中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (3): 473-481.doi: 10.4103/1673-5374.266059

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

藁本内酯保护PC12细胞免受氧糖剥夺再灌注损伤诱导凋亡的机制

  

  • 出版日期:2020-03-15 发布日期:2020-05-26

Ligustilide protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced apoptosis via the LKB1-AMPK-mTOR signaling pathway

Dan-Yang Zhao1, 2, Dong-Dong Yu3, Li Ren2, Guo-Rong Bi1   

  1. 1 Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China
    2 The First People’s Hospital of Shenyang, Shenyang, Liaoning Province, China
    3 The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, China
  • Online:2020-03-15 Published:2020-05-26
  • Contact: Guo-Rong Bi, MD,bigr@sj-hospital.org.

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摘要：

最近的研究证实自噬对脑损伤具有一定的保护作用，藁本内酯是一种从传统中药川芎中分离出来的生物活性物质，具有保护神经细胞的药理作用，但是藁本内酯的神经保护作用是否通过影响细胞自噬的机制而发挥，目前尚不清楚。(1)实验以1×10-5-1×10-9M藁本内酯干预PC12细胞0，3，12，24h，以MTS检测检测细胞增殖活性，发现以1 × 10-6M藁本内酯干预3h对细胞增殖的影响最为明显；（2）将PC12细胞以1 × 10-6M藁本内酯预干预3h，然后于无葡萄糖和血清的DMEM培养基中95% N2，5% CO2环境培养4h，然后正常培养16h，建立氧糖剥夺再灌注损伤模型。以MTS检测检测细胞增殖活性，以流式细胞仪检测细胞凋亡，以western blot检测细胞在凋亡相关蛋白Bcl-2和Bax、自噬相关蛋白Beclin-1和LC3-II以及LKB1-AMPK-mTOR信号通路相关蛋白的表达，同时以免疫荧光染色检测细胞中LC3-II的免疫阳性反应，并通过电镜观察自噬体的形成；（3）结果发现，藁本内酯可增加氧糖剥夺再灌注损伤的PC12细胞凋亡明显减少，Bcl-2，Beclin-1，LC3-II表达增加，Bax表达降低，LC3-II免疫阳性反应增加，自噬体数量增加，LKB1-AMPK-mTOR信号通路被激活。但是在氧糖剥夺再灌注损伤前加入自噬抑制剂3-甲基腺嘌呤或Dorsomorphin，藁本内酯干预的细胞中LKB1-AMPK-mTOR信号通路活化减弱；（4）结果表明，藁本内酯的神经保护作用是通过活化氧糖剥夺再灌注损伤的PC-12细胞中的LKB1-AMPK-mTOR信号通路，促进自噬而实现的。

orcid: 0000-0002-6805-4430 (Dan-Yang Zhao)

关键词: 藁本内酯, PC12细胞, 自噬, 细胞凋亡, Bcl-2, Bax, Beclin-1, LC3-II, AMPK, mTOR, 神经再生

Abstract: Autophagy has been shown to have a protective effect against brain damage. Ligustilide (LIG) is a bioactive substance isolated from Ligusticum chuanxiong, a traditional Chinese medicine. LIG has a neuroprotective effect; however, it is unclear whether this neuroprotective effect involves autophagy. In this study, PC12 cells were treated with 1 × 10–5–1 × 10–9 M LIG for 0, 3, 12 or 24 hours, and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Treatment with 1 × 10–6 M LIG for 3 hours had the greatest effect on cell proliferation, and was therefore used for subsequent experiments. PC12 cells were pre-treated with 1 × 10–6 M LIG for 3 hours, cultured in 95% N2/5% CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours, and then cultured normally for 16 hours, to simulate oxygen-glucose deprivation/reoxygenation (OGD/R). Cell proliferation was assessed with the MTS assay. Apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins, Bcl-2 and Bax, autophagy-related proteins, Beclin 1 and microtubule-associated protein l light chain 3B (LC3-II), and liver kinase B1 (LKB1)-5′-adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway-related proteins were assessed by western blot assay. Immunofluorescence staining was used to detect LC3-II expression. Autophagosome formation was observed by electron microscopy. LIG significantly decreased apoptosis, increased Bcl-2, Beclin 1 and LC3-II expression, decreased Bax expression, increased LC3-II immunoreactivity and the number of autophagosomes, and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R. The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG. Taken together, our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.

Key words: AMPK, apoptosis, autophagy, Bax, Bcl-2, Beclin 1, LC3-II, ligustilide, mTOR, PC12 cells