中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (12): 2119-2128.doi: 10.4103/1673-5374.241463

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

迷迭香酸对缺血性卒中神经保护作用的机制

  

  • 收稿日期:2018-08-07 出版日期:2018-12-15 发布日期:2018-12-15
  • 基金资助:

    中国国家自然科学基金项目（81571292，81601152），河北省自然科学基金项目（H2017206338）

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling

Hai-Ying Cui1, 2, Xiang-Jian Zhang1, 2, Yi Yang1, 2, Cong Zhang1, 2, Chun-Hua Zhu1, 2, Jiang-Yong Miao1, 2, Rong Chen1, 2   

  1. 1 Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China
    2 Hebei Key Laboratory of Vascular Homeostasis and Hebei Collaborative Innovation Center for Cardiocerebrovascular Disease, Shijiazhuang, Hebei Province, China
  • Received:2018-08-07 Online:2018-12-15 Published:2018-12-15
  • Contact: Xiang-Jian Zhang, MD, PhD, zhang6xj@aliyun.com
  • Supported by:

    This work was generously supported by the National Natural Science Foundation of China, No. 81571292 (to XJZ), 81601152 (to YY), and the Natural Science Foundation of Hebei Province of China, No. H2017206338 (to RC).

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摘要：

已证实迷迭香酸对缺血性卒中具有神经保护作用，但其确切机制尚不清楚。实验将北京维通利华实验动物技术有限公司提供的CD-1小鼠右侧大脑中动脉闭塞1h，再灌注24h，建立实验性缺血性卒中模型，然后以10，20，40mg/kg的迷迭香酸腹腔内注射进行干预。以改良的Longa评分方法评估神经功能缺陷，以TTC染色检测脑梗死体积，以TUNEL染色检测缺血脑组织中细胞的凋亡，以羟胺法测量缺血脑组织中超氧化物岐化酶的水平，以黄嘌呤-黄嘌呤氧化酶法测量缺血脑组织中丙二醛水平，以Western blot分析缺血脑组织中血红素加氧酶1，核因子红细胞2相关因子2，Bcl-2，Bax，phospho-Ser473 Akt和Akt的表达水平，以定量实时PCR分析定量实时PCR分析核因子红细胞2相关因子2，血红素加氧酶1，Bcl-2和Bax mRNA表达水平，以分光光度法测量缺血脑组织中血红素加氧酶1酶的活性。结果显示：（1）20及40mg/kg迷迭香酸能显著改善实验性缺血性卒中模型小鼠的神经功能，缩小梗死体积，减少细胞凋亡，上调脑组织中Bcl-2蛋白及mRNA的表达，下调Bax蛋白及mRNA的表达，同时增加血红素加氧酶1和核因子红细胞2相关因子2蛋白及mRNA水平，增加超氧化物岐化酶水平，降低丙二醛水平；（2）而缺血前24h腹腔内注射特异性血红素加氧酶1抑制剂锌原卟啉IX(10mg/kg)则能逆转迷迭香酸对血红素加氧酶1活性及Bcl-2和Bax蛋白及mRNA表达的作用。且PI3K/Akt信号通路抑制剂LY294002(10mM)抑制Akt的磷酸化以及核因子红细胞2相关因子2和血红素加氧酶1的表达；（3）结果表明迷迭香酸可通过其抗氧化和抗凋亡特性保护脑免实验性缺血性卒中损伤，且其作用机制与通过PI3K/Akt信号通路上调核因子红细胞2相关因子2和血红素加氧酶1表达有关。

orcid:0000-0003-0114-1668(Xiang-Jian Zhang)

关键词: 脑缺血/再灌注, 迷迭香酸, 细胞凋亡, 氧化损伤, 神经保护, Bcl-2, Bax, 核因子红细胞2相关因子2, 血红素加氧酶1, PI3K/Akt信号通路

Abstract:

Rosmarinic acid (RA) can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice (Beijing Vital River Laboratory Animal Technology, Beijing, China) by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA (20 and 40 mg/kg) greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor (10 mg/kg zinc protoporphyrin IX) reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3K/Akt signaling pathway inhibitor LY294002 (10 mM) inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3K/Akt signaling pathway.

Key words: cerebral ischemia/reperfusion, rosmarinic acid, cellular apoptosis, oxidative injury, neuroprotection, Bcl-2, Bax, Nrf2, heme oxygenase 1, PI3K/Akt signal pathway, neural regeneration