exposed to sevoflurane for different durations and sacrificed to collect the brain and hippocampus tissues were tested for protein levels of ROS,SOD,MDA,GSH and S100βby immunoenzyme-linked adsorption.Mice brain and hippocampal tissue sections were made and MMP-2 and MMP were detected in mouse brain and hippocampus tissue by immunochemical staining and immunofluorescence staining-9,TIMP-1,TIMP-2 and HO-1 expression,and detect the nuclear translocation of Nrf2.After HE staining,examine the damage of hippocampal neurons by transmission electron microscope,and analyze PKC/Nrf2 and MMP-2 by western blot.MMP-9 expression,using in situ zymography to detect gelatinase activity and PCR to detect ZO-1 expression.Results:Compared with the CON group,hippocampal neuron damage was significant in the sevoflurane group,and ligustrazine treatment could significantly improve hippocampal neuron damage induced by sevoflurane;compared with the CON group,MMP-2 after sevoflurane treatment,MMP-9 expression and gelatinase activity were significantly increased,ZO-1 expression was significantly reduced,BBB permeability was significantly increased,and TIMP-1 and TIMP-2 expressions were not significantly changed;compared with SEV group,ligustrazine could inhibit Gelatinase activity promotes ZO-1 expression and improves BBB physiological status.Compared with S+S and SEV groups,ROS and BMD levels in the CON group and S+L group were significantly reduced,MDA and GSH activities were significantly increased,and PKCα,Nrf2 and HO-1 expression and nuclear translocation of Nrf2 increased significantly.Conclusion:Ligustrazine can improve the BBB permeability,protect the hippocampus from oxidative stress,and improve the histopathological structure of the hippocampus through the PKCα/Nrf2 signaling pathway and inhibition of gelatinase activity.