ORC ID , Dong-Dong Yu2, Li Ren3, Guo-Rong Bi MD 4">
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Year : 2020  |  Volume : 15  |  Issue : 3  |  Page : 473-481

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

1 Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China; The First People's Hospital of Shenyang, Shenyang, Liaoning Province, China
2 The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, China
3 The First People's Hospital of Shenyang, Shenyang, Liaoning Province, China
4 Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China

Correspondence Address:
Guo-Rong Bi
Shengjing Hospital of China Medical University, Shenyang, Liaoning Province
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1673-5374.266059

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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.

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