IC is a troubling disease characterized by a complex pathogenesis involving numerous associated conditions. A majority of patients experience anxiety, depression, and other obstacles, which contribute to the complexity and chronicity of treatment. Moreover, misdiagnosis of IC as urinary tract infection, urethral syndrome, pelvic inflammatory disease, or overactive bladder[46] can lead to inappropriate treatment, worsening the patient's state. Enhancing the diagnostic precision of IC is crucial, necessitating the development of more sensitive and specific markers. Increased ROS production in IC/BPS patients is well documented. Jiang et al[47, 48] showed elevated levels of urinary OS biomarkers (8-OHdG, 8-isoprostane), while Ener et al[49] found that the total antioxidant capacity of serum samples from IC/BPS patients was significantly lower than that of controls. These findings, along with the complex interactions between OS, inflammation, and immunity, suggest a new research direction. Therefore, the quest for novel diagnostic markers linked to OS is paramount. These markers not only offer fresh insights for early IC detection but also lay the groundwork for tailored treatment strategies.
TFs serve as major orchestrators of biological processes, exerting control over the expression of multiple gene targets and forming intricate feedback loops. During the early stages of diseases like IC, numerous genes, including many TFs, undergo significant alterations. Previous research has highlighted the involvement of several TFs, such as E2F1, JUN, and TP53[50, 51], in the occurrence process of IC, aligning with our findings. In our study, the TFs associated with hub genes, as predicted, hold promise as novel candidate genes for investigating the regulation of IC pathophysiological processes in future studies. Meanwhile, miRNA research represents a burgeoning area of interest across various scientific disciplines. A wealth of evidence now underscores the critical role of miRNAs in immune system development, as well as their contributions to innate and adaptive responses. miRNAs such as mir-155, mir-181a, mir-146a, mir-150, mir-223, and mir-17-92 have been demonstrated to play regulatory roles in immune cell development, differentiation, and function[52, 53]. Moreover, in a mouse model of cyclophosphamide-induced cystitis, miRNAs like mir-34c-5p, mir-34b-3p, mir-212-3p, mir-449a-5p, and mir-21a-3p, as well as mir-376b-3p, mir-376b-5p, and mir-409-5p, have shown involvement in inflammation and smooth muscle function over the medium term[54]. However, the specific mechanisms by which the miRNAs identified in our study exert their effects on IC remain incompletely elucidated, warranting further investigation into their roles in IC pathology.
In this study, we identified four diagnostic marker genes (BMP2, MMP9, CCK, and NOS3) significantly associated with IC using a combination of DEGs analysis, WGCNA, PPI networks, and machine learning. The ANN model, ROC analysis, and nomogram diagnostic model demonstrated that these markers have excellent discriminatory ability in distinguishing the IC group from controls. These results suggest that BMP2, MMP9, CCK, and NOS3 are significantly involved in the pathological processes of IC and have potential as diagnostic markers and therapeutic targets for this disease. BMP2(bone morphogenetic protein 2), a member of the TGF-β superfamily, is crucial for bone and cartilage development and repair[55]. Recent research indicates that BMP2 overexpression in osteoblasts (specifically MC3T3-E1) inhibits apoptosis, diminishes ROS production, and lowers the secretion of TNF-α, IL-6, and macrophage colony-stimulating factor (M-CSF)[56]. Consequently, BMP2 is deemed beneficial, aligning with our observation of reduced BMP2 expression in the IC patient group. While BMP2 has been extensively studied in orthopedic diseases, its role in IC remains uncertain. Subsequent investigations ought to delve into the precise mechanisms and therapeutic potential of BMP2 in IC, offering novel insights and treatment modalities. MMP9 (Matrix Metalloproteinase-9) is a member of the Zn2+-dependent enzyme family known as matrix metalloproteinases (MMPs). It is involved in various physiological processes, including embryonic development, tissue remodeling, and wound healing[57]. MMP9 not only contributes to tissue remodeling and inflammation but is also implicated in various autoimmune disorders such as systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, rheumatoid arthritis, multiple sclerosis, polymyositis, and atherosclerosis[58]. Recent literature reports a significant elevation in MMP9 expression in a cyclophosphamide-induced rat model of IC[59, 60], correlating with processes such as inflammatory response, cell migration, and tissue damage. This aligns with our discovery of elevated MMP9 expression in the IC patient group. Furthermore, MMPs are integral to the extracellular matrix proteasome and influence the remodeling and degradation of tight junctions (TJs). For instance, certain MMPs family genes, including MMP9, MMP7, and MMP2, have been shown to decrease the expression of TJ proteins[61, 62], implicating MMP-9 in diverse pathogenic mechanisms of IC and suggesting its potential as a therapeutic target. CCK (Cholecystokinin) functions as both a neuropeptide and gut hormone, regulating pancreatic enzyme secretion, gastrointestinal motility, and satiety signaling. It is released by endocrine cells of the small intestine and various neurons in the gastrointestinal tract and central nervous system following ingestion [63]. A significant portion of current research has focused on its regulatory functions within the digestive, nervous, and endocrine systems. While bladder diseases remain poorly understood, our analysis suggests that CCK may contribute to IC occurrence through its impact on OS and immune responses. Consequently, conducting a comprehensive investigation into the mechanisms underlying CCK's involvement in bladder diseases, particularly its modulation of OS in bladder tissues, holds promise for identifying novel therapeutic targets. NOS3(Nitric Oxide Synthase 3), a subtype of nitric oxide synthase (NOS) referred to as eNOS, exhibits significantly higher levels in Hunner type IC bladder samples compared to non-Hunner IC bladder samples, indicating potential involvement of eNOS in the divergent pathogenesis of these IC subtypes[64]. eNOS, an enzyme crucial for NO production. NO serves as a signaling molecule with diverse physiological functions and extensive implications in both physiological and pathophysiological contexts. Its roles encompass the regulation of vascular tone, promotion of angiogenesis in wound healing, modulation of inflammatory responses, and involvement in pathologies such as ischemic cardiovascular disease and malignancies[65]. Under normal conditions, eNOS synthesizes substantial quantities of NO, contributing to the maintenance of homeostasis between the endothelium and adjacent tissues[66]. Similar to endothelial tissue, the urothelial epithelium of the lower urinary tract possesses NOS and produces NO. Inflammation resulting from chronic irritation or infection leads to increased NO production, and the upregulation of NOS in response to chronic inflammation may serve as an adaptive mechanism to enhance spinal nociceptive or reflexive responses triggered by nociceptive inputs from the bladder[67]. This phenomenon has been verified in a study by M V Souza-Fiho et al[68].
Previous studies have established that IC development involves the infiltration of various immune cells, including T cells, B cells, plasma cells, macrophages, neutrophils, and mast cell[10, 69]. Our study conducted a comprehensive analysis of the infiltration extent of 22 immune cell types using the CIBERSORT algorithm. We observed differences in immune cell infiltration levels between the IC and control groups, consistent with previous findings. Furthermore, we discovered associations between the four genes and T cell CD4 memory resting, T cell CD4 memory activated, and eosinophils. CD4+ T cells play multifaceted roles in regulating immune responses, naive CD4+ T cells can differentiate into various subpopulations, including Th1, Th2, Th17, Th22, Tfh and CTLs, each with distinct phenotypes and protective functions. These subpopulations are crucial in pathogen response, immune regulation, and maintaining immune homeostasis[70]. CD4+ T cells play a critical immunoregulatory role in the occurrence and progression of IC[10], they recruit and activate other immune cells through the secretion of various cytokines and chemokines, thereby contributing significantly to the immune response in IC and research has indicated a significant increase in dysfunctional CD4+ T cells in IC patients[71]. Eosinophilic primarily maintain the tissue microenvironment during normal development and regulate host innate and adaptive immune responses[72]. Eosinophils express and release epithelial-mesenchymal transition mediators, including TGF-β, basic fibroblast growth factor, platelet-derived growth factor, matrix metalloproteinases, and vascular endothelial growth factor. They also release other repair/remodeling factors, such as nerve growth factor, neuropeptides, and cytokines like IL-1β and IL-6[73]. Despite extensive study, the specific role of eosinophils in IC remains unclear. The existing research suggests that eosinophils may play a role in the pathological process of IC, particularly through allergy-related mechanisms. Some scholars[74, 75] speculate that allergic reactions could potentially trigger IC, with eosinophils playing a significant role in this response. They found significantly elevated levels of eosinophilic protein X in the morning urine of interstitial cystitis patients compared to controls, along with occasional eosinophil infiltration in the submucosal layer upon bladder tissue biopsy. Additionally, researchers[64] have observed more severe or moderate eosinophilic infiltration in Hunner IC bladder specimens compared to non-Hunner IC specimens. Therefore, increased eosinophils may contribute to the progression of IC. Further studies are necessary to elucidate the specific functions of eosinophils in IC disease progression and symptom presentation. To investigate the potential biological functions of the four genes in IC, we found, using GSEA, that they are associated with primary immunodeficiency and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. PPARs are ligand-dependent transcription factors belonging to the nuclear hormone receptor superfamily. PPARα is one of its subtypes. PPARα is expressed in macrophages, dendritic cells (DCs), B cells, and T cells, actively participating in various aspects of immune regulation by modulating cytokine production in DCs and T cells, as well as lymphocyte proliferation[76]. Furthermore, research has confirmed that PPARα exhibits anti-inflammatory effects by inhibiting the NF-κB signaling pathway and reducing the expression of inducible NOS, cyclooxygenase-2, and TNF-α. This finding further demonstrates the association between OS, inflammation, and immunity. Additionally, the PPAR signaling pathway is considered a viable therapeutic target for treating chronic pain and its stress-related psychiatric complications[77]. Therefore, further investigation into the role of these four genes in the PPAR signaling pathway in IC is crucial.
Through screening, we identified Decitabine as the most promising drug candidate for the treatment of IC. Decitabine (5-aza-2'-deoxycytidine, DAC) is a DNA methyltransferase inhibitor with a wide range of antimetabolic and anticancer activities. Recent studies have found that Decitabine alleviates symptoms of acute respiratory distress syndrome through its anti-inflammatory and antioxidant properties and by inhibiting the mitogen-activated protein kinase (MAPK) pathway[78]. Additionally, Chang Su et al[79] found that Decitabine increased the release of the inhibitory factor IL-10, decreased the pro-inflammatory factor IL-17, activated CD4+ Foxp3+ T cells, and elevated levels of zonula occludens 1 and occludin, thereby reducing symptoms of ulcerative colitis. Therefore, Decitabine has potential therapeutic value for the treatment of IC.
There are several limitations to our study. First, it involved secondary data mining and analysis of a previously published dataset, where differences in dataset selection and analysis methods could influence outcomes. Second, due to the absence of clinical samples, validation was limited to the cellular level. Third, the impact of these oxidative stress-related markers on inflammatory and immune responses, contributing to IC occurrence, remains unclear. Therefore, future studies should include diverse populations and larger sample sizes, employing both in vitro and in vivo experiments for validation.