Identification of CCL8, MMP25, and PLP1 as Potential Diagnostic Markers for Interstitial Cystitis/Bladder Pain Syndrome

Identification of CCL8, MMP25, and PLP1 as Potential Diagnostic Markers for Interstitial Cystitis/Bladder Pain Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of CCL8, MMP25, and PLP1 as Potential Diagnostic Markers for Interstitial Cystitis/Bladder Pain Syndrome Changqin Jiang, Guangjie Ji, Jing Chen, Xiao Li, Xiaoling Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9276619/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Purpose Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic syndrome characterized by lower urinary tract symptoms, yet its pathogenesis and diagnostic markers remain unclear. Methods Bladder mucosal tissues from 10 IC/BPS patients and 11 controls underwent whole transcriptome sequencing. Differentially expressed genes and their biological functions were analyzed using GO, KEGG, GSEA, and WGCNA. Hub genes (CCL8, MMP25, PLP1) were validated by internal samples and an external dataset (GSE11873). Immune infiltration was assessed via ssGSEA, and LASSO regression identified hub genes. Results Differentially expressed genes in IC/BPS were enriched in cytokine receptor binding and extracellular matrix synthesis pathways. Increased infiltration of central memory CD8 + T cells (Tcm CD8 + cells) was observed. CCL8, MMP25, and PLP1 were significantly associated with IC/BPS. Conclusion The pathogenesis of IC/BPS may be related to the activation of cytokine receptor binding pathways and the upregulation of Tcm CD8 + cells via CCL8-dependent regulation. This provides new targets and directions for future basic and clinical studies of IC/BPS. Bladder Pain Syndrome Interstitial Cystitis CCL8 immune infiltration biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Interstitial cystitis (IC), also known as bladder pain syndrome (BPS), is a clinical syndrome manifested by urinary frequency, urinary urgency, and pain or discomfort in the suprapubic bladder area. Individuals diagnosed with IC/BPS are predominantly female, with an estimated male-to-female ratio of about 1:10 1 . IC/BPS significantly affects quality of life, as persistent pelvic pain, sexual avoidance, and disrupted sleep interfere with daily functioning and occupational activities. In severe cases, repeated nighttime awakenings increase the risk of falls and related injuries, including fractures. Comorbid anxiety or depression occurs frequently in individuals with IC/BPS, and the suicide rate in this population is reported to be approximately fourfold higher than that of the general population 2 . The etiology of IC is still unclear, which poses a great challenge for diagnosis and treatment. The current treatments of IC/BPS presented limited efficacy and high recurrence rates 3 . Therefore, there is an urgent need for in-depth research on the molecular biology of IC/BPS pathogenesis to provide potentially valuable targets for IC/BPS. The clinical symptoms of patients with IC/BPS are similar to many urological conditions, such as overactive bladder and chronic prostatitis, which leads to easy underdiagnosis and misdiagnosis in many patients. Molecular markers have the potential to enhance the accuracy of clinical diagnosis in IC/BPS and, therefore, inform the selection of effective therapeutic strategies. Current research, however, has largely concentrated on markers associated with the HIC subtype, which represents only a limited proportion of the IC/BPS population. This indicates the need for broader investigations aimed at identifying molecular markers applicable to the wider IC/BPS patient cohort. Therefore, this study aims to elucidate the key molecular factors and pathways involved in the pathogenesis and progression of IC/BPS using sequencing data derived from patient tissues. The resulting insights are expected to clarify disease mechanisms and support the development of targeted therapeutic approaches. Materials and methods Patient selection and sample collection The participants in this study were 10 IC/BPS patients and 11 control patients collected from the department of Urology of The First Affiliated Hospital of Anhui Medical University from November 1st, 2021, to February 28th, 2022. Diagnosis of IC/BPS was based on the American Urological Association (AUA) Guidelines 2022 4 . Control group patients were selected based on the presence of overactive bladder or bladder neck obstruction identified during bladder neck examination, with no evident intravesical lesions. Clinical and pathological data for all participants were obtained from the electronic medical records, including baseline clinical characteristics, comorbidities, and pathological findings. For all IC/BPS patients, sufficient bladder mucosal tissue was collected from lesions showing significant bleeding following cystoscopic water dilation at a pressure of 80 cmH 2 O. Correspondingly, comparable bladder tissue samples were obtained from control patients under direct cystoscopic visualization. All tissue samples were promptly frozen and preserved in liquid nitrogen for later analysis. Written informed consent was obtained from each participant. RNA sequencing and data processing In this study, 21 samples were sequenced using the BGISEQ platform, yielding an average of approximately 6.64 Gb of bases per sample. The mean alignment rate to the reference genome was 90.15%, while the average gene mapping rate was 48.62%, resulting in the identification of 18,622 genes. To assess gene distribution across different expression levels, the number of genes within distinct FPKM ranges was calculated. Low-quality reads were removed from the raw data to ensure the reliability of subsequent analyses. Clean reads were then aligned to the reference genome GCF_000001405.39_GRCh38.p13 using HISAT and Bowtie2. The external validation dataset GSE11783 was downloaded from the GEO datasets. Differentially expressed gene (DEG) analysis Differential mRNA expression between 10 IC/BPS and 11 control samples was analyzed using the “edgeR” R package. The resulting DEGs were visualized with the “pheatmap” R package. GO and KEGG Pathway Enrichment Analyses To investigate potential downstream mechanisms, functional enrichment analysis was performed on all differentially expressed RBPs. The “clusterProfiler” R package was used to carry out and visualize the downstream analyses, including GO and KEGG. Gene set enrichment analysis (GSEA) and Gene set variant analysis GSEA and GSVA were performed by using “GSEABase” and “GSVA” R packages in R 4.0 software, and debugging parameters according to the instruction manual. Weighted gene correlation network analysis (WGCNA) The methodology involves constructing gene co-expression networks from RNA expression measurements, producing network structures characterized by scale-free properties 5 . Initially, transcriptomic profiles and clinical information from 21 bladder tissue specimens were obtained for network analysis. Genes with missing values in the dataset were excluded from further examination. Following the calculation of gene variance across individual specimens, only those genes showing non-zero standard deviation were retained. Following, hierarchical clustering analysis identified two specimens as outliers based on their clustering patterns, which were then eliminated from the study. Following, the R-based WGCNA package was employed to generate correlation networks. A soft-thresholding parameter of β = 8 was selected to achieve scale-free topology characteristics. The gene expression matrix underwent transformation into an adjacency matrix, which was further converted into a Topological Overlap Matrix (TOM). Gene grouping relied on hierarchical clustering with average linkage, using topological overlap measurements as distance metrics. The hybrid dynamic tree-cutting algorithm identified distinct modules, with a predefined constraint requiring each module to encompass at least 30 genes. Following the identification of gene modules using the dynamic shear method, the feature vector values (eigengenes) for each module were calculated. Cluster analysis was then performed on the modules, and those displaying closer similarity were merged into new modules, with a height threshold set at 0.25. The analysis yielded 23 distinct modules, with genes failing to cluster into any defined module assigned to a grey category. To evaluate module-phenotype associations, Gene triat Significance (GtS) values were derived through correlation analysis between eigengenes and three tumor microenvironment metrics: stromal composition, immune cell presence, and purity levels. Zero GtS values denote no meaningful gene-trait relationship. Immune infiltration evaluation ssGSEA was employed to quantify the abundance of 28 distinct immune cell populations 6 . Gene signatures defining each immune cell subset were obtained from previous literature 7 . The resulting ssGSEA scores reflect the collective expression trends of cell type-specific gene sets within individual samples, indicating either coordinated upregulation or downregulation. Drug sensitivity and immunotherapy response prediction Predicted drug responses to 138 targeted therapies were obtained through pRRophetic-based IC 50 calculations, referencing the Genomics of Drug Sensitivity in Cancer database. This analysis aimed to assess potential treatment efficacy in IC/BPS patients. Furthermore, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm ( http://tide.dfci.harvard.edu/ ) was employed to predict immunotherapeutic response outcomes. Immunohistochemistry (IHC) All paraffin-embedded tissues were cut into 5 µm sections, and the slides were prepared. Following deparaffinization, heat-induced epitope retrieval was performed by immersing tissues in 10 mM sodium citrate buffer (pH 6.0) at boiling temperature for 10 minutes. Endogenous peroxidase activity was quenched by treating tissue sections with 3% hydrogen peroxide for 20 minutes. Nonspecific binding was blocked through a 1-hour incubation with 10% goat serum at ambient temperature. Sections were then exposed to CCL8 primary antibody overnight at 4°C, followed by detection using a polymer-based detection system (PV-9000, ZSGB-Bio, China) per manufacturer's protocols. Two independent pathologists evaluated staining patterns, assigning scores ranging from negative to strong based on signal intensity and distribution. Statistical analysis Student t-test and Wilcoxon tests were used to test statistical significance between two variables with or without a normalized distribution. The Kruskal-Wallis test was applied to determine the statistical significance between three or more variables. Kaplan-Meier survival distributions were compared using log-rank statistics to evaluate group differences. R software (version 4.0) executed all statistical computations. A significance threshold of p < 0.05 was applied throughout the analysis. Results DEG in IC/BPS To systematically elucidate the molecular alterations underlying IC/BPS, a comprehensive identification of DEGs and hub genes was performed. Using the “edgeR” R package for rigorous bioinformatics analysis of transcriptomic data from all samples, 152 genes with significant expression changes out of 17,744 genes detected were identified. As illustrated in Fig. 1 , the DEG profile comprised 37 up-regulated and 115 down-regulated genes, indicating significant transcriptional alterations in IC/BPS and establishing a strong basis for further network analyses. Functional Enrichment Analysis of DEGs Functional characterization of DEGs in IC/BPS was achieved through separate GO and KEGG enrichment analyses for genes showing increased versus decreased expression. It was found that the overexpressed genes are associated with the biological progress term of leukocyte chemotaxis, neutrophil chemotaxis, and neutrophil migration, cellular component term of secretory granule membrane, and molecular function term of receptor-ligand activity, cytokine receptor binding, and signaling receptor activator activity according to GO analysis (Fig. 2 A). KEGG enrichment analysis identified four significantly enriched pathways in IC/BPS, namely cytokine-cytokine receptor interaction, chemokine signaling, viral protein interactions with cytokines and their receptors, and human cytomegalovirus infection pathways ( Fig. 2 B ). The down-regulated genes show strong enrichment in biological processes associated with ribonucleoprotein complex biogenesis, ncRNA metabolism, and ncRNA processing, as well as cellular components related to the mitochondrial inner membrane, mitochondrial protein complexes, and ribosomal subunits. They are also enriched for molecular functions linked to catalytic activity acting on RNA, structural constituents of the ribosome, and electron transfer activity (Fig. 2 C). The KEGG analysis also revealed ten pathways that were significantly down-regulated in IC/BPS, including those associated with Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Fig. 2 D). These results outline a pathogenic framework in which increased innate and adaptive immune activity is closely associated with the suppression of core cellular metabolic and biosynthetic pathways. Pathway Enrichment Analysis Using GSEA and GSVA To further explore the potentially related signal pathway of DEGs, the GSEA and GSVA were used. Three pathways were found to be significantly upregulated in IC/BPS, including NABA_MATRISOME, NABA_MARISOME_ASSOCIATED, and REACTOME_SIGNALING_BY_GPCR (Fig. 3 A). GSVA analysis showed that six signaling pathways were significantly upregulated in IC/BPS, including TNF-α signaling via apoptosis, NF-κB, IL6–JNK–STAT3 signaling, inflammatory response, IL2–STAT5 signaling, and KRAS signaling. The MYC target V1 pathway was significantly down-regulated in IC/BPS (Fig. 3 B). In summary, these results highlight a dysregulated network of pro-inflammatory and matrisome-associated pathways as central to IC/BPS pathology. Key modules identification by WGCNA To explore genes associated with reduced infilteration of immune cells in IC/BPS, WGCNA was carried out for the transcriptome data of all 21patient samples. After deleting outlier samples and choosing a soft threshold of 8, 21 gene modules were constructed in our scale-free network (Fig. 4 A-B). Combining the clinical characteristics, including grouping information with gene co-expression network, it was identified that the modules greenyellow and black were significantly associated with IC/BPS (Fig. 4 C). Intersection analysis between module black, module greenyellow, and DEGs revealed that module greenyellow contained 17 upregulated genes, while module black harbored 25 downregulated genes (Fig. 4 D). Immune cell infiltration evaluation in IC/BPS patients Immune landscape profiling of IC/BPS tissues revealed distinct shifts in particular cell subsets. IC/BPS samples displayed significantly elevated frequencies of central memory CD8 + T cells, eosinophils, and T helper 17 (Th17) cells compared to control tissues (Fig. 5 ). The co-enrichment of these cells points to a potent, synergistic effector response capable of driving chronic inflammation and tissue remodeling, which may contribute to the persistent symptoms and disease chronicity observed in IC/BPS. Identification and validation of hub genes in IC/BPS LASSO logistic regression was employed on the 17 upregulated genes identified through overlap analysis of the green-yellow module and upregulated DEGs to identify critical drivers of IC/BPS pathogenesis (Fig. 6 A-B). This analysis identified three hub genes, including CCL8, MMP25, and PLP1, as significantly associated with IC/BPS. Their mRNA expression levels were further validated using the internal patient samples and an external dataset (GSE11873). The results confirmed that CCL8 expression was significantly elevated in IC/BPS compared to controls (Fig. 6 C-G). Bleomycin resistance was also found to be highly associated with IC/BPS patients (Fig. 6 H). These findings not only establish CCL8, MMP25, and PLP1 as strong molecular signatures of IC/BPS but also integrate key aspects of its pathology, linking dysregulated immune chemotaxis, matrisome remodeling, and specific T-cell responses into a more coherent disease model. Discussion There is currently no definitive clinical diagnostic method for IC/BPS, and diagnosis relies on excluding other conditions through assessment of patient-reported symptoms, physical examination, and laboratory evaluations. IC/BPS clinical symptoms are extremely complex and are affected by many diseases, such as depression, asthma, autoimmune diseases, and inflammatory bowel disease. Clinical assessment of IC/BPS relies on five widely utilized symptom scales: the Interstitial Cystitis Symptom Index (ICSI) 8 . Interstitial Cystitis Problem Index (ICPI) 9 , Wisconsin Interstitial Cystitis Scale (UW-IC) 9 , the Pain, Urgency, Frequency Score (PUF) 10 , and Bladder Pain Interstitial Cystitis Symptom Score (BPIC-SS) 11 . However, it is worth noting that none of the scales is specific enough to diagnose IC/BPS alone and can only be used as an adjunct to clinical diagnosis. Cystoscopic examination, with or without hydrodistension, effectively diagnoses IC/BPS when Hunner's lesions are present; however, these distinctive pathological features appear in only 16% of the total IC/BPS patient population during anesthetic cystoscopy 12 . Cystoscopy is also an invasive procedure and is more likely to increase patient pain if hydrodistension is required. There are numerous treatments available for IC/BPS, ranging from conservative therapeutics with few side effects to major abdominal surgery. In IC/BPS, treatments should be individualized, comprehensive, and systematic for each patient to achieve the best outcomes. All patients receive initial conservative management strategies, including educational counseling, dietary adjustments, scheduled voiding protocols, stress reduction methods, psychological interventions, and selected physical therapy modalities. Meanwhile, there are some medications suggested for IC/BPS treatment, like oral pentosane polysulfate (PPS), intravesical dimethylsulfoxide, etc 13 , 14 . In terms of minimally invasive surgical procedures, hydrodistension with low-pressure and short-duration remains one of the most common therapies for IC/BPS, showing about 30–54% efficacy rates in the first month 15 . For IC/BPS patients with Hunner’s lesions, endoscopic treatment of Hunner’s lesions is recommended. Research by Peeker and colleagues demonstrated that 90% of patients experienced symptom improvement following Hunner's lesion excision, with 40% maintaining relief beyond three years 16 . For transurethral ablation, recommended safety parameters include neodymium: yttrium-aluminum-garnet laser application, minimal bladder distension, reduced energy settings, and brief coagulation intervals of 1–3 seconds to minimize perforation risk to the bladder or bowel 17 , 18 . Intravesical botulinum toxin A (BTX-A) has been demonstrated to provide significant improvement in pain, urinary symptoms, as well as quality of life in IC/BPS based on several previous observational studies. However, patients must be counseled regarding BTX-A's potential adverse effects and therapeutic outcomes, particularly the risk of urinary retention requiring catheterization 19 – 23 . Some novel therapies are emerging for the treatment of IC/BPS, including hyperbaric oxygen 24 – 26 , sildenafil 27 , monoclonal antibodies 28 , 29 , cannabinoids 30 – 32 , and intravesical liposomes 33 , 34 . However, the efficacy of these treatment modalities still needs to be validated by further studies, and the side effects deserve caution, so there is a clear need to identify more effective and specific treatment targets for IC/BPS. In this study, CCL8, MMP25, and PLP1 were found to play a vital role in IC/BPS. Chemokines play a key role in the immune and inflammatory response to disease development 35 , 36 . CCL8 was found to be highly expressed in IC/BPS and could be used as a potential diagnostic marker. The primary function of CCL8 in immune regulation is to recruit a variety of immune cells, including eosinophils, monocytes, natural killer cells, T lymphocytes, basophils, and mast cells 37 – 39 . Previous studies have shown that tumor cells can promote CCL8 secretion by fibroblasts in tumor-adjacent tissues and that the pro-metastatic activity of CCL8 is highly correlated with prognosis 40 . In addition to tumors, CCL8 plays a crucial role in inflammatory diseases. Harini et al. have shown that the expression of CCL8 was elevated in synovial fluids from patients with osteoarthritis, which results in monocyte recruitment and tissue damage 41 . Similarly, the circulating level of CCL8 presented high sensitivity and specificity for identifying active Crohn’s disease 42 . MMP25, a member of the matrix metalloproteinases family, which is involved in multiple cellular and tissue microenvironments, is anchored to the cell membrane by the glycosyl-phosphatidyl inositol anchoring domain. In previous studies, the overexpression of MMP25 was found in multiple types of human diseases, including cancers, such as glioblastomas 43 , colon 44 , urothelial 45 , and prostate cancers 46 . Furthermore, the expression of MMP25 was highly correlated with the biological progress of the immune microenvironment. Huang et al. have shown that both the classical and alternative activation of macrophages promote the mRNA expression level of MMP25 47 . Moreover, the NF-κB signaling pathway and proinflammatory molecules secretion were reduced in mmp25-null mice, which suggested that MMP25 could be associated with immune-related diseases 48 . It is well known that IC/BPS is a neuroimmune disease, and this study found that PLP1 was highly expressed in the bladder tissue of IC/BPS patients, suggesting that PLP1 plays a role in disease progression. PLP1 is a transmembrane proteolipid protein that constitutes the major structural element of myelin. It is essential for proper myelin compaction, structural stability, long-term maintenance of the sheath, and supporting oligodendrocyte maturation and axonal integrity. Variants in the PLP1 gene are recognized as the primary genetic basis of Pelizaeus-Merzbacher disease and spastic paraplegia type 2. Our findings may suggest that PLP1-related neurofibrillary cell demyelination in bladder tissue may be associated with IC/BPS development. Meanwhile, in recent research, Peng et al. have found that several immune cell types including Tcm CD4 + cell, regulatory T cell, activated B cell, memory B cell, and neutrophils increased significantly in IC/BPS bladder tissues, and CD8 effector T cells, Th17 cell, follicular helper T cells, and macrophages decreased significantly in IC/BPS 49 . There are some limitations of this study. Firstly, the results of this study still need to be confirmed in a larger sample of IC/BPS patients with high expression and diagnostic efficacy of CCL8, MMP25, and PLP1. Then, the specific role of CCL8 in the development of IC/BPS disease and the related molecular biological mechanisms still need to be further explored. Lastly, the results of this study can be further analyzed in conjunction with high-throughput single-cell sequencing data. Conclusion The pathogenesis of IC/BPS may be associated with activation of cytokine receptor binding pathways and upregulation of Tcm CD8 + cells through CCL8-dependent regulation, providing new targets and directions for future basic and clinical research in IC/BPS. Abbreviations IC Interstitial cystitis BPS Bladder pain syndrome IC/BPS Interstitial cystitis/bladder pain syndrome AUA American Urological Association DEG Differentially expressed gene GSEA Gene set enrichment analysis WGCNA Weighted gene correlation network analysis TOM Topological Overlap Matrix GtS Gene triat Significance TIDE Tumor Immune Dysfunction and Exclusion IHC Immunohistochemistry ICSI Interstitial Cystitis Symptom Index ICPI Interstitial Cystitis Problem Index BPIC-SS Bladder Pain Interstitial Cystitis Symptom Score Declarations Acknowledgments We sincerely thank all the participants in this study for their time and contributions. Funding This study was supported by the National Natural Science Foundation of China (82170787), Distinguished Young Scholar of Anhui Colleges (2021-108-10), and Science Foundation for Outstanding Young Scholar of Anhui Colleges (2022AH020073). Author information Affiliations Department of Urology, The First Affiliated Hospital of Anhui Medical University; Institute of Urology & Anhui Province Key Laboratory of Genitourinary Diseases, Anhui Medical University, Hefei, China. Changqin Jiang, Guangjie Ji, Jing Chen, Xiao Li, Xiaoling Li, Li Zhang, Zongyao Hao, Chaozhao Liang Contributions Chaozhao Liang: Conceptualization, Funding acquisition; Zongyao Hao: Conceptualization, Data curation, Writing - review and editing; Li Zhang: Data curation, Project administration, Writing - review and editing; Changqing Jiang: Formal analysis, Investigation, Methodology, Writing - original draft; Guangjie Ji: Validation, Visualization, Writing - original draft; Jing Chen: Methodology, Validation; Xiao Li: Methodology; Xiaoling Li: Methodology. Corresponding authors Correspondence to Chaozhao Liang and Changqin Jiang. Ethics approval and informed consent For the AHMU-IC cohort, the research contents and programs were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (PJ2022-11-33), and patient consent for the retrospective cohorts was waived. Clinical trial number Not applicable. 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Nuttall RK, Pennington CJ, Taplin J, Wheal A, Yong VW, Forsyth PA, et al. Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol Cancer Res . 2003;1(5):333-45. Wallard MJ, Pennington CJ, Veerakumarasivam A, et al. Comprehensive profiling and localisation of the matrix metalloproteinases in urothelial carcinoma. Br J Cancer . 2006;94(4):569-77. Riddick AC, Shukla CJ, Pennington CJ, et al. Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. Br J Cancer . 2005;92(12):2171-80. Huang WC, Sala-Newby GB, Susana A, Johnson JL, Newby AC. Classical macrophage activation up-regulates several matrix metalloproteinases through mitogen activated protein kinases and nuclear factor-kappaB. PLoS One . 2012;7(8):e42507. Soria-Valles C, Gutierrez-Fernandez A, Osorio FG, et al. MMP-25 Metalloprotease Regulates Innate Immune Response through NF-kappaB Signaling. J Immunol . 2016;197(1):296-302. Peng L, Jin X, Li BY, et al. Integrating single-cell RNA sequencing with spatial transcriptomics reveals immune landscape for interstitial cystitis. Signal Transduct Target Ther . 2022;7(1):161. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 01 May, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 24 Apr, 2026 Editor invited by journal 10 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9276619","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634142990,"identity":"e9b14455-008c-48a5-9c23-c3f55f27120c","order_by":0,"name":"Changqin Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDAC5gMMBxIMJHj42ZsPfADyE8CiPPi0sCUwHnhQYSEn2XMscQaxWpgPPjhTYWxwI8eQOC0Gx3gMDiS2SSQ23Mj52MzDYJenOyOB8cHbNgZ5c0JaGnvebgRqSS42u5HAbDi3jcFwZwMOLfd7IFqa2XO3P+ZhYE7cdiOBTZq3jSHB4AABW9oYch4CbakHaWH/TVBLwhkJYx6OHEaglsNgW5jxaZE8xlZwIKFCQk6C55hh4xyD44nbzjxslpxzTsJwAw4tfMeYN3/8YVDHY3+8+WHDm4rqxG3Hkw9+eFNmI4/LFoUDHAZwDhMPmM3YACQksKsHAvkG9gdwDuMPnOpGwSgYBaNgJAMAiuNn8i1LBWIAAAAASUVORK5CYII=","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Changqin","middleName":"","lastName":"Jiang","suffix":""},{"id":634142992,"identity":"594ff968-fad4-44ec-b63e-58b9679f9072","order_by":1,"name":"Guangjie Ji","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guangjie","middleName":"","lastName":"Ji","suffix":""},{"id":634142999,"identity":"f63d0044-0747-4fee-a55a-869bc6e3997c","order_by":2,"name":"Jing Chen","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Chen","suffix":""},{"id":634143007,"identity":"918a4a11-147c-42c2-8461-2b96e514f295","order_by":3,"name":"Xiao Li","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Li","suffix":""},{"id":634143011,"identity":"01c8336a-d2c8-49f0-9feb-e19c7d7d02ee","order_by":4,"name":"Xiaoling Li","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Li","suffix":""},{"id":634143023,"identity":"d45328fe-55cf-4f90-87ca-65db686587c1","order_by":5,"name":"Li Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""},{"id":634143024,"identity":"98f2f649-a779-47c2-aa1f-441f74060e1d","order_by":6,"name":"Zongyao Hao","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zongyao","middleName":"","lastName":"Hao","suffix":""},{"id":634143025,"identity":"0f08f9b9-ca01-478d-8aea-2a1efca58cc3","order_by":7,"name":"Chaozhao Liang","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chaozhao","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2026-03-31 08:10:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9276619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9276619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108977194,"identity":"f469556f-a92f-4aa6-a570-830cc25b864d","added_by":"auto","created_at":"2026-05-11 11:30:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104080,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs in the IC/BPS and control samples. Volcano plot of DEGs (A). Heatmap plot of DEGs distribution in all samples (B).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/7a0f70942122ce6347ec68f5.jpg"},{"id":108837855,"identity":"b2c6b0a5-3c93-478a-b45f-76aca982f442","added_by":"auto","created_at":"2026-05-09 00:18:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214185,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG analysis of up- and down-regulated DEGs. Bubble plot in GO analysis of up-regulated DEGs (A). Bubble plot in KEGG analysis of up-regulated DEGs (B). Bubble plot in GO analysis of down-regulated DEGs (C). Bubble plot in KEGG analysis of down-regulated DEGs (D).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/489fcfb3cb6d11567191be42.jpg"},{"id":108977155,"identity":"389ed7fb-0abd-4b6b-b209-c60350064622","added_by":"auto","created_at":"2026-05-11 11:30:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133264,"visible":true,"origin":"","legend":"\u003cp\u003eGene set analysis in IC/BPS.GSEA (A) and GSVA (B).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/50d000e118e416b88b4faec3.jpg"},{"id":108837859,"identity":"90cf1236-fc8e-4d66-a2ad-b07995cd2102","added_by":"auto","created_at":"2026-05-09 00:18:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80118,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of key modules by WGCNA.Optimizing the soft threshold to make sure there is a scale-free network (A). Cluster dendrogram of module construction (B). Correlation between modules and clinical features (C). Intersection of up- and down-regulated genes and the most related modules (D).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/6953a3bdce947c053235d653.jpg"},{"id":108837857,"identity":"bdf2c0ef-67f0-4717-ad4c-81104a866086","added_by":"auto","created_at":"2026-05-09 00:18:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174562,"visible":true,"origin":"","legend":"\u003cp\u003eImmune cell infiltration evaluation in IC/BPS patients. Heatmap of different types of immune cells in the AHMU-IC cohort (A). Boxplot of comparison of different types of immune cells between IC/BPS patients and the control group (B).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/cae95d75a0e538f82f175f51.jpg"},{"id":108837858,"identity":"b2eca9fc-6b50-45a0-8cbe-34ee76e0f619","added_by":"auto","created_at":"2026-05-09 00:18:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77672,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and validation of hub genes in IC/BPS. Variance trace plot of genes associated with IC/BPS (A). Cross-validation plot (B). Validation the expression of CCL8 (C), MMP25 (D), and PLP1 (E) in AHMU-IC cohort. Validation of CCL8 expression in GSE11783 (F). IHC of CCL8 in AHMU-IC cohort (G). Bleomycin sensitivity in the IC/BPS and control group (H).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/dbdea09a5d5a4fcde28f26a2.jpg"},{"id":109252447,"identity":"a446e4ec-ec91-40ef-ae39-d6ef9627f9fc","added_by":"auto","created_at":"2026-05-14 09:26:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1014299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9276619/v1/c48fc836-eff6-47f2-8b4c-3f33f0cd55d5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIdentification of CCL8, MMP25, and PLP1 as Potential Diagnostic Markers for Interstitial Cystitis/Bladder Pain Syndrome \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInterstitial cystitis (IC), also known as bladder pain syndrome (BPS), is a clinical syndrome manifested by urinary frequency, urinary urgency, and pain or discomfort in the suprapubic bladder area. Individuals diagnosed with IC/BPS are predominantly female, with an estimated male-to-female ratio of about 1:10\u003csup\u003e1\u003c/sup\u003e. IC/BPS significantly affects quality of life, as persistent pelvic pain, sexual avoidance, and disrupted sleep interfere with daily functioning and occupational activities. In severe cases, repeated nighttime awakenings increase the risk of falls and related injuries, including fractures. Comorbid anxiety or depression occurs frequently in individuals with IC/BPS, and the suicide rate in this population is reported to be approximately fourfold higher than that of the general population\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The etiology of IC is still unclear, which poses a great challenge for diagnosis and treatment. The current treatments of IC/BPS presented limited efficacy and high recurrence rates\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, there is an urgent need for in-depth research on the molecular biology of IC/BPS pathogenesis to provide potentially valuable targets for IC/BPS.\u003c/p\u003e \u003cp\u003eThe clinical symptoms of patients with IC/BPS are similar to many urological conditions, such as overactive bladder and chronic prostatitis, which leads to easy underdiagnosis and misdiagnosis in many patients. Molecular markers have the potential to enhance the accuracy of clinical diagnosis in IC/BPS and, therefore, inform the selection of effective therapeutic strategies. Current research, however, has largely concentrated on markers associated with the HIC subtype, which represents only a limited proportion of the IC/BPS population. This indicates the need for broader investigations aimed at identifying molecular markers applicable to the wider IC/BPS patient cohort.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to elucidate the key molecular factors and pathways involved in the pathogenesis and progression of IC/BPS using sequencing data derived from patient tissues. The resulting insights are expected to clarify disease mechanisms and support the development of targeted therapeutic approaches.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient selection and sample collection\u003c/h2\u003e \u003cp\u003e The participants in this study were 10 IC/BPS patients and 11 control patients collected from the department of Urology of The First Affiliated Hospital of Anhui Medical University from November 1st, 2021, to February 28th, 2022. Diagnosis of IC/BPS was based on the American Urological Association (AUA) Guidelines 2022\u003csup\u003e4\u003c/sup\u003e. Control group patients were selected based on the presence of overactive bladder or bladder neck obstruction identified during bladder neck examination, with no evident intravesical lesions. Clinical and pathological data for all participants were obtained from the electronic medical records, including baseline clinical characteristics, comorbidities, and pathological findings. For all IC/BPS patients, sufficient bladder mucosal tissue was collected from lesions showing significant bleeding following cystoscopic water dilation at a pressure of 80 cmH\u003csub\u003e2\u003c/sub\u003eO. Correspondingly, comparable bladder tissue samples were obtained from control patients under direct cystoscopic visualization. All tissue samples were promptly frozen and preserved in liquid nitrogen for later analysis. Written informed consent was obtained from each participant.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA sequencing and data processing\u003c/h3\u003e\n\u003cp\u003eIn this study, 21 samples were sequenced using the BGISEQ platform, yielding an average of approximately 6.64 Gb of bases per sample. The mean alignment rate to the reference genome was 90.15%, while the average gene mapping rate was 48.62%, resulting in the identification of 18,622 genes. To assess gene distribution across different expression levels, the number of genes within distinct FPKM ranges was calculated. Low-quality reads were removed from the raw data to ensure the reliability of subsequent analyses. Clean reads were then aligned to the reference genome GCF_000001405.39_GRCh38.p13 using HISAT and Bowtie2. The external validation dataset GSE11783 was downloaded from the GEO datasets.\u003c/p\u003e\n\u003ch3\u003eDifferentially expressed gene (DEG) analysis\u003c/h3\u003e\n\u003cp\u003eDifferential mRNA expression between 10 IC/BPS and 11 control samples was analyzed using the \u0026ldquo;edgeR\u0026rdquo; R package. The resulting DEGs were visualized with the \u0026ldquo;pheatmap\u0026rdquo; R package.\u003c/p\u003e\n\u003ch3\u003eGO and KEGG Pathway Enrichment Analyses\u003c/h3\u003e\n\u003cp\u003eTo investigate potential downstream mechanisms, functional enrichment analysis was performed on all differentially expressed RBPs. The \u0026ldquo;clusterProfiler\u0026rdquo; R package was used to carry out and visualize the downstream analyses, including GO and KEGG.\u003c/p\u003e\n\u003ch3\u003eGene set enrichment analysis (GSEA) and Gene set variant analysis\u003c/h3\u003e\n\u003cp\u003eGSEA and GSVA were performed by using \u0026ldquo;GSEABase\u0026rdquo; and \u0026ldquo;GSVA\u0026rdquo; R packages in R 4.0 software, and debugging parameters according to the instruction manual.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWeighted gene correlation network analysis (WGCNA)\u003c/h2\u003e \u003cp\u003eThe methodology involves constructing gene co-expression networks from RNA expression measurements, producing network structures characterized by scale-free properties\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Initially, transcriptomic profiles and clinical information from 21 bladder tissue specimens were obtained for network analysis. Genes with missing values in the dataset were excluded from further examination. Following the calculation of gene variance across individual specimens, only those genes showing non-zero standard deviation were retained. Following, hierarchical clustering analysis identified two specimens as outliers based on their clustering patterns, which were then eliminated from the study. Following, the R-based WGCNA package was employed to generate correlation networks. A soft-thresholding parameter of β\u0026thinsp;=\u0026thinsp;8 was selected to achieve scale-free topology characteristics. The gene expression matrix underwent transformation into an adjacency matrix, which was further converted into a Topological Overlap Matrix (TOM). Gene grouping relied on hierarchical clustering with average linkage, using topological overlap measurements as distance metrics. The hybrid dynamic tree-cutting algorithm identified distinct modules, with a predefined constraint requiring each module to encompass at least 30 genes. Following the identification of gene modules using the dynamic shear method, the feature vector values (eigengenes) for each module were calculated. Cluster analysis was then performed on the modules, and those displaying closer similarity were merged into new modules, with a height threshold set at 0.25. The analysis yielded 23 distinct modules, with genes failing to cluster into any defined module assigned to a grey category. To evaluate module-phenotype associations, Gene triat Significance (GtS) values were derived through correlation analysis between eigengenes and three tumor microenvironment metrics: stromal composition, immune cell presence, and purity levels. Zero GtS values denote no meaningful gene-trait relationship.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmune infiltration evaluation\u003c/h3\u003e\n\u003cp\u003essGSEA was employed to quantify the abundance of 28 distinct immune cell populations\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Gene signatures defining each immune cell subset were obtained from previous literature\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The resulting ssGSEA scores reflect the collective expression trends of cell type-specific gene sets within individual samples, indicating either coordinated upregulation or downregulation.\u003c/p\u003e\n\u003ch3\u003eDrug sensitivity and immunotherapy response prediction\u003c/h3\u003e\n\u003cp\u003ePredicted drug responses to 138 targeted therapies were obtained through pRRophetic-based IC\u003csub\u003e50\u003c/sub\u003e calculations, referencing the Genomics of Drug Sensitivity in Cancer database. This analysis aimed to assess potential treatment efficacy in IC/BPS patients. Furthermore, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tide.dfci.harvard.edu/\u003c/span\u003e\u003cspan address=\"http://tide.dfci.harvard.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to predict immunotherapeutic response outcomes.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eAll paraffin-embedded tissues were cut into 5 \u0026micro;m sections, and the slides were prepared. Following deparaffinization, heat-induced epitope retrieval was performed by immersing tissues in 10 mM sodium citrate buffer (pH 6.0) at boiling temperature for 10 minutes. Endogenous peroxidase activity was quenched by treating tissue sections with 3% hydrogen peroxide for 20 minutes. Nonspecific binding was blocked through a 1-hour incubation with 10% goat serum at ambient temperature. Sections were then exposed to CCL8 primary antibody overnight at 4\u0026deg;C, followed by detection using a polymer-based detection system (PV-9000, ZSGB-Bio, China) per manufacturer's protocols. Two independent pathologists evaluated staining patterns, assigning scores ranging from negative to strong based on signal intensity and distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStudent t-test and Wilcoxon tests were used to test statistical significance between two variables with or without a normalized distribution. The Kruskal-Wallis test was applied to determine the statistical significance between three or more variables. Kaplan-Meier survival distributions were compared using log-rank statistics to evaluate group differences. R software (version 4.0) executed all statistical computations. A significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied throughout the analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDEG in IC/BPS\u003c/h2\u003e \u003cp\u003eTo systematically elucidate the molecular alterations underlying IC/BPS, a comprehensive identification of DEGs and hub genes was performed. Using the \u0026ldquo;edgeR\u0026rdquo; R package for rigorous bioinformatics analysis of transcriptomic data from all samples, 152 genes with significant expression changes out of 17,744 genes detected were identified. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the DEG profile comprised 37 up-regulated and 115 down-regulated genes, indicating significant transcriptional alterations in IC/BPS and establishing a strong basis for further network analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Enrichment Analysis of DEGs\u003c/h2\u003e \u003cp\u003eFunctional characterization of DEGs in IC/BPS was achieved through separate GO and KEGG enrichment analyses for genes showing increased \u003cem\u003eversus\u003c/em\u003e decreased expression. It was found that the overexpressed genes are associated with the biological progress term of leukocyte chemotaxis, neutrophil chemotaxis, and neutrophil migration, cellular component term of secretory granule membrane, and molecular function term of receptor-ligand activity, cytokine receptor binding, and signaling receptor activator activity according to GO analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). KEGG enrichment analysis identified four significantly enriched pathways in IC/BPS, namely cytokine-cytokine receptor interaction, chemokine signaling, viral protein interactions with cytokines and their receptors, and human cytomegalovirus infection pathways \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e The down-regulated genes show strong enrichment in biological processes associated with ribonucleoprotein complex biogenesis, ncRNA metabolism, and ncRNA processing, as well as cellular components related to the mitochondrial inner membrane, mitochondrial protein complexes, and ribosomal subunits. They are also enriched for molecular functions linked to catalytic activity acting on RNA, structural constituents of the ribosome, and electron transfer activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The KEGG analysis also revealed ten pathways that were significantly down-regulated in IC/BPS, including those associated with Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, and Huntington\u0026rsquo;s disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results outline a pathogenic framework in which increased innate and adaptive immune activity is closely associated with the suppression of core cellular metabolic and biosynthetic pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePathway Enrichment Analysis Using GSEA and GSVA\u003c/h2\u003e \u003cp\u003eTo further explore the potentially related signal pathway of DEGs, the GSEA and GSVA were used. Three pathways were found to be significantly upregulated in IC/BPS, including NABA_MATRISOME, NABA_MARISOME_ASSOCIATED, and REACTOME_SIGNALING_BY_GPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). GSVA analysis showed that six signaling pathways were significantly upregulated in IC/BPS, including TNF-α signaling \u003cem\u003evia\u003c/em\u003e apoptosis, NF-κB, IL6\u0026ndash;JNK\u0026ndash;STAT3 signaling, inflammatory response, IL2\u0026ndash;STAT5 signaling, and KRAS signaling. The MYC target V1 pathway was significantly down-regulated in IC/BPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In summary, these results highlight a dysregulated network of pro-inflammatory and matrisome-associated pathways as central to IC/BPS pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eKey modules identification by WGCNA\u003c/h2\u003e \u003cp\u003eTo explore genes associated with reduced infilteration of immune cells in IC/BPS, WGCNA was carried out for the transcriptome data of all 21patient samples. After deleting outlier samples and choosing a soft threshold of 8, 21 gene modules were constructed in our scale-free network (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Combining the clinical characteristics, including grouping information with gene co-expression network, it was identified that the modules greenyellow and black were significantly associated with IC/BPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Intersection analysis between module black, module greenyellow, and DEGs revealed that module greenyellow contained 17 upregulated genes, while module black harbored 25 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmune cell infiltration evaluation in IC/BPS patients\u003c/h2\u003e \u003cp\u003eImmune landscape profiling of IC/BPS tissues revealed distinct shifts in particular cell subsets. IC/BPS samples displayed significantly elevated frequencies of central memory CD8\u003csup\u003e+\u003c/sup\u003e T cells, eosinophils, and T helper 17 (Th17) cells compared to control tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The co-enrichment of these cells points to a potent, synergistic effector response capable of driving chronic inflammation and tissue remodeling, which may contribute to the persistent symptoms and disease chronicity observed in IC/BPS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and validation of hub genes in IC/BPS\u003c/h2\u003e \u003cp\u003eLASSO logistic regression was employed on the 17 upregulated genes identified through overlap analysis of the green-yellow module and upregulated DEGs to identify critical drivers of IC/BPS pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). This analysis identified three hub genes, including CCL8, MMP25, and PLP1, as significantly associated with IC/BPS. Their mRNA expression levels were further validated using the internal patient samples and an external dataset (GSE11873). The results confirmed that CCL8 expression was significantly elevated in IC/BPS compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-G). Bleomycin resistance was also found to be highly associated with IC/BPS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These findings not only establish CCL8, MMP25, and PLP1 as strong molecular signatures of IC/BPS but also integrate key aspects of its pathology, linking dysregulated immune chemotaxis, matrisome remodeling, and specific T-cell responses into a more coherent disease model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is currently no definitive clinical diagnostic method for IC/BPS, and diagnosis relies on excluding other conditions through assessment of patient-reported symptoms, physical examination, and laboratory evaluations. IC/BPS clinical symptoms are extremely complex and are affected by many diseases, such as depression, asthma, autoimmune diseases, and inflammatory bowel disease. Clinical assessment of IC/BPS relies on five widely utilized symptom scales: the Interstitial Cystitis Symptom Index (ICSI)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Interstitial Cystitis Problem Index (ICPI)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, Wisconsin Interstitial Cystitis Scale (UW-IC)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, the Pain, Urgency, Frequency Score (PUF)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and Bladder Pain Interstitial Cystitis Symptom Score (BPIC-SS)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, it is worth noting that none of the scales is specific enough to diagnose IC/BPS alone and can only be used as an adjunct to clinical diagnosis. Cystoscopic examination, with or without hydrodistension, effectively diagnoses IC/BPS when Hunner's lesions are present; however, these distinctive pathological features appear in only 16% of the total IC/BPS patient population during anesthetic cystoscopy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Cystoscopy is also an invasive procedure and is more likely to increase patient pain if hydrodistension is required.\u003c/p\u003e \u003cp\u003eThere are numerous treatments available for IC/BPS, ranging from conservative therapeutics with few side effects to major abdominal surgery. In IC/BPS, treatments should be individualized, comprehensive, and systematic for each patient to achieve the best outcomes. All patients receive initial conservative management strategies, including educational counseling, dietary adjustments, scheduled voiding protocols, stress reduction methods, psychological interventions, and selected physical therapy modalities. Meanwhile, there are some medications suggested for IC/BPS treatment, like oral pentosane polysulfate (PPS), intravesical dimethylsulfoxide, etc\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In terms of minimally invasive surgical procedures, hydrodistension with low-pressure and short-duration remains one of the most common therapies for IC/BPS, showing about 30\u0026ndash;54% efficacy rates in the first month\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For IC/BPS patients with Hunner\u0026rsquo;s lesions, endoscopic treatment of Hunner\u0026rsquo;s lesions is recommended. Research by Peeker and colleagues demonstrated that 90% of patients experienced symptom improvement following Hunner's lesion excision, with 40% maintaining relief beyond three years\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For transurethral ablation, recommended safety parameters include neodymium: yttrium-aluminum-garnet laser application, minimal bladder distension, reduced energy settings, and brief coagulation intervals of 1\u0026ndash;3 seconds to minimize perforation risk to the bladder or bowel\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Intravesical botulinum toxin A (BTX-A) has been demonstrated to provide significant improvement in pain, urinary symptoms, as well as quality of life in IC/BPS based on several previous observational studies. However, patients must be counseled regarding BTX-A's potential adverse effects and therapeutic outcomes, particularly the risk of urinary retention requiring catheterization\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Some novel therapies are emerging for the treatment of IC/BPS, including hyperbaric oxygen\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, sildenafil\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, monoclonal antibodies\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, cannabinoids\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and intravesical liposomes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the efficacy of these treatment modalities still needs to be validated by further studies, and the side effects deserve caution, so there is a clear need to identify more effective and specific treatment targets for IC/BPS.\u003c/p\u003e \u003cp\u003eIn this study, CCL8, MMP25, and PLP1 were found to play a vital role in IC/BPS. Chemokines play a key role in the immune and inflammatory response to disease development\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. CCL8 was found to be highly expressed in IC/BPS and could be used as a potential diagnostic marker. The primary function of CCL8 in immune regulation is to recruit a variety of immune cells, including eosinophils, monocytes, natural killer cells, T lymphocytes, basophils, and mast cells\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that tumor cells can promote CCL8 secretion by fibroblasts in tumor-adjacent tissues and that the pro-metastatic activity of CCL8 is highly correlated with prognosis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In addition to tumors, CCL8 plays a crucial role in inflammatory diseases. Harini \u003cem\u003eet al.\u003c/em\u003e have shown that the expression of CCL8 was elevated in synovial fluids from patients with osteoarthritis, which results in monocyte recruitment and tissue damage\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Similarly, the circulating level of CCL8 presented high sensitivity and specificity for identifying active Crohn\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMMP25, a member of the matrix metalloproteinases family, which is involved in multiple cellular and tissue microenvironments, is anchored to the cell membrane by the glycosyl-phosphatidyl inositol anchoring domain. In previous studies, the overexpression of MMP25 was found in multiple types of human diseases, including cancers, such as glioblastomas\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, colon\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, urothelial\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and prostate cancers\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Furthermore, the expression of MMP25 was highly correlated with the biological progress of the immune microenvironment. Huang \u003cem\u003eet al.\u003c/em\u003e have shown that both the classical and alternative activation of macrophages promote the mRNA expression level of MMP25\u003csup\u003e47\u003c/sup\u003e. Moreover, the NF-κB signaling pathway and proinflammatory molecules secretion were reduced in mmp25-null mice, which suggested that MMP25 could be associated with immune-related diseases\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that IC/BPS is a neuroimmune disease, and this study found that PLP1 was highly expressed in the bladder tissue of IC/BPS patients, suggesting that PLP1 plays a role in disease progression. PLP1 is a transmembrane proteolipid protein that constitutes the major structural element of myelin. It is essential for proper myelin compaction, structural stability, long-term maintenance of the sheath, and supporting oligodendrocyte maturation and axonal integrity. Variants in the PLP1 gene are recognized as the primary genetic basis of Pelizaeus-Merzbacher disease and spastic paraplegia type 2. Our findings may suggest that PLP1-related neurofibrillary cell demyelination in bladder tissue may be associated with IC/BPS development. Meanwhile, in recent research, Peng \u003cem\u003eet al.\u003c/em\u003e have found that several immune cell types including Tcm CD4\u003csup\u003e+\u003c/sup\u003e cell, regulatory T cell, activated B cell, memory B cell, and neutrophils increased significantly in IC/BPS bladder tissues, and CD8 effector T cells, Th17 cell, follicular helper T cells, and macrophages decreased significantly in IC/BPS\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are some limitations of this study. Firstly, the results of this study still need to be confirmed in a larger sample of IC/BPS patients with high expression and diagnostic efficacy of CCL8, MMP25, and PLP1. Then, the specific role of CCL8 in the development of IC/BPS disease and the related molecular biological mechanisms still need to be further explored. Lastly, the results of this study can be further analyzed in conjunction with high-throughput single-cell sequencing data.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe pathogenesis of IC/BPS may be associated with activation of cytokine receptor binding pathways and upregulation of Tcm CD8\u003csup\u003e+\u003c/sup\u003e cells through CCL8-dependent regulation, providing new targets and directions for future basic and clinical research in IC/BPS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterstitial cystitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBladder pain syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIC/BPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterstitial cystitis/bladder pain syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Urological Association\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eGSEA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eGene set enrichment analysis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eWGCNA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eWeighted gene correlation network analysis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eTOM\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTopological Overlap Matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGtS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene triat Significance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTIDE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor Immune Dysfunction and Exclusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eImmunohistochemistry\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterstitial Cystitis Symptom Index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterstitial Cystitis Problem Index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBPIC-SS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBladder Pain Interstitial Cystitis Symptom Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all the participants in this study for their time and contributions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82170787), Distinguished Young Scholar of Anhui Colleges (2021-108-10), and Science Foundation for Outstanding Young Scholar of Anhui Colleges (2022AH020073).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Urology, The First Affiliated Hospital of Anhui Medical University; Institute of Urology \u0026amp; Anhui Province Key Laboratory of Genitourinary Diseases, Anhui Medical University, Hefei, China.\u003c/p\u003e\n\u003cp\u003eChangqin Jiang, Guangjie Ji, Jing Chen, Xiao Li, Xiaoling Li, Li Zhang, Zongyao Hao, Chaozhao Liang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChaozhao Liang: Conceptualization, Funding acquisition;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZongyao Hao: Conceptualization, Data curation, Writing - review and editing;\u003c/p\u003e\n\u003cp\u003eLi Zhang: Data curation, Project administration, Writing - review and editing;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChangqing Jiang: Formal analysis, Investigation, Methodology, Writing - original draft;\u003c/p\u003e\n\u003cp\u003eGuangjie Ji: Validation, Visualization, Writing - original draft;\u003c/p\u003e\n\u003cp\u003eJing Chen: Methodology, Validation;\u003c/p\u003e\n\u003cp\u003eXiao Li: Methodology;\u003c/p\u003e\n\u003cp\u003eXiaoling Li: Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Chaozhao Liang and Changqin Jiang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and informed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the AHMU-IC cohort, the research contents and programs were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (PJ2022-11-33), and patient consent for the retrospective cohorts was waived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDavis NF, Brady CM, Creagh T. 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Integrating single-cell RNA sequencing with spatial transcriptomics reveals immune landscape for interstitial cystitis. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e. 2022;7(1):161.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bladder Pain Syndrome, Interstitial Cystitis, CCL8, immune infiltration, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-9276619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9276619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eInterstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic syndrome characterized by lower urinary tract symptoms, yet its pathogenesis and diagnostic markers remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBladder mucosal tissues from 10 IC/BPS patients and 11 controls underwent whole transcriptome sequencing. Differentially expressed genes and their biological functions were analyzed using GO, KEGG, GSEA, and WGCNA. Hub genes (CCL8, MMP25, PLP1) were validated by internal samples and an external dataset (GSE11873). Immune infiltration was assessed via ssGSEA, and LASSO regression identified hub genes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDifferentially expressed genes in IC/BPS were enriched in cytokine receptor binding and extracellular matrix synthesis pathways. Increased infiltration of central memory CD8\u003csup\u003e+\u003c/sup\u003e T cells (Tcm CD8\u003csup\u003e+\u003c/sup\u003e cells) was observed. CCL8, MMP25, and PLP1 were significantly associated with IC/BPS.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe pathogenesis of IC/BPS may be related to the activation of cytokine receptor binding pathways and the upregulation of Tcm CD8\u003csup\u003e+\u003c/sup\u003e cells \u003cem\u003evia\u003c/em\u003e CCL8-dependent regulation. This provides new targets and directions for future basic and clinical studies of IC/BPS.\u003c/p\u003e","manuscriptTitle":"Identification of CCL8, MMP25, and PLP1 as Potential Diagnostic Markers for Interstitial Cystitis/Bladder Pain Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-09 00:18:21","doi":"10.21203/rs.3.rs-9276619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-01T12:34:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145277399126609581489466354004218939862","date":"2026-04-25T00:22:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T12:28:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-10T20:59:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T02:17:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T02:16:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Urology","date":"2026-03-31T07:59:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94e166fc-5b9d-427e-876b-4039e5bb9d03","owner":[],"postedDate":"May 9th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-01T12:34:53+00:00","index":71,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-09T00:18:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-09 00:18:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9276619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9276619","identity":"rs-9276619","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}