Diabetes, characterized by chronically elevated blood glucose levels, primarily manifests as Type 2 diabetes mellitus (T2DM), accounting for over 90% of all diabetes cases (Zheng et al., 2018). T2DM is associated with various complications, and one of the most severe is osteoporosis (Ferrari et al., 2018; Khosla et al., 2021). Osteoporosis is a metabolic bone disease that reduces bone mass, impairs bone regeneration, and increases fracture risk (Compston, 2018; Veronese et al., 2019; Yang et al., 2020).

Diabetic osteoporosis is linked to factors such as persistent hyperglycemia, disturbances in calcium and phosphorus metabolism, and oxidative stress (Ferrari et al., 2018; Pscherer et al., 2016). However, effective treatments targeting bone regeneration are currently lacking, necessitating the exploration of new therapeutic approaches (Mohsin et al., 2019; Sheu et al., 2022). In addition to oxidative stress and calcium/phosphorus absorption disorders, recent research suggests that excess intracellular iron may contribute to diabetic osteoporosis (Tsay et al., 2010; Yaribeygi et al., 2020; Zhang et al., 2019). While iron is essential for the body, excessive absorption can be harmful.

Ferroptosis, a newly discovered form of regulated cell death in 2012, is characterized by iron-induced lipid peroxidation (Dixon et al., 2012; Jiang et al., 2021). The primary mechanism involves the oxidation of unsaturated fatty acids on the cell membrane, resulting in lipid peroxidation and a reduction in intracellular antioxidants like glutathione (GSH) and glutathione peroxidase 4 (GPX4) (Forcina and Dixon, 2019; Liang et al., 2022; Tang et al., 2021). Iron homeostasis is crucial in diabetes, with disrupted iron balance reported in diabetic patients (Jehn et al., 2007; Wang et al., 2015). Excess intracellular iron can lead to reactive oxygen species (ROS) generation via the Fenton reaction, triggering ferroptosis (Baba et al., 2018; Liu et al., 2022). Limiting iron intake has been found to delay early kidney disease progression in diabetic rats (Cooksey et al., 2010; Matsumoto et al., 2013). We hypothesize that ferroptosis in osteoblasts contributes to bone regeneration disorders and aim to investigate the specific mechanisms involved.

Hypoxia-inducible factor 1 (HIF1) is oxygen-regulated, playing a crucial role in gene expression regulation under hypoxic conditions and high glucose, facilitating cellular adaptation and survival (Wang et al., 1995). HIF1 is expressed under low oxygen conditions (5% O2). However, synthesized HIF1 protein undergoes rapid degradation through an oxygen-dependent ubiquitin-proteasome degradation pathway (Ke and Costa, 2006). Impaired degradation of HIF1α affects the transcription of downstream genes involved in iron metabolism (Chung et al., 2012). Studies have demonstrated that HIF1α overexpression leads to increased expression of transferrin (Tf), transferrin receptor (Tf receptor) (Rolfs et al., 1997; Tacchini et al., 1999), and divalent metal transporter proteins (DMT1) (Qian et al., 2011), resulting in the excessive influx of free iron ions into cells and the induction of ferroptosis. Furthermore, high glucose conditions have been found to upregulate HIF1α expression (Li et al., 2012, 2023), and elevated HIF1α expression has been linked to increased endothelial cell permeability under high glucose conditions (Yan et al., 2012). These findings suggest that high glucose levels may enhance HIF1α activity, leading to the excessive influx of free iron ions.

Vitamin D is a fat-soluble vitamin, and ED-71, a novel active form of vitamin D3, is widely recognized as a standard drug for osteoporosis treatment (Christakos et al., 2016; Rong et al., 2022a). ED-71 is known for its role in regulating calcium and phosphorus metabolism, which is crucial for promoting bone mineralization (Salum et al., 2013; Saponaro et al., 2020; Sergeev, 2016). Epidemiologic surveys have shown that individuals with low serum vitamin D levels have a higher risk of suffering from type 2 diabetes mellitus (Hong et al., 2021). ED-71 supplementation has also shown potential in reducing the risk of diabetes (Breslavsky et al., 2013). Moreover, ED-71 regulates iron metabolism and iron ion pathways (Cheng et al., 2021; Malczewska-Lenczowska et al., 2018). However, the specific impact of ED-71 on iron metabolism regulation in the context of diabetic osteoporosis remains unclear, warranting further investigation into its pharmacological mechanisms.

In our study, we investigated the influence of ED-71 in bone regeneration and the potential mechanisms of osteogenesis in vitro and vivo T2DM models. Our research findings indicate that ED-71 treatment modulates the HIF1α signaling pathway, upregulates GPX4 expression, protects osteoblasts from Ferroptosis in T2DM, and improves bone regeneration.