周小青 曹泽标 刘旺华 等

摘要：目的 探讨丹龙醒脑方对局灶性脑缺血再灌注大鼠侧脑室室管膜下区（SVZ）神经干细胞（NSCs）增殖与c-myc、c-jun表达的关系。方法 采用线栓法制备大脑中动脉栓塞局灶性脑缺血再灌注模型。150只雄性SD大鼠随机分为假手术组、模型组和丹龙醒脑方小、中、大剂量组。于再灌注24 h后，丹龙醒脑方各剂量组予相应剂量药液灌胃，模型组和假手术组予等体积蒸馏水灌胃。1次/d，连续7 d。再灌注1、3、7 d采用m-NSS法进行神经功能缺损评分，再灌注7 d取缺血侧SVZ脑组织，采用Brdu免疫组化检测NSCs增殖，RT-qPCR、Western blot分别检测c-jun、c-myc mRNA和蛋白的表达。结果 与假手术组比较，其余各组神经功能缺损评分明显升高（P<0.01）；再灌注3、7 d，与模型组比较，丹龙醒脑方各剂量组神经功能缺损评分明显降低（P<0.01）。与假手术组比较，其余各组Brdu阳性细胞率明显升高；与模型组比较，丹龙醒脑方各剂量组Brdu阳性细胞率亦明显升高（P<0.01）。丹龙醒脑方各剂量组较假手术组、模型组c-jun、c-myc蛋白及mRNA表达均明显增强（P<0.05，P<0.01）。结论 丹龙醒脑方能改善脑缺血后神经功能，促进SVZ NSCs增殖，其机制可能与增强c-jun和c-myc表达及延长表达持续时间有关。

关键词：丹龙醒脑方；脑缺血再灌注；侧脑室室管膜下区；神经干细胞增殖；c-jun表达；c-myc表达；大鼠

DOI：10.3969/j.issn.1005-5304.2016.04.013

中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2016）04-0049-05

Effects of Danlong Xingnao Formula on Proliferation of Neural Stem Cells in SVZ and Expressions of c-jun and c-myc in Cerebral Ischemia Reperfusion Rats ZHOU Xiao-qing1，2， CAO Ze-biao1，3， LIU Wang-hua1，2， CHEN Ping-ting1，3， LI Hua1，2， CHEN Yu-wen1 （1. Institute of Diagnostics Traditional Chinese Medicine， Hunan University of Chinese Medicine， Provincial Key Laboratory of Diagnostics in Chinese Medicine， Changsha 410208， China； 2. Collaborative Center for Research and Innovation of Digital Chinese Medicine， Hunan University of Chinese Medicine， Changsha 410208， China； 3. The First Affiliated Hospital of Hunan University of Chinese Medicine， Changsha 410007， China）

Abstract： Objective To study the relationship of proliferation of neural stem cells （NSCs） in SVZ and the expressions of c-jun and c-myc in rats with middle cerebral artery occlusion/reperfusion （MCAO/R） injury model administrated by Danlong Xingnao Formula. Methods The focal cerebral ischemia reperfusion injury models were prepared by longa method. Totally 150 male SD rats were randomly divided into sham-operation group， cerebral model group， Danlong Xingnao Formula low-， medium-， and high-dose groups. The treatment groups were given corresponding dose of Danlong Xingnao Formula， while the sham-operation group and model group were given the same amount of distilled water 24 h after modeling by gavage， once a day， 7 days in a row. 1 d， 3 d and 7 d after reperfusion， modified Neurological Severity Scores （m-NSS） was used to grade neurologic impairment. 7 d after

基金项目：国家自然科学基金（81373702、81473567、81202632）；教育部博士点基金（20124323120003）；湖南省自然科学基金（13JJ3097）；湖南省教育厅科研项目（14B134、15K092）

通讯作者：刘旺华，E-mail：lwhww@aliyun.com

reperfusion taken to the SVZ brain tissue of ischemia side， Brdu immunohistochemical method was used to record the BrdU positive cells number. The hippocampal c-jun， c-myc mRNA and protein expressions were determined respectively by RT-qPCR method and Western blot method. Results Grades of neurologic impairment in others groups were improved obviously than sham-operation group （P<0.01）； 3 d， and 7 d after reperfusion， grades of neurologic impairment in Danlong Xingnao Formula groups were obviously lower compared with model group （P<0.05， P<0.01）. Brdu positive cell rates in others groups increased obviously compared with sham-operation group； Compared with model group， Brdu positive cell rates in Danlong Xingnao Formula groups increased obviously （P<0.01）. The expressions of c-jun and c-myc protein and mRNA in Danlong Xingnao Formula groups improved obviously than sham-operation group and model group （P<0.01）. Conclusion Danlong Xingnao Formula can improve the neural function after cerebral ischemia and stimulate the proliferation of NSCs， and the mechanism may be related to activating the expression of c-jun and c-myc and extending the duration.

Key words： Danlong Xingnao Formula； cerebral ischemia reperfusion； SVZ； proliferation of NSCs； c-jun expression； c-myc expression； rats

脑血管疾病发生后的主要病理变化为神经元大量丢失导致脑功能缺失。神经干细胞（NSCs）以其自我更新和多向分化潜能为脑血管病的治疗开辟了新的方向。丹龙醒脑方由丹参、三七、地龙、淫羊藿、菟丝子等组成，具有活血通络、补肾生髓之功，临床治疗缺血性脑血管病疗效较好。前期研究表明，丹龙醒脑方能促进NSCs增殖[1]，其机制可能与增强Wnt/β-catenin信号通路上游因子Wnt-3α、β连环蛋白（β-catenin）的表达有关[2]，但对通路下游因子尚未探讨。本实验采用线栓法制备大脑中动脉栓塞局灶性脑缺血再灌注（MCAO/R）大鼠模型，观察侧脑室室管膜下区（SVZ）5-溴脱氧尿嘧啶核苷（Brdu）阳性细胞和通路下游因子c-jun、c-myc的表达，进一步探讨丹龙醒脑方促进NSCs增殖的机制。……