Naringenin Ameliorates Iron Overload-Associated Osteoporosis via Tfeb/p62/Nrf2-Mediated Antioxidation

Postmenopausal women, elderly individuals, and transfusion-dependent patients are prone to bone marrow iron overload, which is closely associated with iron overload-associated osteoporosis (IOOP). Currently, the treatment of IOOP mainly focuses on promoting iron efflux and alleviating iron-induced damage, but the intervention value of natural active ingredients remains unclear. Naringenin (NAR), as a natural flavonoid, can regulate bone metabolism, yet its role and mechanism in IOOP have not been elucidated. In this study, an in vitro model was established by inducing MC3T3-E1 cells with ferric ammonium citrate (FAC), and an in vivo IOOP model was constructed by inducing mice with iron dextran to investigate the effects and mechanisms of NAR. The results showed that NAR improved the alkaline phosphatase (ALP) activity and mineralization capacity of FAC-induced iron-overloaded cells, upregulated the expression of Collagen I (Col1a1) and runt-related transcription factor 2 (Runx2), reduced the accumulation of reactive oxygen species (ROS) and lipid peroxide (LPO), attenuated mitochondrial membrane potential (MMP) impairment, and inhibited apoptosis. In in vivo experiments, NAR restored the density and quantity of trabecular bone in iron-overloaded mice. Mechanistically, RNA sequencing indicated that the effect of NAR was associated with transcription factor EB (Tfeb)-dependent transcription: NAR promoted Tfeb nuclear translocation and upregulated p62 transcription under iron overload conditions. Co-immunoprecipitation (Co-IP) demonstrated that NAR enhanced the binding of p62 to kelch-like ECH associated protein 1 (Keap1), while increasing Nrf2 phosphorylation and upregulating its key effector genes HO-1 and NQO1. Functional validation showed that the Nrf2 antagonist ML385 or siRNA could block the effects of NAR without affecting Tfeb expression, whereas the Tfeb inhibitor eltrombopag simultaneously inhibited Nrf2 expression and NAR-induced effects. In conclusion, NAR alleviates iron overload-induced oxidative damage and osteogenic disorders via the Tfeb/p62/Nrf2 pathway, suggesting that it may serve as a potential therapeutic agent for IOOP.