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This study delved into the role of the PI3K/AKT signaling pathway and cuproptosis in steroid-induced osteonecrosis of the femoral head (SIONFH), assessing the therapeutic potential of the PI3K agonist 740Y-P. We analyzed femoral head specimens from SIONFH patients using DIA proteomics, identifying differentially expressed proteins linked to cuproptosis. In vitro, MC3T3-E1 cells treated with dexamethasone (DEX) exhibited hallmarks of cuproptosis, including downregulation of DLAT, PDHB, SLC25A3, and FDX1, increased copper ions, and reduced osteogenic potential, as shown by decreased ALP activity and RUNX2/BMP2 expression. The PI3K/AKT pathway’s modulation of FDX1 was key to cuproptosis regulation; activating it with 740Y-P restored FDX1 levels and partially recovered osteogenic capacity. An in vivo rat model of SIONFH treated with 740Y-P demonstrated improved bone parameters, reversed osteogenic suppression, and upregulated PI3K/AKT/FDX1 expression, validating the pathway’s role in cuproptosis and the agonist’s therapeutic potential for treating SIONFH and glucocorticoid-associated bone disorders.

Osteonecrosis of the femoral head (ONFH) is a refractory orthopedic condition characterized by bone cell ischemia, necrosis, bone trabecular fracture, and clinical symptoms such as pain, femoral head collapse, and joint dysfunction that can lead to disability. The disability rate of ONFH is very high, which imposes a significant economic burden on both families and society. Steroid-associated osteonecrosis of the femoral head (SANFH) is the most common type of ONFH. However, the pathogenesis of SANFH remains unclear, and it is an urgent challenge for orthopedic surgeons to explore it. In this paper, the pathogenesis of SANFH and its related signaling pathways were briefly reviewed to enhance comprehension of the pathogenesis and prevention of SANFH.

teroid-induced osteonecrosis of the femoral head (SONFH) is a severe bone disorder caused by long-term glucocorticoid administration and is characterized by osteoblast apoptosis. Oleuropein (OLP), a natural compound with anti-inflammatory and antioxidant properties, has demonstrated anti-apoptotic potential in bone-related diseases. However, its therapeutic role in SONFH has not yet been elucidated. This study aimed to investigate the therapeutic effects of OLP on SONFH and elucidate its underlying molecular mechanisms. In vitro, MC3T3-E1 osteoblasts treated with methylprednisolone (MPS) were co-incubated with OLP. Cell viability was assessed using a CCK-8 assay and live/dead cell staining. In vivo, a rat SONFH model was established with lipopolysaccharide and MPS, followed by OLP treatment. Bone microstructure was analyzed by micro-computed tomography and histopathological staining (H&E, Masson, Goldner). Network pharmacology and proteomics analyses were used to identify key targets and pathways related to the effects of OLP on SONFH. Apoptosis was examined with flow cytometry, TUNEL staining and ELISA. Protein and mRNA expression levels of relevant targets and pathways were examined with western blotting and quantitative real-time polymerase chain reaction. OLP significantly reversed MPS-induced osteoblast apoptosis and enhanced cell viability. In SONFH rats, OLP reduced empty lacunae, restored trabecular bone structure, and improved collagen organization. Molecular docking confirmed OLP’s binding to AKT and Bcl2. Network pharmacology and proteomics highlighted apoptosis regulation, PI3K-AKT signaling, and cell cycle control as key mechanisms. Furthermore, OLP activated the PI3K-AKT-Bcl2 pathway, increasing p-PI3K, p-AKT, and Bcl2 levels while decreasing Caspase3 and Caspase9. OLP also promoted osteogenesis (upregulated ALP and Runx2) and angiogenesis (increased vWF and CD31). OLP alleviates SONFH by inhibiting osteoblast apoptosis and promoting osteogenesis and angiogenesis through activation of the PI3K-AKT-Bcl2 pathway. These findings support OLP as a promising natural compound for early intervention in SONFH.

The onset of osteonecrosis of the femoral head (ONFH) is intimately associated with the extensive administration of glucocorticoids (GCs). Long‐term stimulation of GCs can induce oxidative stress in both osteoclasts (OCs) and osteoblasts (OBs), resulting in the disturbance of bone remodelling. An alkaloid named crebanine (CN) demonstrates pharmacological properties including anti‐inflammation and reactive oxygen species (ROS) modulation. Our objective is to assess the therapeutic potential of CN in treating ONFH and elucidate the associated underlying mechanisms. The network pharmacology analysis uncovered that CN played a role in regulating ROS metabolism. In vitro, CN demonstrated its ability to reduce the dexamethasone (DEX)‐stimulated generation of OCs and suppress their resorptive function by downregulating the level of osteoclast marker genes. Concurrently, CN also mitigated DEX‐induced damage to OBs, facilitating the restoration of osteoblast marker gene expression, cellular differentiation and function. These effects were achieved by CN augmenting the antioxidant system to reduce intracellular ROS levels. Furthermore, in vitro results were corroborated by micro‐CT and histological data, which also showed that CN attenuated MPS‐induced ONFH in mice. This study highlights the therapeutic potential of CN in counteracting GCs‐induced ONFH.

Survival and stemness of bone marrow mesenchymal stem cells (BMSCs) in osteonecrotic areas are especially important in the treatment of early steroid-induced osteonecrosis of the femoral head (ONFH). We had previously used BMSCs to repair early steroid-induced ONFH, but the transplanted BMSCs underwent a great deal of stress-induced apoptosis and aging in the oxidative-stress (OS) microenvironment of the femoral-head necrotic area, which limited their efficacy. Our subsequent studies have shown that under OS, massive accumulation of damaged mitochondria in cells is an important factor leading to stress-induced apoptosis and senescence of BMSCs. The main reason for this accumulation is that OS leads to upregulation of protein 53 (P53), which inhibits mitochondrial translocation of Parkin and activation of Parkin’s E3 ubiquitin ligase, which decreases the level of mitophagy and leads to failure of cells to effectively remove damaged mitochondria. However, P53 downregulation can effectively reverse this process. Therefore, we upregulated Parkin and downregulated P53 in BMSCs. We found that this significantly enhanced mitophagy in BMSCs, decreased the accumulation of damaged mitochondria in cells, effectively resisted stress-induced BMSCs apoptosis and senescence, and improved the effect of BMSCs transplantation on early steroid-induced ONFH.

The imbalance between osteogenesis and adipogenesis in the femoral head is a major pathogenic mechanism underlying glucocorticoid (GC)-induced osteonecrosis of the femoral head (GIONFH), yet its specific molecular pathogenesis remains elusive. Dexras1 has been reported to mediate GC-induced osteogenesis-adipogenesis imbalance in osteoporosis, but its functional role and related mechanisms in GIONFH remain unclear. Here, we first demonstrated that Dexras1 expression was upregulated in rat models of GIONFH. Using Dexras1-knockout (KO) rats, radiographic and histological assessments demonstrated that Dexras1 ablation attenuated the osteonecrosis severity and restored the osteogenesis-adipogenesis balance in the femoral head. Results from RT-PCR, western blotting, alkaline phosphatase staining, and Oil Red O staining showed that Dexras1 KO promoted osteogenesis while inhibiting adipogenesis in bone marrow mesenchymal stem cells (BMSCs), whereas Dexras1 overexpression exerted the opposite effects. Additionally, TUNEL, Cell Counting Kit-8, and flow cytometry assays revealed that Dexras1 had no impact on BMSC viability in the rat GIONFH model. Dihydroethidium (DHE) staining and superoxide dismutase (SOD) activity assays further confirmed that Dexras1 was not involved in GC-induced oxidative stress in the femoral head. Finally, we confirmed that the upregulation of peroxisome proliferator-activated receptor γ (PPARγ) and downregulation of Wnt signaling pathways were potential mechanisms underlying Dexras1-mediated osteogenesis-adipogenesis imbalance by RNA sequencing and some in vitro experiments. Collectively, our results demonstrate that Dexras1 is critical for GIONFH development, as it mediates the imbalance between osteogenesis and adipogenesis.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a serious bone disease commonly seen in patients on long-term glucocorticoid therapy. Although statins have shown some efficacy in improving lipid metabolism, their efficacy in the treatment of SONFH remains limited. PPARγ inhibitors may enhance the efficacy of statins through several mechanisms. This study aims to investigate how PPARγ inhibitors may enhance the effects of statins in the treatment of SONFH by directly inhibiting apoptosis and indirectly modulating lipoprotein subfractions. We first treated osteoblasts in vitro with high concentrations of hormones to simulate the SONFH environment. We then treated the cells with either the PPARγ inhibitor GW9662, the statin lovastatin, or a combination of both. We assessed cell proliferation and apoptosis using CCK-8, flow cytometry and Western blotting. We then established a SONFH rabbit model using high doses of methylprednisolone and lipopolysaccharide. The rabbits were randomly divided into four groups: control group, lovastatin group, GW9662 group and combination therapy group. We observed hip joint MRI before treatment, after 4 weeks of treatment, and 4 weeks after stopping treatment. We performed hematoxylin-eosin staining of the femoral head and analysed serum lipoprotein subfractions using VAP technology. In addition, we used quantitative polymerase chain reaction (qPCR) to analyse the expression of genes related to lipid metabolism at week 3. In vitro experiments showed that both GW9662 and lovastatin effectively inhibited hormone-induced apoptosis. In the animal studies, imaging and pathological results showed that the progression of SONFH was slower in the combination therapy group than in the other groups. VAP analysis showed that the lovastatin group had disturbed lipoprotein subfractions at the fourth week after stopping treatment, while the combination therapy group had more stable lipoprotein subfractions. PPARγ inhibitors significantly enhance the efficacy of statins in the treatment of SONFH by directly inhibiting apoptosis and indirectly modulating lipoprotein subfractions. These findings provide new insights into the clinical management of SONFH and suggest that combination therapy may be an effective strategy.

Autophagy may play an important role in the occurrence and development of glucocorticoid‐induced osteonecrosis of the femoral head (GC‐ONFH). Lithium is a classical autophagy regulator, and lithium can also activate osteogenic pathways, making it a highly promising therapeutic agent for GC‐ONFH. We aimed to evaluate the potential therapeutic effect of lithium on GC‐ONFH. For in vitro experiments, primary osteoblasts of rats were used for investigating the underlying mechanism of lithium's protective effect on GC‐induced autophagy levels and osteogenic activity dysfunction. For in vivo experiments, a rat model of GC‐ONFH was used for evaluating the therapeutic effect of oral lithium on GC‐ONFH and underlying mechanism. Findings demonstrated that GC over‐activated the autophagy of osteoblasts and reduced their osteogenic activity. Lithium reduced the over‐activated autophagy of GC‐treated osteoblasts through PI3K/AKT/mTOR signalling pathway and increased their osteogenic activity. Oral lithium reduced the osteonecrosis rates in a rat model of GC‐ONFH, and restrained the increased expression of autophagy related proteins in bone tissues through PI3K/AKT/mTOR signalling pathway. In conclusion, lithium can restrain over‐activated autophagy by activating PI3K/AKT/mTOR signalling pathway and up‐regulate the expression of genes for bone formation both in GC induced osteoblasts and in a rat model of GC‐ONFH. Lithium may be a promising therapeutic agent for GC‐ONFH. However, the role of autophagy in the pathogenesis of GC‐ONFH remains controversial. Studies are still needed to further explore the role of autophagy in the pathogenesis of GC‐ONFH, and the efficacy of lithium in the treatment of GC‐ONFH and its underlying mechanisms.

It has been demonstrated that both hUCMSC-exo and Cyasterone exhibit protective effects against steroid-induced osteonecrosis of the femoral head (SIONFH). Additionally, studies have shown that CTSD N-glycosylation influences BMSC apoptosis. Based on these findings, we aim to investigate the mechanism of hUCMSCs-exo@Cyasterone in the Dex-induced BMSCs model of SIONFH, focusing on its regulatory role in CTSD N-glycosylation during apoptosis. The SIONFH cell model was induced by dexamethasone (Dex) at a concentration of 10-6mol/L. Experiments with hUMSCs-exo@Cyasterone and CTSD mutants were performed in the BMSC model to analyze proliferation, apoptosis, lysosomal pH, lysosomal membrane permeability, and lysosomal colocalization. Additionally, the expression of apoptosis-related proteins in BMSCs and CTSD in lysosomes and the cytoplasm were examined. MTT, AO staining, EDU staining, flow cytometry, and confocal microscopy revealed that hUMSCs-exo@Cyasterone attenuated the proliferation of Dex-induced BMSCs and reduced the lysosomal membrane permeability. It also decreased the expression level of apoptosis-related proteins including BID, Caspase-3, and Caspase-1, as well as the levels of CTSD in lysosomal and cytoplasm. CTSD-N258A inhibited BMSC apoptosis, enhanced the protective effect of hUMSCs-exo@Cyasterone, and promoted the lysosomal localization of CTSD and the lysosomal membrane permeability. Moreover, CTSD-N258A helped suppress the expression of apoptosis-related proteins and reduced CTSD expression in cytoplasm and lysosomes. hUMSCs-exo@Cyasterone mitigates apoptosis in Dex-induced BMSCs, a cell model of steroid-induced femoral head necrosis, by modulating the N-glycosylation modification of CTSD-N258A.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a severe bone disease associated with long-term glucocorticoid use, characterized by impaired bone metabolism and vascular insufficiency. Bilobalide (BB), a natural sesquiterpene from Ginkgo biloba, exhibits anti-apoptotic, antioxidant, and pro-angiogenic properties, yet its role in SONFH remains unclear. We integrated network pharmacology and molecular docking to predict the targets and pathways of BB in SONFH. Key targets were validated using molecular docking software. For in vivo experiments, a rat SONFH model was established using methylprednisolone (MPS), and BB was administered orally. Micro-CT, H&E staining, TUNEL assay, and immunohistochemistry were employed to evaluate bone microstructure, apoptosis, and the expression of osteogenic and angiogenic markers. Immunofluorescence was used to assess HIF-1α expression in rat femoral head tissues. For in vitro experiments, MC3T3-E1 osteoblasts were treated with dexamethasone(DEX) and BB. Cell viability was detected using the CCK-8 assay, and the protein levels of the HIF-1α and ERK pathways were examined by Western blot. Network pharmacology identified 94 common targets between BB and SONFH, with enrichment in HIF-1 and ERK signaling pathways. Molecular docking confirmed strong binding affinities between BB and core targets. In MPS-induced rats, BB treatment significantly improved bone mineral density, trabecular microstructure, and reduced osteocyte apoptosis. BB also upregulated HIF-1α, Runx2, OCN, CD31, and VEGF expression, indicating enhanced osteogenesis and angiogenesis. In vitro, BB rescued dexamethasone-induced suppression of osteoblast viability and upregulated the ERK/HIF-1α pathway. Bilobalide attenuates SONFH progression by activating the ERK/HIF-1α signaling pathway, promoting osteogenesis and angiogenesis, and reducing osteocyte apoptosis. These findings highlight BB as a promising candidate for SONFH prevention and support the utility of network pharmacology in mechanistic natural product research.