Discussion
Despite our understanding of known risk factors, such as obesity, mechanical abnormalities, genetic predisposition, and age, the exact cause of OA remains unclear ( Deveza, Zankl & Hunter, 2023 ). Thus, current therapeutic approaches for OA primarily focus on symptom relief rather than specific treatment ( Makarczyk et al., 2021 ). Previous studies on OA have focused mainly on the synovium, articular cartilage, and meniscus while neglecting the role of subchondral bone in OA development ( Weber, Chan & Wen, 2019 ; Chen et al., 2020 ). However, recent evidence suggests that abnormal metabolism of subchondral bone is associated with articular cartilage degeneration ( Chen et al., 2017a ). Furthermore, an increasing number of research studies have indicated that the immune system plays a crucial role in the development of OA ( Fahy et al., 2015 ; Kalaitzoglou, Griffin & Humphrey, 2017 ). However, the connection between immune cells and subchondral bone disturbance remains ambiguous. Bioinformatics, which combines molecular biology and information technology, holds immense importance in uncovering the molecular mechanisms underlying various diseases. This study used bioinformatics to further understand the differential gene expression and immune infiltration profiles between lesioned and preserved subchondral bone in OA. We identified dysregulated genes associated with OA progression in lesioned and preserved subchondral bone in OA patients. STMN2 and POSTN were the most upregulated genes, and LEP and APOB were the most downregulated. The most enriched pathway associated with the upregulated genes was the Wnt signaling pathway, while the most enriched pathway associated with the downregulated genes was tyrosine metabolism. JUN, TNF, IL-1 β , and LEB were identified as hub genes. LRRC15 and Col11A1 were upregulated and coexpressed in multiple OA tissues. In addition, immune infiltration analysis revealed that many immune cells demonstrated different infiltration abundance scores.

Previous studies have focused on articular cartilage degeneration and overlooked the role of the subchondral bone and synovium. In recent years, increasing studies have demonstrated that the knee joint is an organ and that OA can affect subchondral bone, the synovium, and other tissues ( Aspden & Saunders, 2019 ; Mao et al., 2021 ; Du et al., 2023 ). Subchondral bone links the joint to the diaphyseal bone, provides mechanical support, provides some nutrition, and removes metabolic waste products ( Schilling, 2017 ). Subchondral bone remodeling begins in the early stages of cartilage degeneration. The earliest signs of OA on MRI scanning in the subchondral bone are bone marrow lesions (BMLs; excessive water signals in bone). Subchondral bone remodeling is thought to arise due to mechanical overload ( Donell, 2019 ). The pathological changes in subchondral bone during OA include angiogenesis, de novo bone formation, sensory innervation invasion, bone cysts, sclerosis, and osteophyte formation ( Wang et al., 2017a ; Wang et al., 2017b ; Zhu et al., 2019 ; Zhou et al., 2020 ). Evidence suggests that abnormal bone remodeling may contribute to the development of OA and could be a target for OA therapy ( Chen et al., 2017a ; Hu et al., 2021 ; Kon et al., 2021 ).

The most upregulated genes were stathmin2 (STMN2) and periostin (POSTN), and the most downregulated genes were leptin (LEP) and APOB. STMN2 is associated with sensory neuron growth and contributes to regenerating axons after nerve injury ( Dubový et al., 2018 ). STMN2 may also be involved in OA-related pain. POSTN is a 90-kDa member of the fasciclin family that can induce the expression of proinflammatory factors, such as MMP-9, MMP-10, and MMP-13, leading to extracellular matrix degeneration ( Ohta et al., 2014 ). LEP is a peptide hormone containing 167 amino acids ( Yang et al., 2019 ) that promotes the differentiation of osteoblasts under normal conditions ( Xu et al., 2016 ). LEP expression was increased in OA subchondral osteoblasts and partially elevated the expression levels of alkaline phosphatase, osteocalcin release, collagen1, and TGF- β 1. Given these contradictory findings, the disturbance of osteoblast, osteoclast, and subchondral bone remodeling under OA conditions ( Mutabaruka et al., 2010 ) requires further study.

Among the upregulated DEGs, the most enriched pathways were the Wnt and PI3K-Akt signaling pathways. The Wnt signaling pathway can directly affect subchondral bone cartilage and synovial tissue and has been demonstrated to play important roles in pathology ( Zhou et al., 2017 ; Wang et al., 2019 ). AKT phosphorylation in subchondral bone can promote osteogenic differentiation and osteoblastic proliferation, resulting in aberrant bone formation. In addition, targeting these pathways may alleviate OA development. In contrast, PI3K/AKT inhibition reduces subchondral bone sclerosis by decreasing osteogenesis ( Lin et al., 2018 ). Suppressing the PI3K-Akt signaling pathway can also enhance autophagy and reduce inflammation in chondrocytes ( Xue et al., 2017 ). Pharmaceutically targeting this pathway is a promising approach for OA treatment ( Sun et al., 2020 ).

Regarding the downregulated DEGs, the most enriched pathways included tyrosine metabolism and AMP-activated protein kinase (AMPK) signaling. AMPK mainly regulates energy balance and metabolism. AMPK dysregulation is associated with multiple age-related diseases, including atherosclerosis, cardiovascular disease, diabetes, cancer, neurodegenerative diseases, and OA ( Jeon, 2016 ; Wang et al., 2020 ). Upregulation of phosphorylated and total AMPK expression in articular cartilage can limit OA development and progression in OA animal models ( Zhou et al., 2017a ; Zhou et al., 2017b ; Li et al., 2020 ).

Ten hub genes were identified in the PPI network, and the proteins encoded by these genes, including JUN, TNF, IL-1 β , LEB, CXCL8, and FN1, were the key nodes in the PPI network. These proteins are associated with OA progression, especially JUN, IL-1 β , and TNF ( Rhee et al., 2017 ; Zhao et al., 2019 ; Ahmad et al., 2020 ). JUN is a major component of the transcription factor activator protein-1 (AP-1) family ( Atsaves et al., 2019 ) and can mediate catabolic transcription and cell apoptosis/death. Jun also plays an important role in the TNF and IL-17 signaling pathways ( Cai et al., 2020 ).

LRRC15 and COL11A1 were identified as coexpressed genes in subsequent multiple-tissue dataset analyses and experiments. We propose that these two genes, newly associated with OA, play important roles in the disease pathogenesis. LRRC15, a type I membrane protein comprising 581 amino acids with no obvious intracellular signaling domains, is upregulated by the proinflammatory cytokine TGF β in cancer-associated fibroblasts ( Ben-Ami et al., 2020 ). LRRC15 is highly expressed on stromal fibroblasts and tumor cells, such as those in melanoma, sarcoma, and glioblastoma ( Purcell et al., 2018 ). ABBV-085 is an antibody drug that targets LRRC15 and has been widely studied in antitumor research ( Hingorani et al., 2021 ). Therefore, ABBV-085 may be effective for treating OA. Elevated LRRC15 expression in early OA may indicate chondrocyte activation, promoting disease progression and leading to permanent changes in the phenotype and response of OA chondrocytes. This effect can facilitate the transformation of chondrocyte function and phenotype by regulating the stress response and imbalanced remodeling of the extracellular matrix. Further research is necessary to gain a deeper understanding of how LRRC15 contributes to cartilage damage regulation and the signaling pathways related to chondrocyte function ( Singh et al., 2021 ). In addition, LRRC15 has been found to be significantly downregulated in OVX mice and upregulated upon osteogenic induction in a p65-dependent manner ( Wang et al., 2018 ). Collagen XI, which encodes the α 1 chain of type XI collagen, is essential for collagen fibril formation in articular cartilage ( Blaschke et al., 2000 ). Primary type II collagen with an alpha 1 collagen XI ( α 1(XI)) chain structured into collagen fibrils forms a network in the cartilage ECM that contributes to the retention of proteoglycans and tensile strength in cartilage tissue ( Holyoak et al., 2018 ). Many studies have shown that COL11A1 may increase genetic susceptibility to OA ( Wang et al., 2017a ; Wang et al., 2017b ; Styrkarsdottir et al., 2018 ; Fernández-Torres et al., 2020 ). Changes in COL11A1 can affect chondrocyte maturation and collagen fiber thickness, potentially increasing the risk of various types of OA ( Jacobsen et al., 2024 ). COL11A1 is in the same Wnt/ β -catenin signaling pathway as GDF5 and has been identified as a candidate gene for OA ( Fernández-Torres et al., 2018 ).

Inflammation and angiogenesis can alter the process of subchondral bone modeling ( Weber, Chan & Wen, 2019 ). Immune infiltration analyses revealed that the infiltration of ly endothelial cells, mv endothelial cells, and endothelial cells was significantly positively correlated. There was also a positive correlation between CD4+ T cells and CD4+ Tems. Fewer adipocytes and endothelial cells infiltrated lesioned versus preserved samples, while MSCs, myocytes, plasma cells, Th2 cells, ly endothelial cells, and mv endothelial cells infiltrated more in lesioned samples.