Necrosis of the Femoral Head (NFH) arises from a multitude of etiologies leading to the disruption or damage of blood circulation in the femoral head (Chen et al., 2022). This results in varying degrees of necrosis in bone cells, bone marrow hematopoietic cells, adipocytes, and other vital components of the femoral head, subsequently inducing alterations in its structure, and manifesting as hip pain, dysfunction, and other symptoms (Tan et al., 2012). Once femoral head collapse occurs in a patient, the progression of the disease becomes challenging to reverse, ultimately evolving into severe hip arthritis within a few years, necessitating total hip arthroplasty (Crim et al., 2021; George and Lane, 2022). The damage to joints and associated tissues in Necrosis of the Femoral Head (NFH) typically precedes the manifestation of clinical symptoms (Larson et al., 2018). While Magnetic Resonance Imaging (MRI) is capable of early NFH diagnosis, the majority of patients are diagnosed in the intermediate or advanced stages of the disease, owing to challenges in early detection of NFH and its rapid progression (Liu et al., 2017; Yuan et al., 2022). Consequently, predicting and preemptively preventing osteonecrosis in high-risk groups for Necrosis of the Femoral Head (NFH) is of paramount importance.

Insufficient studies focused on correlation between NFH and immune inflammation. The bone marrow produces a spectrum of immune cells and factors. Meanwhile immune cells, in turn, regulate bone formation and resorption by secreting relevant cytokines, maintaining the dynamic equilibrium of bone mass (Tsukasaki and Takayanagi, 2019). This denotes a bidirectional regulatory relationship between the two systems (Cai et al., 2022). Notably, the previous research has identified anomalous immune cell infiltration in the peripheral blood samples of patients with NFH (Ma et al., 2019). Furthermore, investigations have substantiated that in the initial stages of Necrosis of the Femoral Head (NFH), osteocytes and osteoblasts are more susceptible to apoptosis than to mere cell necrosis (Calder et al., 2004). Consequently, this study aims to analyze the distribution of immune cell molecules in NFH using high-throughput expression profiling technology. This analysis, in conjunction with examining gene expression patterns associated with cell death, seeks to elucidate the immune mechanisms underlying NFH and identify novel therapeutic targets.

NFH is a progressive condition characterized by an inadequate blood supply to the necrotic region, culminating in osteocyte mortality. This sequence of events precipitates the progressive collapse of the femoral head and subsequent joint destruction, ultimately progressing to secondary hip arthritis (Cui et al., 2021; Motta et al., 2022). Inflammation and osteocyte death are the key pathogenic factors of NFH. (Konarski et al., 2022); however, the precise molecular mechanisms underlying their contribution to NFH remain obscure. Post-onset of NFH, necrotic tissue discharges copious cytokines and chemokines, orchestrating the infiltration of inflammatory cells into the affected region, disrupting the existing immune equilibrium, and culminating in extensive osteocyte demise, which leads to the collapse of the femoral head. Consequently, the pathogenesis of NFH is intimately associated with immune infiltration (Ma et al., 2019; Wang et al., 2021). M1 macrophages have been demonstrated to be closely associated with the progression of NFH(Cheng et al., 2023).

In this study, a total of 134 DEGs were obtained based on the intersection of differentially expressed genes in the GSE123568 data set and CDGs in the published literature. GO results showed that DEGs were significantly enriched in apoptosis, necroptosis, and inflammation-related pathways (e.g., interleukin-1-mediated signaling pathway, tumor necrosis factor-mediated signaling pathway). Previous studies have shown that the initial death in the tissue from patients with osteonecrosis, is the apoptosis of osteoblasts and osteocytes, rather than their simple necrosis of osteoblasts and osteocytes (Calder et al., 2004; Plotkin et al., 2007). KEGG enrichment results showed that these genes were mainly related to Apoptosis, Necroptosis, TNF signaling pathway, NOD-like receptor signaling pathway, PI3K-Akt signaling pathway and et al. NOD-like receptor signaling pathway is closely related to immune response. Research has substantiated that M1 macrophages demonstrate a significant correlation with the progression of NFH (Cheng et al., 2023). Furthermore, it has been observed that in macrophages, aberrant activation of the TLR4/NLRP3/GSDMD signaling pathway induces pyroptosis, resulting in atypical accumulation of inflammatory cells in the affected region, thereby markedly exacerbating the inflammatory response (Xu et al., 2021).

We next, constructed the PPI network of differentially expressed genes involved in NFH by STRING database, and 14 hub genes including BCL2L1, BIRC2, NFKBIA, XIAP, CFLAR, AKT1, BIRC3, IKBKB, RIPK1, CASP8, TNFRSF1A, IL1B, CASP1 and STAT3 were identified. KEGG enrichment analysis was used to discuss the molecular mechanism of these 14 hub genes. Reveals these genes were mainly enriched in Apoptosis, TNF signaling pathway, NOD-like receptor signaling pathway, F-kappa B signaling pathway and necrotosis. The enriched pathways basically revolve around ' apoptosis ' and ' necroptosis '. Related studies have found that the level of tumor necrosis factor-α (TNF-α) in the serum of patients with NFH is higher than that of normal people, and the high level of TNF-α promotes the apoptosis of osteoblasts (Zheng et al., 2020). The activation and propagation of the NF-kappa B signaling pathway are pivotal in the formation and activation of osteoclasts (Wu et al., 2019). Excessive activation of osteoclasts can compromise the structural integrity and strength of the femoral head, potentially resulting in its collapse and necrosis (Chen et al., 2020). The expression levels of these 14 Hub genes were identified from the GSE123568 dataset. BCL2L1 and BIRC2 were significantly downregulated, and the remaining 12 were significantly upregulated. The results of the GSE123568 dataset and the GSE74089 dataset were consistent. Finally, we validated 14 biomarkers by collecting clinical samples. The analysis revealed that five of these molecules exhibited significant statistical relevance, and their expression trends were consistent with our previous findings.

BCL2-xL (gene/transcript name BCL2L1) is a member of the BCL-2 family and functions as a prevalent anti-apoptotic protein, promoting cellular survival (Opferman and Kothari, 2018). A study demonstrated that, compared to wild-type, transgenic mice overexpressing BCL-XL exhibited an increase in both trabecular and cortical bone volume, and a concurrent reduction in osteoblast apoptosis (Moriishi et al., 2016).

AKT1, a crucial intracellular signaling molecule, encodes a serine/threonine kinase that plays a significant role in regulating the development, strength, and integrity of normal bones by phosphorylating various downstream target proteins (Cho et al., 2001; Mukherjee et al., 2014). Studies have reported that AKT1 not only aids the survival of cells exposed to apoptotic stress but also promotes angiogenesis by influencing various angiogenic factors (Somanath et al., 2006; Sun et al., 2020). The expression of AKT1 in NFH patients might indicate the body’s response to adverse environmental conditions by activating it, thereby facilitating the repair and regeneration of damaged tissues. Additionally, AKT1 plays a vital role in glucose metabolism by phosphorylating and activating key glycolytic enzymes, such as hexokinase and phosphofructokinase-1, thereby accelerating the glycolysis process (Lee et al., 2017). In a study on gastric cancer, cancer cells increased AKT1 expression to enhance their proliferation and glycolytic capacity (Pan et al., 2019). Correspondingly, under hypoxic conditions, as observed in NFH, osteoblasts and osteocytes may overly rely on glycolysis for energy production (Huffman et al., 2007). AKT1’s promotion of glycolysis aids in sustaining the cellular energy requirements in hypoxic environments.

CASP8, a member of the cysteine-aspartic acid protease (caspase) family, plays a pivotal role in apoptosis and immune system functions (Fritsch et al., 2019). It primarily initiates extrinsic apoptosis, whereupon the activation of CASP8 occurs following the binding of death receptors (such as Fas) to their ligands, subsequently activating other caspases (like CASP3), culminating in apoptosis (Orning and Lien, 2021). Moreover, CASP8 significantly influences the activation and maturation of T lymphocytes. Studies have shown that patients with inflammatory bowel disease characterized by hereditary Casp8 deficiency exhibit T lymphocyte dysfunction (Becker et al., 2013; Lehle et al., 2019). In bone metabolism, T lymphocytes regulate the balance between bone resorption and formation through the secretion of OPG and RANKL (David, 2007). In a retrospective clinical study, researchers observed a notable increase in lymphocyte counts and CD3T lymphocytes in NFH patients compared to healthy individuals (Ma et al., 2019), suggesting that alterations in CASP8 might reflect the immune system’s state, particularly T lymphocytes, in NFH. Furthermore, CASP8 also enhances inflammation by activating relevant cytokines (Phillips et al., 2016). Studies have confirmed that CASP8 directly participates in the cleavage and activation of IL-1β during the maturation of IL-1β in dendritic cells and macrophages under endoplasmic reticulum stress (Bossaller et al., 2012; Shenderov et al., 2014).

TNFRSF1A, a receptor for tumor necrosis factor alpha (TNFα), plays a pivotal role in regulating inflammatory responses and apoptosis through mediating TNF-α signal transduction (McDermott et al., 1999). In the context of bone homeostasis, TNFα′s interaction with TNFRSF1A contributes to the promotion of osteoclastogenesis and the suppression of osteoblast activity (Osta et al., 2014). Furthermore, research on peripheral nerve injury revealed that TNFRSF1A, BIRC2, and BIRC3 in Schwann cells (SC) assume an anti-apoptotic function by modulating NFκB signaling pathway in response to inflammatory stimuli, thereby mitigating peripheral nerve damage (Wang et al., 2012).

XIAP, BIRC2, and BIRC3, members of the inhibitor of apoptosis proteins (IAP) family, are crucial in apoptosis inhibition (Jost and Vucic, 2020). In spinal cord injury (SCI) research, it was observed that XIAP overexpression diminished neuronal caspase activity and apoptotic cell death (Reigada et al., 2023), suggesting that XIAP exerts anti-apoptotic effects by curtailing specific caspase functions (Shi, 2004). XIAP may thus have a protective and reparative role in NFH(Mollazadeh et al., 2015).

STAT3, belonging to the STAT protein family, is a key intracellular signaling molecule vital for inflammation, apoptosis, bone tissue repair and regeneration, and angiogenesis (Zhou et al., 2021). Investigations have shown that STAT3 hampers apoptosis by enhancing the expression of Bcl-2 family anti-apoptotic proteins (Caldera et al., 2008)​​. Moreover, it augments osteogenic differentiation of BMSCs and fosters BMSC-mediated angiogenesis (Chen et al., 2021). The heightened expression of STAT3 in NFH suggests its necessity in fostering new blood vessel formation to alleviate ischemia in necrotic regions and aid in repairing damaged areas.

Immune infiltration analysis indicated a higher proportion of Neutrophil cells and Macrophage M1 cells in the NFH group compared to normal individuals. A primary contributor to NFH is diminished blood flow to the femoral head, resulting in local cellular demise and subsequent inflammation (Margraf et al., 2022). Neutrophils are likely recruited to the affected site as part of the inflammatory response, facilitating the removal of necrotic cells and debris. Moreover, research suggests that Neutrophils can express RANKL and OPG, playing roles in both bone and vascular remodeling (Schruefer et al., 2005; Poubelle et al., 2007). This ability might account for their increased presence in NFH. Macrophage M1 cells, predominantly involved in inflammation (Yunna et al., 2020), become more prevalent in advancing stages of NFH. This shift results in heightened local inflammatory mediators (including IL-1β and TNF-α) and accelerates bone cell mortality (Cheng et al., 2023). Studies have demonstrated that human umbilical cord mesenchymal stem cells convert Macrophage M1 to Macrophage M2 via the AKT/mTOR signaling pathway, mitigating necrosis and osteocyte apoptosis in steroid-induced NFH models (Tian et al., 2022). Subsequent to our analysis, four central immune cells (Neutrophil, Mast cell resting, Myeloid dendritic cell activated, Macrophage M0) were identified utilizing LASSO and RFE algorithms. Correlation analysis between 14 key hub genes and these immune cells revealed a strong positive correlation between STAT3 and Neutrophil cells (r = 0.6804, p = 3.525e-05), and a marked negative correlation between XIAP and activated Myeloid dendritic cells (r = −0.3610, p = 0.04003). Prior research has established that STAT3 facilitates the differentiation of bone marrow progenitor cells into Neutrophil cells while inhibiting their transformation into mononuclear macrophages (Zhang et al., 2023). Given STAT3’s directive role and Neutrophil cells’ significant involvement in inflammatory responses (Margraf et al., 2022), this interaction may significantly influence NFH through mechanisms related to inflammation and immune cell dynamics. Activated Myeloid dendritic cells, pivotal components of the immune system, are crucial in modulating both immune and inflammatory responses (Chistiakov et al., 2015). In the context of NFH, they might exacerbate tissue damage by fostering an inflammatory response. XIAP, functioning as an anti-apoptotic protein that impedes apoptosis (Jost and Vucic, 2020), could safeguard bone cells from harm, potentially decelerating NFH progression. An upsurge in XIAP expression might imply reduced apoptosis, thereby preserving bone tissue integrity. Concurrently, this could diminish the activation necessity of bone marrow-derived dendritic cells, as decreased cellular damage might result in lower inflammation levels. The results of our study provide valuable clues for further study of the pathophysiological mechanism of NFH on immune infiltration.

The main limitation of this study lies in the failure to collect and analyze longitudinal data. Due to the high costs of long-term follow-up and the challenges in data completeness caused by individual patient differences, the implementation of the study was difficult. Additionally, the study design did not directly link biomarkers with clinical outcomes and disease severity. It primarily focused on the identification and preliminary characterization of biomarkers without conducting large-scale clinical etiological research. In the future, we will focus on improving the study design by conducting longitudinal data collection to explore the temporal dynamics of gene expression and immune responses in disease progression, strengthen the association analysis with clinical outcomes, and enhance subsequent clinical etiological research.

In summary, this study utilized bioinformatics analysis to investigate immune cell types in NFH, identifying several hub genes. Experimental validation confirmed that CASP8, TNFRSF1A, AKT1, XIAP and STAT3 can serve as potential biomarkers for early diagnosis, understanding of pathogenic mechanisms, and therapeutic strategy development in NFH. Furthermore, our research also explored the associations between these hub genes and immune cells.