Metformin prevents PFKFB3‑related aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts by regulating AMPK/mTOR pathway

Tang,Ci-Jun; Xu,Ji; Ye,Hai-Yan; Wang,Xue-Bin

doi:10.3892/etm.2021.10013

Metformin prevents PFKFB3‑related aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts by regulating AMPK/mTOR pathway

Authors:
- Ci-Jun Tang
- Ji Xu
- Hai-Yan Ye
- Xue-Bin Wang
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Affiliations: Department of Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China, Department of Emergency, Huashan Hospital, School of Medicine, Fudan University, Shanghai 200040, P.R. China
Published online on: March 31, 2021 https://doi.org/10.3892/etm.2021.10013
Article Number: 581
Copyright: © Tang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Aerobic glycolysis has been shown to contribute to the abnormal activation of lung fibroblasts with excessive collagen deposition in lipopolysaccharide (LPS)-induced pulmonary fibrosis. Targeting aerobic glycolysis in lung fibroblasts might therefore be considered as a promising therapeutic approach for LPS‑induced pulmonary fibrosis. In the present study, the aim was to investigate whether metformin, a widely used agent for treating type 2 diabetes, could alleviate LPS‑induced lung fibroblast collagen synthesis and its potential underlying mechanisms. Different concentrations of metformin were used to treat the human lung fibroblast MRC‑5 cells after LPS challenge. Indicators of aerobic glycolysis in MRC‑5 cells were detected by measuring glucose consumption and lactate levels in culture medium in addition to lactate dehydrogenase activity in cellular lysates. The glucose consumption, lactate levels and the lactate dehydrogenase activity were measured respectively using colorimetric/fluorometric and ELISA kits. The effects of metformin in AMP‑activated protein kinase (AMPK) activation was assessed by mitochondrial complex I activity kits. Collagen I, α‑smooth muscle actin (α‑SMA) and collagen III were used as markers of collagen synthesis, which was measured using western blotting, whereas phosphorylated (p‑) AMPK, AMPK, 6‑phosphofructo‑2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and mTOR were detected by western blotting. Metformin significantly decreased mitochondrial complex I activity and upregulated the expression of p‑AMPK/AMPK protein in a concentration‑dependent manner. Furthermore, the aerobic glycolysis mediated by PFKFB3 and collagen synthesis in LPS‑treated MRC‑5 cells was gradually inhibited with increasing concentrations of metformin. However, this inhibitory role of metformin on PFKFB3‑meditaed aerobic glycolysis and collagen synthesis was prevented by treatments with 3BDO and compound C, which are specific mTOR activator and AMPK inhibitor, respectively. Taken together, the findings from this study suggested that metformin may prevent PFKFB3‑associated aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts via regulating the AMPK/mTOR pathway.

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