Metformin reverses oxidative stress-induced mitochondrial dysfunction in pre-osteoblasts via the EGFR/GSK-3β/calcium pathway

Oxidative stress is one of the main causes of osteoblast apoptosis induced by post-menopausal osteoporosis. The authors previously found that metformin can reverse the loss of bone mass in post-menopausal osteoporosis. The present study aimed to further clarify the effects and mechanisms of action of metformin in post-menopausal osteoporosis under conditions of oxidative stress. Combined with an in-depth investigation using the transcriptome database, the association between oxidative stress and mitochondrial dysfunction in post-menopausal osteoporosis was confirmed. A pre-osteoblast model of oxidative stress was constructed, and the apoptotic rate following the addition of hydrogen peroxide and metformin was detected using CCK-8 assay and Annexin V-FITC/PI staining. Mitochondrial membrane potential was detected using the JC-1 dye, the intracellular calcium concentration was detected using Fluo-4 AM, the intracellular reactive oxygen species (ROS) level was observed using DCFH-DA, and the mitochondrial superoxide level was observed using MitoSOX Red. Bay K8644 was used to increase the level of intracellular calcium. siRNA was used to interfere with the expression of glycogen synthase kinase (GSK)-3β. Western blot analysis was used to detect the expression of mitochondrial dysfunction-related proteins. The results revealed that oxidative stress decreased mitochondrial membrane potential and increased intracellular ROS, mitochondrial superoxide and cytoplasmic calcium levels in pre-osteoblasts; however, metformin improved mitochondrial dysfunction and reversed oxidative stress-induced injury. Metformin inhibited mitochondrial permeability transition pore opening, suppressed the cytoplasmic calcium influx and reversed pre-osteoblast apoptosis by promoting GSK-3β phosphorylation. Moreover, it was found that EGFR was the cell membrane receptor of metformin in pre-osteoblasts, and the EGFR/GSK-3β/calcium axis played a key role in metformin reversing the oxidative stress response of pre-osteoblasts in post-menopausal osteoporosis. On the whole, these findings provide a pharmacological basis for the use of metformin for the treatment of post-menopausal osteoporosis.