Zhao Dan-Yang, Yu Dong-Dong, Ren Li, Bi Guo-Rong
Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China; The First People's Hospital of Shenyang, Shenyang, Liaoning Province, China.
The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, China.
Neural Regen Res. 2020 Mar;15(3):473-481. doi: 10.4103/1673-5374.266059.
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 × 10-1 × 10 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 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 M LIG for 3 hours, cultured in 95% N/5% CO 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.
自噬已被证明对脑损伤具有保护作用。川芎嗪（LIG）是从传统中药川芎中分离出的一种生物活性物质。LIG具有神经保护作用；然而，尚不清楚这种神经保护作用是否涉及自噬。在本研究中，将PC12细胞用1×10⁻¹×10 M的LIG处理0、3、12或24小时，并使用3-（4,5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺基苯基）-2H-四唑（MTS）测定法评估细胞增殖。用1×10 M的LIG处理3小时对细胞增殖的影响最大，因此用于后续实验。将PC12细胞用1×10 M的LIG预处理3小时，在不含葡萄糖或血清的杜氏改良 Eagle培养基中于95%N₂/5%CO₂条件下培养4小时，然后正常培养16小时，以模拟氧糖剥夺/复氧（OGD/R）。用MTS测定法评估细胞增殖。通过流式细胞术检测细胞凋亡。通过蛋白质印迹法评估凋亡相关蛋白Bcl-2和Bax、自噬相关蛋白Beclin 1和微管相关蛋白1轻链3B（LC3-II）以及肝激酶B1（LKB1）-5'-腺苷单磷酸激活蛋白激酶（AMPK）-雷帕霉素靶蛋白（mTOR）信号通路相关蛋白的表达水平。使用免疫荧光染色检测LC3-II的表达。通过电子显微镜观察自噬体的形成。LIG显著降低了OGD/R处理的PC12细胞的凋亡，增加了Bcl-2、Beclin 1和LC3-II的表达，降低了Bax的表达，增加了LC3-II免疫反应性和自噬体数量，并激活了LKB1-AMPK-mTOR信号通路。在OGD/R之前添加自噬抑制剂3-甲基腺嘌呤或 dorsomorphin减弱了LIG处理的细胞中LKB1-AMPK-mTOR信号通路的激活。综上所述，我们的研究结果表明，LIG通过LKB1-AMPK-mTOR信号通路促进自噬并保护PC12细胞免受OGD/R诱导的凋亡。
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