Metformin's mechanism in reducing oxidative stress and promoting bone regeneration in T2DM rat BMMSCs: A focus on NRF2-GPX7 signaling pathway.

Objectives: Metformin (MF) could improve the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and bone regeneration under diabetes mellitus (T2DM) environment, however, its specific mechanism has not been elucidated. The aim of this study was to investigate whether MF inhibited oxidative stress and promoted osteogenic differentiation of T2DM rats BMMSCs (Tr-BMMSCs) through NRF2-GPX7 pathway.

Methods: BMMSCs were extracted from normal and diabetic rats. In vitro, the western blot analysis were first used to determine the effect of MF on the NRF2-GPX7 signaling pathway. Then, the levels of oxidative stress markers and ALP staining, alizarin red staining, immunofluorescence assay were performerd respectively to detect the role of NRF2-GPX7 pathway in the regulating effect of MF on the oxidative stress and osteogenic differentiation of Tr-BMMSCs. In vivo, the newly formed bone was evaluated by micro computed tomography, HE staining, Masson staining and immunohistochemistry through T2DM rat mandibular bone defect model.

Results: In vitro assays revealed that MF significantly promoted the expression of NRF2, NQO1, HO-1 and GPX7. The NRF2 pathway inhibitor (ML385) significantly inhibited the antioxidant and osteogenic promotion effects of MF on Tr-BMMSCs, while GPX7 overexpression effectively reversed the inhibitory effect of ML385. In vivo experiments showed that ML385 completely inhibited the combined promoting effects of MF and Tr-BMMSCs on the mandibular defect regeneration in T2DM rats. However, when MF was used in combination with GPX7 overexpressed Tr-BMMSCs, both new bone mass and OCN expression were significantly increased, indicating that GPX7 overexpression effectively reversed the osteogenesis inhibition of ML385 on Tr-BMMSCs.

Conclusions: MF inhibits oxidative stress level and promots osteogenic differentiation of Tr-BMMSCs and the repair of mandible bone defects of T2DM rats through NRF2-GPX7 pathway.