Pemphigus vulgaris, as a chronic, autoimmune bullous skin disease, manifests with the formation of blisters that develop when autoantibodies compromise the desmosome adhesion structures among keratinocytes [13]. DNA methylation modifications play a vital role in the regulation of gene expression. In general, the higher the degree of CpG methylation, the lower the gene expression levels. In this study, analysis of peripheral blood DNA methylation from patients with pemphigus vulgaris revealed DMRs within the promoter, exon, intron, and downstream gene regions. Such DMRs may contribute to the differential expression of key genes involved in the disease, thereby influencing critical biological processes underlying its pathogenesis.

The GO enrichment analysis revealed that numerous aberrantly methylated genes were enriched in processes like protein, nucleotide, and nucleic acid binding, as well as the negative regulation of transcription by RNA polymerase II and calcium ion binding, among other molecular functions and biological processes. KEGG enrichment analysis indicated that these DE methylated genes were mainly enriched in metabolic pathways such as lipid, coenzyme and vitamin, amino acid, and carbohydrate metabolism, as well as in signaling pathways involving signal transduction, the immune system, and translation processes. Among these genes, 58 aberrantly methylated genes played a biological role via the mTOR signaling pathway.

The integrated analysis of methylation, mRNA, and proteomics data revealed involvement of DE genes in several key biological processes such as platelet activation, innate and inflammatory immune responses, cytokine production, and positive regulation of adhesion plaque assembly. Cellular components of interest were primarily extracellular, such as exosomes of extracellular vesicles, the extracellular space, and the plasma membrane. Molecular functions mainly included protein, immunoglobulin, calcium ion, and receptor binding. Central to the pathogenesis of pemphigus is the production of immunoglobulin (Ig) antibodies targeting keratinocyte surface proteins. The main target antigens are desmoglein 1 and desmoglein 3 of the desmoglein subfamily of cadherin adhesion molecules. IgG antibodies against desmogleins can destroy the adhesion function between keratinocytes, resulting in their separation from each other and blister formation. The analysis revealed disruptions in signaling pathways related to focal adhesion and tight junctions, suggesting that aberrant DNA methylation patterns in the peripheral blood of patients with pemphigus vulgaris may drive the abnormal expression of structural proteins crucial to intercellular junctions, thus compromising cell-cell adhesion.

GO enrichment statistical analysis revealed that DE genes from the two groups were mainly involved in biological functions such as platelet activation and coagulation, cellular structure adhesion, and immunoglobulin binding. KEGG enrichment analysis suggested that the DE genes were mainly concentrated in pathways related to bacterial infection, signal transduction, and immune system function. The KEGG enrichment analysis revealed that the main disrupted pathways were those involved in platelet activation, adhesion plaque, tight junctions, and infectious inflammation.

These results reveal the potential biological processes and pathways driving the observed differences in gene expression between the two groups. Such analyses enhance the current understanding of disease pathogenesis and also offer insights to guide the identification of potential drug targets and novel treatment strategies. For instance, if specific genes or pathways are found to be associated with specific diseases, these can become key targets for drug development. In addition, comparative analyses of gene expression patterns across various diseases or conditions can help identify potential therapeutic interventions.

In this study, an integrative analysis of methylation-associated transcriptomics and proteomics identified abnormally expressed genes, including FGA (fibrinogen alpha chain), VWF (von Willebrand factor), and ACTG1 (Homo sapiens actin gamma 1), all of which are involved in platelet activation. In addition to their prominent function in hemostasis and thrombosis, platelets also serve as immune cells by initiating and regulating inflammation and immune responses [14, 15]. Platelets house a large number of immune-related molecules, and platelet activation can occur through stimulation by thrombin, chemokines, and microbial toxins [16, 17]. At the same time, adhesive and immune receptors—including P-selectin, CD 40 ligands, and toll-like receptors—are expressed on the surface of platelet cells, and they release soluble mediators such as chemokines, cytokines, and antimicrobial peptides [18, 19]. Platelets further interact with endothelial cells and leukocytes (including dendritic cells, T cells, B cells, neutrophils, monocytes, and natural killer cells) via both direct cell-to-cell contact and indirect mechanisms involving soluble mediator secretion [20].

Platelets play a prominent role in inflammatory skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis through several pathological mechanisms. For instance, the formation of platelet-leukocyte complexes enhances leukocyte rolling on the endothelium, leading to the release of inflammatory mediators, including chemokines. This promotes the recruitment of leukocytes into inflamed skin, inhibits monocyte apoptosis, induces neutrophil cytophagy, promotes sensitization, and causes pruritus, collectively regulating inflammation [21,22,23,24]. Furthermore, platelets can also detect bacterial pathogens via interactions involving toll-like receptors, subsequently releasing antimicrobial peptides or clustering around bacteria to eliminate them [25]. Therefore, platelets are integral to both innate and acquired immune responses within the skin, engaging in complex interactions with leukocytes and endothelial cells.

In their retrospective study, Kridin et al. highlighted the crucial role of platelets in pemphigus vulgaris, noting that the mean platelet volume (MPV) of patients was reduced and that MPV was negatively correlated with severity of the disease. They also found that the MPV of patients with laryngeal involvement was lower than those without laryngeal involvement. Compared to healthy controls, MPV values were reduced in patients with pemphigus vulgaris [26]. In the present study, there was a notable increase in gene and protein expression levels related to platelet activation, suggesting that platelet activation plays an important role in the pathogenesis of pemphigus vulgaris.

The primary limitation of this study is the relatively small sample size, with only the minimum number of samples included to enable effective statistical analysis. Larger samples are necessary to validate these findings in future research.

In conclusion, numerous abnormally methylated genes were identified in this study, suggesting that changes in the transcription and expression of these genes may be associated with the pathogenesis of pemphigus vulgaris. Among these genes, FGA, VWF, and ACTG1 were abnormally expressed, supporting the hypothesis that platelet activation may play an important role in the progression of pemphigus vulgaris.