Reducing PDK4 level constitutes a pivotal mechanism for glucocorticoids to impede osteoblastic differentiation through the enhancement of ferroptosis in mesenchymal stem cells

Background: This study mainly explores the possible role and mechanism of pyruvate dehydrogenase kinase 4 (PDK4) in the onset and development of Glucocorticoid-induced osteoporosis (GIOP), and seeks potential targets for the treatment of GIOP.

Methods: Mesenchymal stem cells (MSCs) were treated with osteogenic induction medium. An in vitro osteogenic damage model was established by exposing MSCs to a high concentration (10^{- 6} M) of dexamethasone (DEX). Osteogenic markers were measured with real-time quantitative polymerase chain reaction, western blot, Alkaline Phosphatase staining, and Alizarin Red S staining. Ferroptosis markers were assessed through Reactive Oxygen Species (ROS) fluorescent probe, transmission electron microscopy, and measurement of malondialdehyde (MDA). The potential mechanism was investigated using RT-qPCR, western blot, lysosomal probes, molecular docking, and Other analytical approaches. The role of PDK4 was validated by using a GIOP rat model, micro-computed tomography and Masson's trichrome staining.

Results: High concentrations (10^{- 6} M) of DEX inhibited osteogenic differentiation in C3H10T1/2 cells, and PDK4 exhibited the opposite effect. PDK4 partially reversed the osteogenic inhibitory effect of DEX both in vivo and in vitro. DEX caused mitochondrial shrinkage and disappearance of cristae in C3H10T1/2 cells, as well as an increase in total iron, ROS, MDA contents, and the level of Ferroptosis key factors. These changes were partially weakened by PDK4. The Ferroptosis inhibitor ferrostatin-1 partially blocked the inhibitory effect of DEX, while Ferroptosis inducer RSL3 inhibited osteogenic differentiation and weakened the reversal effect of PDK4. DEX reduced the protein level of PDK4, which was partially weakened by Bafilomycin A1. The molecular docking results showed that DEX can directly bind with PDK4.

Conclusion: PDK4 can enhance the osteogenic differentiation ability of MSCs and bone mass of GIOP rats. DEX may promote the degradation of PDK4 via lysosome pathway, through which to weaken the osteogenic ability of MSCs by increasing Ferroptosis. PDK4 may become a potential target for improving GIOP.

Dexamethasone; Ferroptosis; Glucocorticoid-induced osteoporosis (GIOP); Osteogenic differentiation; Pyruvate dehydrogenase kinase 4.