电针抗氧应激效应细胞内Nrf2/ARE信号调控的研究

The Nrf2/ARE-protein signaling has been newly reported to be one of the most important pathways to regulate the endogenous anti-oxidant system. Our prelimenary studies have also shown that the promotion effect of electro-acupuncture on the neural function repair should be highly correlcated to the regulation of Nrf2/ARE-protein signaling. Here, Oxidative stress model of brain ischemia hypoxia was be set up by Nrf2 knock-out mice and SD rats. The confocal laser microscopy, immunohistochemistry, RT-PCR, and transmission electron microscopy were used to investigate the electro-acupuncture anti-oxidant stree effcts on different brain neurons of model animal, especially for the distribution and content change of Nrf2 signaling and the transcription and expression of downstream genes/proteins of anti-oxidant, anti-microbial proinflammatory, and pro-/anti-apoptosis. The results show that, based on the anti-oxidant stress of the electro-acupuncture, the transcription and expression of multiple target genes/proteins on downstream pathways can be overally regulated through the Nrf2/ARE signaling pathway, which is helpful for inhibiting the oxidantive damage, inflammation and apoptosis created in the anima model and achieving the ultimate aim of neural function repair. The present studies regarding the correlation between Nrf2/ARE signaling pathway and the anti-oxidant stress effect of the electro-acupuncture would provide the scientific evidence for the electro-acupuncture anti-oxidant stress mechanism, and open new possibilities of trerapeutic targes and strategies for the neural function repair.

Nrf2/ARE蛋白信号调节是最新发现的体内内源性抗氧化系统最重要通路之一,前期我们研究已表明电针促进神经功能修复的整体调衡与此信号所介导的作用密切相关。本课题拟采用SD大鼠和Nrf2基因敲除小鼠,建立脑缺血缺氧的氧化应激模型,并利用激光共聚焦、免疫组化、RT-PCR、透射电镜等技术手段，分析电针对模型动物不同脑区神经元在电针抗氧应激作用下Nrf2信号的分布及含量变化，及下游抗氧化、抑/促炎、促/抗凋亡等靶基因、蛋白的转录及表达，进一步证实电针可能通过调控Nrf2/ARE信号途径达到整体调节下游多条通路的靶基因、蛋白的转录及表达，进而抑制模型造成的氧化损伤、炎症反应及细胞凋亡，最终达到抗氧应激促进神经功能修复的目的。本课题创新地提出电针抗氧应激效应可能与Nrf2/ARE信号通路促发诱导下游相关基因有关，为电针抗氧应激机理的进一步深入提供科学依据，也为神经功能修复提供新的治疗靶点和方法。

背景：研究表明针灸可通过多层次、多途径、多靶点作用于多个病理环节，从整体上有效抑制或阻断脑缺血再灌注级联损伤效应，对脑缺血神经元起到保护作用。而Nrf2/ARE信号是近年新发现的机体抵抗伤害性刺激的防御性转导通路之一，与抗炎、抗凋亡、抗氧化的关系密切。.方法及结果：课题采用SD大鼠和Nrf2基因敲除小鼠, 将实验动物随机分为假手术组和用于造模动物，采用经改良的经典Logna线栓法将后者成功制备成大脑中动脉局灶性脑缺血再灌注损伤模型，并随机分为模型组及电针组，根据标准穴位图谱，电针组动物电针“百会”、“大椎”两穴。结果发现，脑缺血再灌注模型动物脑组织中Nrf2-ARE信号通路存在动态变化，可能和其损伤程度有关；Nrf2基因敲除处理可加重脑缺血再灌注模型的脑损伤；电针可明显提高模型大鼠和WT模型小鼠神经行为学评分，不论是TTC和HE染色，还是锥体细胞等超微结构均呈现出损伤减轻，趋向正常的态势；形成以Bcl-2占优势的Bcl-2/Bax二聚体，抑制Bax的促凋亡作用，降低缺血再灌注区的细胞凋亡，促进细胞的存活；调控脑缺血再灌注机体脑组织内γ-GCS蛋白及mRNA的表达，保持机体谷胱甘肽系统在一较高的动态平衡状态；降低TNF-α和升高IL-4蛋白及mRNA的表达，提高IL-4/TNF-α比值，从而抑制缺血再灌注区的炎症反应；但电针对Nrf2基因敲除小鼠模型的改善明显弱于野生型小鼠。.结论：电针可能通过调控Nrf2/ARE信号途径达到整体调节下游相关靶基因、蛋白的转录及表达，进而抑制脑缺血再灌注模型造成的氧化损伤、炎症反应及细胞凋亡，最终达到减少神经损伤，促进功能修复的目的。.科学意义：本研究证实了Nrf2信号通路对脑缺血再灌注后脑损伤的保护作用，为临床应用Nrf2保护剂或激活Nrf2信号通路保护脑缺血患者提供实验依据，也为电针对脑缺血再灌注神经元保护性信号转导机制研究指出新的研究方向。