Metformin alleviates β‑glycerophosphate‑induced calcification of vascular smooth muscle cells via AMPK/mTOR‑activated autophagy

Qiu,Xiaobo; Xu,Qing; Xu,Tianhua; Wan,Pengzhi; Sheng,Zitong; Han,Yiran; Yao,Li

doi:10.3892/etm.2020.9490

Metformin alleviates β‑glycerophosphate‑induced calcification of vascular smooth muscle cells via AMPK/mTOR‑activated autophagy

Authors:
- Xiaobo Qiu
- Qing Xu
- Tianhua Xu
- Pengzhi Wan
- Zitong Sheng
- Yiran Han
- Li Yao
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Affiliations: Department of Nephrology, The First Hospital of China Medical University, Shenyang, Liaoning 110000, P.R. China, Department of Orthopaedics, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China
Published online on: November 19, 2020 https://doi.org/10.3892/etm.2020.9490
Article Number: 58
Copyright: © Qiu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate the effect of metformin on β‑glycerophosphate‑induced calcification of vascular smooth muscle cells (VSMCs) and the possible mechanisms underlying this. Using an established VSMC calcification model, VSMCs were first treated with β‑glycerophosphate, before metformin, 3‑methyladenine and compound C were added to the cell cultures in different combinations. Calcium deposition in the cells was examined by Alizarin Red S staining and using the O‑cresolphthalein complexone method. To assess the occurrence of autophagy, autophagosomes inside the cells were studied using a transmission electron microscope and green fluorescent microtubule‑associated protein 1 light chain 3 (LC3) puncta were examined using a fluorescent microscope. Additionally, protein expression levels of α‑smooth muscle actin (α‑SMA), runt‑related transcription factor 2 (RUNX2), LC3II/I, beclin 1 and 5' adenosine monophosphate‑activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway‑associated proteins were determined by western blot analysis. Metformin increased the number of autophagosomes, green fluorescent LC3 puncta and the levels of LC3II/I, beclin 1, α‑SMA and phosphorylated (p)‑AMPK in the VSMCs that were treated with β‑glycerophosphate when compared to controls; whereas, calcium deposition and the expression levels of RUNX2 and p‑mTOR were found to be decreased. Treating the VSMCs with 3‑methyladenine or compound C reversed the effects of metformin. The results of the present study suggested that metformin may alleviate β‑glycerophosphate‑induced calcification of VSMCs, which may be attributed to the activation of AMPK/mTOR signaling pathway‑dependent autophagy.

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