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 (6): 989-998.doi: 10.4103/1673-5374.233441

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

葛根素干预减轻自噬保护大脑免受缺血再灌注损伤的途径

  

  • 收稿日期:2018-04-25 出版日期:2018-06-15 发布日期:2018-06-15
  • 基金资助:

    国家自然科学基金(编号81202625)、中国三峡大学心血管与脑血管病转运医学重点实验室开放基金(2016xnxg101)

Puerarin protects rat brain against ischemia/ reperfusion injury by suppressing autophagy via the AMPK-mTOR-ULK1 signaling pathwa

Jin-Feng Wang1, Zhi-Gang Mei1, Yang Fu2, Song-Bai Yang3, Shi-Zhong Zhang1, Wei-Feng Huang1, Li Xiong4, Hua-Jun Zhou5, Wei Tao1, Zhi-Tao Feng1   

  1. 1 Third-Grade Pharmacological Laboratory on Chinese Medicine Approved by State Administration of Traditional Chinese Medicine, Medical College of China Three Gorges University, Yichang, Hubei Province, China;
    2 Xiangyang Hospital of Traditional Chinese Medicine, Xiangyang, Hubei Province, China;
    3 Yichang Hospital of Traditional Chinese Medicine, Clinical Medical College of Traditional Chinese Medicine, China Three Gorges University, Yichang, Hubei Province, China;
    4 Department of Medicine and Therapeutics, the Chinese University of Hong Kong, Hong Kong Special Administrative Region, China;
    5 The Institute of Neurology, the First College of Clinical Medical Sciences, China Three Gorges University, Yichang, Hubei Province, China
  • Received:2018-04-25 Online:2018-06-15 Published:2018-06-15
  • Contact: Zhi-Gang Mei, M.D. or Zhi-Tao Feng, M.D.,meizhigang@ctgu.edu.cn or fengzhitao2008@126.com.
  • Supported by:

    This work was supported by the National Natural Science Foundation of China, No. 81202625; the Open Fund of Key Laboratory of Cardiovascular and Cerebrovascular Diseases Translational Medicine, China Three Gorges University, China, No. 2016xnxg101.

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摘要：

葛根素能抑制自噬减轻脑缺血再灌注损伤，而且有证据证实，AMPK-mTOR信号通路能够通过与Ulk1的相互作用调节自噬。为此，实验希望观察到葛根素干预能够通过AMPK-mTOR-Ulk1信号通路调节自噬，在大鼠脑缺血再灌注模型中发挥神经保护作用。实验建立右侧大脑中动脉栓塞脑缺血损伤大鼠模型，造模前7 d腹腔注射葛根素50，100 mg/kg/d，末次给药30 min后造模。再灌注24 h后，各组大鼠进行Longa评分和脑梗死体积检测，以透射电镜观察自噬小体形成情况，以免疫荧光染色和western blot法检测缺血侧海马与自噬调节相关的LC3，Beclin-1和p62蛋白的表达，以及与AMPK-mTOR-Ulk1信号通路相关的AMPK，mTOR和Ulk1蛋白表达。结果发现，葛根素能显著降低脑缺血再灌注损伤大鼠Longa评分，减少脑梗死体积，减轻脑缺血再灌注损伤后缺血侧海马CA1区的自噬体形成，并呈剂量依赖性。同时，葛根素50，100 mg/kg预处理能显著降低脑缺血再灌注损伤大鼠缺血侧海马Beclin-1，LC3-II/LC3-I，p-AMPK和pS317-Ulk1蛋白的表达，上调p62蛋白表达。100 mg/kg葛根素上调大鼠缺血侧海马p-mTOR和pS757-Ulk1蛋白表达更显著。上述研究数据提示，葛根素对脑缺血再灌注损伤具有良好的保护作用，其作用机制可能与激活APMK-mTOR-Ulk1信号通路减轻自噬有关。

orcid:0000-0002-9099-7099(Zhi-Gang Mei)
        0000-0003-1107-944X(Zhi-Tao Feng)

关键词: 葛根素, 自噬, 脑缺血再灌注, AMPK-mTOR-Ulk1信号通路, 微管相关蛋白1轻链3, 选择性自噬接头蛋白, 缺血性卒中, 腺苷酸活化蛋白激酶, 中药, 大脑中动脉栓塞, 神经再生

Abstract:

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKmTOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-mTOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62,AMPK, mTOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin (50 or 100 mg/kg) reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and pS317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-mTOR and pS757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-mTOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

Key words: nerve regeneration, puerarin, autophagy, cerebral ischemia/reperfusion, AMPK-mTOR-ULK1 signaling pathway, light chain 3, p62, ischemic stroke, AMPK/mTOR, traditional Chinese medicine, middle cerebral artery occlusion, neural regeneration