Currently, the pathogenesis of FS remains poorly understood, characterized by a complex interplay of inflammatory responses, pro-inflammatory cytokines, neural and vascular alterations, fibrosis, metabolic factors, and immune dysregulation [16]. Consequently, there was a pressing need for further elucidation of the underlying pathogenic mechanisms. The emergence of high-throughput RNA-Seq technology and advancements in bioinformatics methodologies had provided avenues for identifying potential core genes implicated in these mechanisms. MR, serving as a genetic epidemiological approach, offered a means to overcome inherent limitations in traditional observational studies. In this study, we harnessed the publicly available dataset GSE140731, linked to FS, to conduct a novel combined analysis integrating DEGs and WGCNA. This integrated approach enabled the identification of co-expressed genes associated with FS and elucidation of their functional roles. Subsequently, we innovatively integrated the eQTLs of these co-expressed genes with FS-related GWAS, leading to the identification and experimental validation of four genes implicated in promoting FS onset and progression: ADAMTS1, NR4A2, PARD6G, and SMKR1. Finally, we assessed the diagnostic utility of these four core genes and investigated their association with immune infiltration. To the best of our knowledge, this was the first study to employ such a multifaceted approach in identifying biomarkers for FS.
In that investigation, a comprehensive bioinformatics methodology was employed to identify 295 co-expressed genes by intersecting the screened DEGs with key modules identified through WGCNA. Results from GO and KEGG enrichment analyses revealed that those co-expressed genes primarily participated in biological processes related to extracellular matrix organization, collagen metabolism, and endoplasmic reticulum function. Moreover, KEGG pathway analysis unveiled significant enrichment in pathways including cytokine-cytokine receptor interaction, TNF signaling, and AGE-RAGE signaling in diabetic complications. Noteworthy pathways such as relaxin signaling, ECM-receptor interaction, interleukin-17 (IL-17) signaling, and Toll-like receptor signaling were also prominently enriched. Previous studies by Yang et al. and Yano et al. had implicated interleukin-6 (IL6) and advanced glycation end-products (AGEs), respectively, in the fibrotic process of FS. Specifically, IL6 had been shown to promote fibrosis via the PI3K-Akt signaling pathway, while AGEs contributed to fibrosis through the activation of NF-kB signaling by binding to RAGE [17, 18]. Additionally, relaxin, a peptide hormone, had been identified as capable of inhibiting myofibroblast activation in inflammation and fibrosis by activating multiple signaling pathways [19]. Furthermore, research by Moeed et al. had indicated that IL-17 produced by T cell subsets within joint capsule tissue induced fibrosis and inflammation of fibroblasts by upregulating its signaling receptor expression [20]. Consistent with those findings, previous pathological studies had characterized FS histologically by the presence of type I and type III collagen matrices populated with fibroblasts and myofibroblasts [21–23]. Clinically, the symptoms of pain and restricted range of motion observed in patients were largely attributed to chronic inflammation and fibrosis of the joint capsule, aligning with the results of our investigation.
ADAMTS1, a protein with multifaceted biological functions, plays a crucial role in various processes such as extracellular matrix organization, angiogenesis, and inflammation regulation [24, 25]. Notably, ADAMTS1 is involved in the regulation of extracellular matrix organization and fibrosis, making it a key player in these mechanisms [26]. Studies conducted on mouse cardiac tissue have demonstrated that the cysteine-rich acidic secreted protein leads to age-dependent collagen deposition in the heart by upregulating ADAMTS1 levels [27]. Moreover, increased expression of ADAMTS1 in rat kidney tissue has been associated with fibrosis and inflammation [28]. In mouse muscle tissue, silencing ADAMTS1 expression has shown promise in alleviating muscle fibrosis [29]. In addition to its role in fibrosis, ADAMTS1 has been implicated in the regulation of angiogenesis. Research on mouse kidney tissue has suggested that ADAMTS1 may promote the formation of unstable blood vessels by modulating the signaling mediated by VEGFR2 [30]. Similarly, in mouse skin tissue, ADAMTS1 has been found to participate in pathological angiogenesis induced by VEGF-A/VPF [31]. However, the involvement of the ADAMTS1 gene in FS remains unexplored. Our findings indicate a significant causal relationship between elevated ADAMTS1 expression and the occurrence of FS. We postulate that under pathological conditions, ADAMTS1 promotes the development of FS by inducing local collagen deposition, fibrosis, and angiogenesis.
NR4A2, belonging to the nuclear receptor superfamily, acted as a ligand-activated transcription factor with diverse roles in various organs [32]. Studies had indicated the regulatory effects of NR4A2 on pro-inflammatory cytokine expression and fibrosis in different diseases [32, 33]. For instance, in a mouse model of multiple sclerosis, abnormal NR4A2 expression enhanced the promoter activity of interleukin-17 and interferon-gamma genes, resulting in excessive cytokine production [34]. Similarly, aberrant NR4A2 expression in mouse liver tissue induced inflammation and fibrosis [32]. However, there was a scarcity of studies investigating the association between NR4A2 and FS. In our MR analysis, we observed a causal relationship between increased NR4A2 expression and the occurrence of FS. We hypothesized that elevated NR4A2 expression might trigger FS by promoting the expression of inflammatory factors and fibrosis. The PAR6 protein family, initially identified in Caenorhabditis elegans and fruit flies, was known to be essential for establishing cell polarity [35]. In mammals, there were three homologs of Par6: PARD6A, PARD6B, and PARD6G. Notably, specific gene knockout studies had suggested that PARD6G was involved in regulating osteoblast proliferation and differentiation, thereby implicating it in cell proliferation [36]. However, the functions of PARD6G and SMKR1 genes in the context of FS remained largely unexplored. Further research was needed to elucidate the precise roles of these two genes in the pathogenesis of FS.
Then, we performed a comprehensive assessment of immune cell infiltration in FS using CIBERSORT. The results indicated that Tregs and Macrophages M1 were highly infiltrated in the synovial tissues of FS patients. Our experimental results showed that the expressions of these makers about these immune cells were increased, further demonstrating the enrichment of specific immune cell infiltrations of FS. In previous reports, multiple immune cells including B lymphocytes, T lymphocytes, macrophages and mast cells were present in the synovium of FS [37], which was consistent with our analysis. During the early stages of wound repair, unstimulated Macrophages M0 were converted to macrophages M1 to produce the cytokines such as TNF-α and nitric oxide, which amplified the inflammatory response and promoted myofibroblast proliferation and fibroblast recruitment [38]. Therefore, we speculate that M1 macrophages play a role in promoting chronic inflammatory responses and accelerating fibrosis in the early stages of FS. Another study reported that it has been shown that Tregs can secrete the pro-fibrotic cytokine transforming growth factor beta [39]. Boveda-Ruiz et al. found that Tregs played a role in promoting pulmonary fibrosis in a mouse model, but exerted a suppressive role in the late stage of the disease [40], Similar complex functions of Tregs during fibrosis progression have been observed in other studies [41]. Therefore, we speculate that the role of Tregs may be involved throughout the development of FS, exerting either protective or pathogenic effects at different stages of FS development. Additionally, we investigated the pivotal involvement of immune infiltrating cells and core genes in the context of FS. Our findings unveiled a significant positive correlation between ADAMTS1 and plasma cells (r = 0.39, p = 0.0057). Prior research had highlighted the crucial role of plasma cells as critical effectors in fibrosis progression, particularly in mouse lung tissue [41]. Therefore, these results provided further evidence supporting the potential influence of ADAMTS1 on the advancement of fibrosis in FS through the regulation of plasma cell function. This finding underscored the significance of immune infiltrating cells and their interplay with core genes in the pathogenesis of FS.
However, this study had several limitations that must be acknowledged. Firstly, the GSE140731 dataset utilized in this research was obtained from a public database, and there was a lack of clear definition regarding the staging of FS patients. This ambiguity in patient staging might potentially impact the interpretation of the obtained results. Furthermore, the precise molecular mechanisms underlying the pathogenesis of FS, specifically concerning the upregulation of ADAMTS1, NR4A2, PARD6G and SMKR1, necessitated further investigation through future animal and cell experiments. These additional studies would contribute to a more comprehensive understanding of the role and function of these genes in FS pathology.