Although there is a strong correlation between AS and HLA-B27, the fact that many individuals with human leukocyte antigen B27 never develop AS suggests that there can be other factors that influence the onset and development of AS [12], and that diagnosis is often delayed due to insidious onset and lack of tissue biomarkers [7]. In this study, biological roles and signaling pathways were examined for seven core genes using bioinformatics analysis.
Many previous studies found that some immune-related pathways are significantly enhanced in AS patients[13, 14], in the same way, n our study, the results of functional enrichment analysis showed that DEGs were significantly associated with immune-related functions and inflammatory signals, enriched KEGG pathways are cytolytic granule, leukocyte activation The enriched KEGG pathways are cytolytic granule, leukocyte activation, immune receptor activity, regulation of cell killing, and adaptive immune response; the enriched KEGG pathways are Apoptosis, Hematopoietic cell lineage. These results suggest that the immune response plays a key role in the course of AS.
The immune microenvironment needs to be focused on in the diagnosis and treatment of diseases. In order to further explore the potential mechanism of key genes in AS, using the CIBERSORT algorithm, we found that T cells CD4 naive, Neutrophils levels were significantly increasing in the disease group samples. Our study also found that most of the key genes were positively correlated with Neutrophils, Macrophages M0 and negatively correlated with NK cells resting ,T cells CD8, further confirmed that immune disorders are important for the progression of AS.
CD4 + T cells have been implicated in the pathogenesis of many inflammatory arthritis and produce the proinflammatory cytokine IL-17 (Th17). [15]. The reciprocal influence between CD4 + T cells and human leukocyte antigen B27 leads to a cascade of chemokines and cytokines that promote inflammatory responses and bone erosion in AS [16]. Previous studies have confirmed naive CD4+/CD8 + T cells are increased, while memory CD4+/CD8 + T cells and terminally differentiated CD4+/CD8 + cells are decreased in AS patients [17], and the course of disease was positively correlated with the initial CD4 + level [18]. TNF-α inhibitor, a clinically used anti-AS drug, acts by increasing the proportion of negative regulatory T cells and decreasing the proportion of naive CD4 + T cells in AS patients. (e.g., Tregs and Bregs) in inflammation [17, 19]. Leukocytes are an important component of the innate immune system and usually play a key role in the chronic inflammatory response of autoimmune diseases [20], and GSEA results indicated that most key genes were enriched in the leukocyte migration pathway, consistent with previously reported results [21, 22].
According to previous reports, glycogen phosphorylase (PYGL) and Complement component 5a receptor (C5aR1) promote the inflammatory response in psoriasis [23, 24]. Neutrophils can store large amounts of glycogen, and inhibition of PYGL reduces the number of neutrophil extracellular traps [25].C5a/C5aR1 is essential for neutrophil re-recruitment in tissues in response to immunoglobulin autoantibody deposition [26], and results from Zheng et al. showed that C5a/C5aR1 signaling enhanced recruitment of plasmacytoid dendritic cells, monocytes and neutrophils [24]. Sortilin-related receptor 1 (SORL1) is now known to be closely related to the pathogenesis of familial and sporadic Alzheimer's disease [27–29]. Recently, Jiang et al [30] reported that SORL is also closely related to AS and may be a key factor in the pathogenesis of AS. In this study, The neutrophil count of AS patients was significantly higher than that of the control group, and the number of neutrophils was positively correlated with the expression of SORL. which is consistent with our findings. In the past, desmoglein (DYSF) was mainly studied as one of the most common subgroups causing Dysferlinopathy [31], and a recent study [32] reported that DYSF can be used as a DNA methylation driver gene to promote monocyte activation, thereby promoting the occurrence of atherosclerotic cardiovascular disease, suggesting that transcriptional activator 3 (STAT3) may be an upstream regulator promoting DYSF. Th17 cells can secrete proinflammatory cytokines, and their differentiation is regulated by STAT3 [33]. Several studies have shown that the imbalance of IL-23/IL-17 axis of Th17 cells can promote the occurrence and development of a variety of inflammatory diseases. [6, 34]. Recently, Liang et al [35] found that Osteoblast differentiation and bone formation in AS may be regulated by STAT3 and SPI1 through MAPK signaling pathway, JAK/STAT and Wnt receptors, and interestingly, GSEA results showed that DYSF is highly enriched in MAPK signaling pathway, and the strong correlation between STAT3 and DYSF suggests that DYSF in AS inflammatory response may likewise play an important role. The SPI1 gene encoding PU.1 has been shown to affect the differentiation and function of multiple myeloid cells [36–39], It plays a key role in the human immune system and has been shown to be involved in the pathogenesis of many immune-related tumor diseases [40, 41]. Recently, many researchers have found that PU.1 plays an important role in the transcriptional control of certain immune cells and the susceptibility to immune diseases. For example, PU.1 can facilitate the progression of rheumatoid arthritis by fibroblast-like synoviocytes and inhibiting macrophages [42] and promotes the expression of pro-inflammatory cytokines by inhibiting miR-150 in autoimmune encephalitis macrophages [43]. Over and above the previously mentioned important role of SPI1 in osteoblast differentiation and function reported by Liang et al [35], It has been reported that SPI1 can regulate the differentiation of dental pulp stem cells by inhibiting the expression of noggin [44]. This may explain the occurrence of ectopic ossification of AS, which in turn leads to spinal fusion. Brain acid-soluble protein 1 (BASP1) is highly expressed in brain tissue and promotes brain tissue development by participating in axon regeneration. Basp1 is a membrane-bound protein in essence. [45, 46], is closely associated with several tumor diseases [47–50], plays a role in apoptosis, differentiation and transcriptional regulation, and is a potential tumor suppressor [51–54]. As an enzyme, ANPEP (CD13) plays an important role in the pathogenesis of multiple inflammatory diseases by regulating the activity of several cytokines through cleavage of the N terminus and by cutting down the polypeptides bound to MHC II involved in antigen processing, which can regulate the development and activity of immune cells [55].
By differential analysis of known ankylosing spondylitis morbigenous genes in the Gene Cards database, we found that the expression of IL10, IL17A, IL23R, TLR4, TNF, and TNFRSF1A genes differed between the two groups of patients, with BASP1 expression levels significantly and positively correlated with TNFRSF1A expression levels (Pearson r = 0.77), and SORL1 was significantly negatively correlated with TNF (Pearson r=-0.3). A total of 85 mirnas and 234 mRNA-miRNA relationship pairs were obtained by reverse prediction of 7 core genes through the mircode database, which requires more studies to explore the specific molecular mechanisms involved.
Genetics plays a significant role in the pathogenesis of AS, and in recent years there has been an increasing focus on genetic factors in AS, leading to the discovery of several drugs [56]. The Gene Atlas genetic mapping database uses the UK Biobank cohort to document relevancy between hundreds of traits and millions of variants, with ankylosing spondylitis having the greatest SNP genetic power, and a high susceptibility [57]. Disease-associated variants can be identified through genome-wide association studies (GWAS), and GWAS have recorded more than 100 motifs including those involved in antigen presentation, Th17 response, macrophages and T cells, especially HLA-B27 and ERAP1, with strong associations with AS pathogenesis [58–60]. It is difficult to identify the SNP sites of pathogenic genes, which limits the translation of genetic research results to clinical practice [61]. In this study, we analyzed GWAS data in ankylosing spondylitis and identified the pathogenic regions of seven core genes in ankylosing spondylitis, described the key SNP loci distributed in the enriched regions, and demonstrated the SNP pathogenic regions corresponding to the seven genes.
We used the Connectivity Map database to predict potentially useful drugs in AS treatment and found that the expression profiles of ibuprofen, forskolin, bongkrek-acid, and cimaterol drug perturbations were most significantly negatively correlated with the expression profiles of disease perturbations, These results indicate that these drugs have an inhibitory effect on the progression of AS.