Steroid-induced osteonecrosis of the femoral head (SONFH) is a skeletal disorder caused by prolonged or excessive glucocorticoid use, characterized by the progressive necrosis of femoral head cells. This can ultimately lead to degenerative arthritis and collapse of the femoral head (1). Each year, approximately 20,000 to 30,000 new cases of SONFH are reported globally (2). The disease progresses rapidly and is associated with a high disability rate, resulting in a substantial economic burden on patients and potentially diminishing their long-term quality of life (3). Due to its destructive nature, early intervention in SONFH is crucial. Current treatment strategies primarily involve conservative therapies such as anticoagulants, vasodilators, statins, bisphosphonates, and physical therapy, along with surgical options like core decompression and total hip replacement (4). Although total hip replacement offers a relatively effective solution for advanced cases, postoperative complications and the need for joint revisions exacerbate the patient’s burden, placing additional strain on social healthcare systems (5). Therefore, there is an urgent need for novel therapeutic strategies for SONFH.

In China, traditional Chinese medicine (TCM) has a long history of treating SONFH and plays a significant role in preventing bone cell death and structural damage to the femoral head caused by the condition (6). Shenggu Zaizao Wan (SZW), composed of Epimedium brevicornu Maxim, Davallia trichomanoides Blume, Astragalus membranaceus, Cinnamomi ramulus, Wolfiporia cocos, Rhizoma alismatis, Salvia miltiorrhiza, Panax notoginseng, Crataegus pinnatifida Bunge, and Psoralea corylifolia L., has shown significant clinical efficacy in treating the early and intermediate stages of SONFH. Recent studies suggest that the active components of these herbs are widely used for treating fractures, joint diseases, and various chronic orthopedic conditions, particularly exhibiting notable effects on SONFH (7, 8). Icariin, the active compound in Epimedium, exerts therapeutic effects on femoral head necrosis through anti-inflammatory actions, reduction of vascular damage, prevention of osteocyte apoptosis, promotion of bone marrow mesenchymal stem cell proliferation, reduction of oxidative stress, and inhibition of adipocyte differentiation (9, 10). Additionally, astragaloside IV can alleviate the progression of SONFH by inhibiting endoplasmic reticulum stress (11). Active ingredients such as tanshinone I (TsI) and tanshinone IIA have shown positive effects in antioxidation and antithrombosis (12, 13). Other components also provide vascular protective effects and promote bone repair (14, 15). In recent years, network pharmacology has achieved significant progress in exploring the therapeutic mechanisms of TCM formulas, identifying effective components, and uncovering therapeutic targets. Compared to previous studies on single components, research on the interactions between multiple components and targets offers a more accurate reflection of the in situ conditions in clinical practice (16). Despite these advancements, the treatment of SONFH with SZW remains based on TCM theory (17) and lacks modern pharmacological research, leaving its molecular mechanisms unclear. Identifying new targets and molecular mechanisms of SZW and its active components in the treatment of SONFH is of significant importance.

In this study, transcriptomic data related to SONFH were obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were identified. Disease targets associated with SONFH and targets of SZW acting on SONFH were sourced from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Herbal Encyclopedia for Research on Bioactivity and Chemical Constituents (HERB), and GeneCards databases. These targets were then intersected with DEGs to identify candidate genes. Machine learning and expression validation methods were employed to screen potential biomarkers. By integrating the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and the protein–protein interaction (PPI) network, the potential molecular mechanisms of these biomarkers in SONFH were elucidated. Finally, molecular docking was used to evaluate the binding affinity of biomarkers to the active ingredients in SZW, verifying the molecular mechanisms of SZW and its active components in the treatment of SONFH. The findings have significant theoretical and practical implications for improving clinical treatments and developing innovative targeted therapeutic agents.

SONFH is a destructive osteoarticular disease caused by long-term and excessive glucocorticoid use, resulting in femoral head collapse, hip pain, and functional impairment (33). The treatment of SONFH presents numerous challenges, highlighting the need for new therapeutic strategies. Using network pharmacology, this study initially identified 283 targets and 124 active components, with Inhibitor of Nuclear Factor Kappa B Kinase Subunit Beta (IKBKB) and Protein Kinase C Alpha (PRKCA) identified as key biomarkers for the therapeutic effects of SZW on SONFH. Additionally, the potential molecular mechanisms of these biomarkers in SONFH were analyzed through KEGG enrichment analysis and PPI network construction. GSEA results suggested that IKBKB and PRKCA may be involved in SONFH progression through pathways related to ribosomes, spliceosomes, and other mechanisms. Immune infiltration analysis revealed that activated CD8⁺ T cells, central memory CD4⁺ T cells, and other immune cell types may significantly impact SONFH. Molecular docking results further demonstrated that certain active ingredients of SZW exhibit strong binding affinities with these biomarkers. Additionally, RT-qPCR analysis showed significant differences in the expression levels of IKBKB and PRKCA between SONFH and control samples. The core innovation of this study lies in the identification of the association between the clinical formula SZW and two key signaling molecules (IKBKB and PRKCA), whose roles in SONFH have not been fully elucidated. This study provides modern scientific evidence for the traditional Chinese herbal compound SZW, laying a theoretical foundation for further mechanistic exploration and optimization of its clinical application.

IKBKB encodes the IKKβ protein, which functions as the central kinase in the NF-κB classical signaling pathway. Along with IKKα and NEMO (IKKγ), IKKβ forms the IKK kinase complex responsible for phosphorylating and degrading the Iκ-Bα protein. This degradation releases RelA/p50 dimers, allowing them to translocate to the nucleus and regulate the expression of target genes (34). Biallelic mutations in IKBKB that result in loss of function cause severe combined immunodeficiency, characterized by reduced T cell populations, developmental abnormalities, and impaired immune responses (35). Additionally, IKBKB plays a pivotal role in B cell maturation, immune responses, cell survival, and the production of antigen-specific antibodies (36). It is also involved in antigen presentation and immune functions in dendritic cells and macrophages, emphasizing its widespread influence on the immune system (37, 38). Previous research has shown that NF-κB signaling mediated by NF-κB-inducing kinase (NIK) is vital for osteoclast differentiation (39). A study further indicated that circIKBKB is upregulated in breast cancer bone metastasis tissues, and its overexpression enhances osteoclastogenesis, promoting the formation of a pre-metastatic niche (40). Moreover, studies on SONFH have demonstrated a significant correlation between IKBKB expression and immune cell function (41). The present study found a notable increase in IKBKB transcription levels in patients with SONFH, and clinical sample validation further corroborated these findings, suggesting that IKBKB plays a role in SONFH progression. Molecular docking analysis in this study revealed that the active components taxifolin and luteolin in SZW have a high binding affinity for the IKBKB protein, suggesting that SZW may regulate IKBKB. Existing literature reports that both taxifolin and luteolin inhibit the activation of the NF-κB signaling pathway (42, 43). Based on the observed upregulation of IKBKB in SONFH and its role in the NF-κB pathway, SZW may target IKBKB through components like taxifolin and luteolin, modulating its kinase activity and preventing excessive NF-κB activation. This could be a potential mechanism through which SZW alleviates inflammation in SONFH, regulates immune cell function, and suppresses abnormal bone resorption. However, this hypothesis requires experimental validation in SONFH models to directly confirm whether SZW and its specific components exert therapeutic effects via IKBKB.

PPRKCA is a member of the serine/threonine-specific protein kinase C family, involved in mediating cell growth and inflammatory responses (44). Overexpression of PRKCA inhibits the secretion of lipopolysaccharide (LPS)-induced inflammatory factors, including IL-1β, TNF-α, and IL-6, thereby alleviating sepsis-induced acute lung injury in mice. PRKCA and its encoded protein kinase (PKCα) primarily regulate cellular proliferation, differentiation, and anti-apoptotic signaling, making them pivotal in tumor formation and chemotherapy resistance (45). PKCα inhibits osteoblast differentiation while promoting osteoblast proliferation (46), highlighting its role in regulating the cellular signaling involved in osteoblast differentiation and proliferation. However, the detailed molecular regulatory mechanisms of PRKCA remain unclear, requiring further investigation, particularly in the context of osteonecrosis of the femoral head. This study identified a significant upregulation of PRKCA expression in both SONFH clinical samples and public transcriptomic data (GSE123568), suggesting its potential involvement in the pathological progression of SONFH. Similarly, molecular docking results revealed high binding affinity between tanshinone IIA, an active component of SZW, and PRKCA. Previous studies have confirmed that tanshinone IIA inhibits osteoclast differentiation and bone resorption (47). Based on these findings, SZW may modulate PRKCA through components such as tanshinone IIA, influencing its downstream signaling pathways. This mechanism could help regulate the balance between osteogenesis and osteoclastogenesis, potentially offering bone-protective effects in SONFH. Future research is needed to further investigate the specific role of PRKCA in SONFH and determine whether it serves as a direct target for SZW’s therapeutic effects, providing additional evidence for SZW in treating SONFH.

According to the GSEA results, IKBKB is significantly enriched in the oxidative phosphorylation pathway, while PRKCA is enriched in the RNA degradation and ubiquitin-mediated proteolysis pathways. Both IKBKB and PRKCA may be closely associated with SONFH through multiple pathways, including the ribosome, spliceosome, proteasome, olfactory transduction, and neuroactive ligand-receptor interactions. The spliceosome, an RNA-protein complex responsible for mRNA splicing, enables alternative splicing, allowing a single gene to produce several proteins through various exon arrangements (48). Circular RNAs (circRNAs), byproducts of precursor mRNAs, act as sponges to regulate miRNA activities, facilitate gene translation via alternative splicing, and interact with RNA-binding proteins (RBPs) (49). Additionally, circRNAs play a role in the physiological and pathological processes of various orthopedic diseases, including osteosarcoma, osteoporosis, and osteoarthritis (50–52). Notably, our findings suggest that spliceosomes may also play a pivotal role in SONFH.

Analysis of peripheral blood immune cell composition revealed significant alterations in the proportions and activity of multiple immune cell subsets in patients with SONFH, including macrophages, neutrophils, activated CD8⁺ T cells, and central memory CD4⁺ T cells. Correlation analysis indicated that activated CD8⁺ T cells exhibited the strongest association with biomarkers, suggesting they may exert a considerable influence on the biomarkers associated with SONFH. Research has demonstrated the presence of neutrophil extracellular traps in the microvascular system surrounding the femoral head in patients with osteonecrosis, suggesting a role for neutrophils in the pathogenesis of SONFH (35). Additionally, studies have shown that macrophages and monocytes serve as sources of osteoclasts, with osteoclast-mediated bone resorption being a critical mechanism in femoral head collapse (53, 54). Previous reports have linked macrophages to bisphosphonate-related osteonecrosis of the jaw and steroid-induced osteonecrosis (55), indicating a close relationship between macrophages and SONFH. A retrospective clinical controlled study revealed that the total lymphocyte count, CD3T cells, Ts cells (CD3CD8), and B-1 cells (CD5CD19) in the peripheral blood of patients with femoral head necrosis were significantly elevated, suggesting an association between the occurrence and progression of femoral head necrosis and immune system imbalances (56). However, this study is based on blood sample analysis, directly reflecting changes in circulating immunity. The involvement of these systemic immune characteristics in the local osteonecrotic process of SONFH requires future validation using lesioned tissue samples or experimental models that more closely replicate the pathological process.

To construct TF-mRNA pairs, two TFs, MLLT1 and BCL11B, were identified as being associated with the key genes IKBKB and PRKCA. Three miRNAs associated with IKBKB, including hsa-miR-503-5P and hsa-miR-196a-5P, were identified, along with twenty-two miRNAs related to PRKCA, such as hsa-miR-24-3P. Furthermore, 260 lncRNAs regulating IKBKB and PRKCA expression were identified, with nine of these lncRNAs serving as effective predictors. In recent years, an increasing number of miRNAs have been implicated in osteonecrosis, with mechanisms such as coagulation disorders, abnormal apoptosis, and lipid metabolism dysregulation identified as contributors (57–59). Additionally, lncRNAs can competitively bind to miRNAs through the “sponge effect,” regulating the proliferation and function of osteoblasts (60). Therefore, these TFs, miRNAs, and lncRNAs may play a significant role in SONFH progression by modulating the expression of biomarkers and influencing disease progression.

This study has several limitations. First, the clinical sample size used for RT-qPCR validation was relatively small, which, although providing preliminary evidence for differential expression, may impact statistical power and limit the generalizability of the findings. Second, the validation dataset (GSE74089) used for trend observation differed from the primary training set in terms of tissue origin and disease scope, potentially introducing contextual interference in the interpretation of gene expression consistency. Additionally, due to research timeline constraints and challenges in sample collection, this study did not establish an intervention group for SZW. As a result, while upregulation of IKBKB and PRKCA was observed in patients with SONFH, it cannot be conclusively confirmed that these factors are the direct therapeutic targets of SZW. Consequently, the related conclusions remain speculative and association-based. Furthermore, although the nomogram diagnostic model demonstrated good discriminatory performance in internal data, the sample size in the training cohort was limited, and independent validation in large, homogeneous clinical cohorts was lacking. The absence of systematic comparisons with existing diagnostic methods further complicates its potential clinical applicability. Therefore, further verification is needed to assess its clinical translation.

To address these limitations, future research will focus on several directions: constructing or utilizing independent SONFH cohorts with well-matched tissues and comprehensive clinical data to validate key biomarkers; designing SZW intervention experiments to determine whether IKBKB and PRKCA serve as response targets of the formula in cellular or animal models; elucidating the specific roles of these two genes in SONFH pathogenesis and progression through gain- and loss-of-function experiments at the mechanistic level; and conducting rigorous external validation of the nomogram model, including comparative evaluations against conventional clinical diagnostic methods, to facilitate its clinical implementation. These follow-up studies are expected to advance the current correlation-based findings into causal mechanistic insights, thereby providing a stronger scientific foundation for the precise diagnosis and targeted therapy of SONFH.

This study leveraged public databases to download SONFH-related transcriptomic data, identifying 1,671 DEGs between the SONFH and control groups. Through network pharmacology, 69 therapeutic targets of SZW for SONFH were identified. The intersection of these 69 targets with the DEGs yielded 11 candidate genes. Machine learning, ROC curve analysis, and expression level validation were subsequently applied to identify two biomarkers (IKBKB and PRKCA) that indicate SZW’s therapeutic effects on SONFH. Finally, the potential molecular mechanisms of these biomarkers as therapeutic targets for SONFH were explored through nomogram construction, GSEA enrichment analysis, immune infiltration analysis, molecular regulatory network analysis, and molecular docking. These findings provide a new theoretical framework for understanding the mechanism of SZW in treating SONFH. However, the effectiveness of these biomarkers in diagnosing and predicting SONFH requires further validation through large-sample clinical trials. Moreover, the regulatory roles of these biomarkers in SONFH need to be experimentally validated in vivo.