Study On The Role And Mechanism Of CXCL13 And S100A8/A9 In Interstitial Cystitis

Background and Objective:Interstitial cystitis/bladder pain syndrome(IC/BPS)is a clinical syndrome of unidentified etiology,characterized by frequent and urgent urination,suprapubic or pelvic pain upon bladder filling,and alleviation following urination.Epidemiological research indicates that IC/BPS is prevalent among middle-aged and elderly women,with an incidence rate of approximately 45-60 per 100,000,exhibiting a yearly increase.The pathogenesis of IC/BPS is intricate,and no specific medication is currently available.Ineffective control of IC/BPS may result in bladder contracture,hydronephrosis,renal function impairment,and necessitate cystectomy in severe cases.The pathogenic origins of IC/BPS are multifaceted.Present theories propose that numerous factors,including infection,alterations in epithelial permeability,autoimmune diseases,neuroinflammation,and urotoxic substances contribute to the pathophysiological process of IC/BPS.Nonetheless,various studies have corroborated that an augmented immune-inflammatory response and regulatory dysfunctions are crucial factors in the persistence and progression of IC/BPS.Consequently,a comprehensive investigation of the enhancement and regulation of immune-inflammatory response mechanisms in IC/BPS bears significant clinical relevance and value for the intervention and treatment of IC/BPS.Due to challenges in acquiring and preserving clinical specimens from IC/ BPS patients,research on the pathogenesis and pathophysiology has been substantially hindered.Procuring and analyzing clinical specimens from IC/BPS patients is essential for elucidating its pathogenesis.An IC/BPS sequencing dataset,encompassing six normal bladder tissue samples,five non-ulcer area samples,and five ulcer area samples,confirmed that inflammatory mediators and damage-associated molecular patterns contribute to the enhancement and irregulation of IC/BPS immune-inflammatory response.Utilizing bioinformatics analysis,TMT proteomics,and single-cell sequencing,this study identified CXCL13,S100A8,and S100A9 as potential key molecules inducing inflammatory damage to bladder tissue in IC/BPS patients.Existing research demonstrates that C XCL13,S100A8,and S100A9 are upregulated in various immune-inflammatory-related diseases,such as systemic lupus erythematosus,rheumatoid arthritis,and Sj?gren’s syndrome.Furthermore,in animal disease models,blocking the expression and activation of CXCL13,S100A8,and S100A9 considerably ameliorates the inflammatory response and tissue damage,suggesting their crucial role in the development and progression of immune-inflammatory-related diseases.However,the expression and mechanism of CXCL13,S100A8,and S100A9 in IC/BPS remain unreported.Therefore,this study fills the research gap by employing clinical specimens,animal models,bioinformatics analysis,TMT proteomics sequencing,single-cell sequencing,and knockout animal techniques.This investigation is anticipated to offer valuable theoretical support for identifying IC/BPS intervention targets.Methods:Sequencing data GSE11783 from IC/BPS patients was employed for bioinformatics analysis,while bladder paraffin sections of IC/BPS patients were gathered for histological examination.An experimental autoimmune cystitis(EAC)animal model of IC/BPS was established using 8-10-week-old female C57BL/6 mice.S100A9 knockout animals(B6/JGpt-S100a9em5Cd9336/Gpt)were utilized for animal experimental research.TMT protein sequencing and single-cell sequencing were applied to EAC mouse bladder tissue protein sequencing.TAK-779(CXCL13/CXCR5 blocker)and Paquinimod(S100A8 and S100A9 blocker)were employed to intervene in EAC animal models.Hematoxyli n and eosin(H&E)staining and mast cell staining were used for histological analysis of bladder tissue in IC/BPS patients and EAC mice.Western blotting was employed to detect the expression of inflammatory factors,apoptosis-related proteins,JNK,TLR4/My D88,NF-κB,and p38 signaling pathway proteins in the bladder tissue of EAC mice.Immunohistochemistry was conducted to determine the expression and distribution of CXCL13,CXCR5,CD4,F4/80,CD68,S100A8,and S100A9 proteins in the bladder of IC/BPS patients and EAC mice.Immunofluorescence was utilized for the colocalization analysis of S100A8 and S100A9 with CD68.TUNEL staining was employed to assess the apoptosis of bladder tissue in EAC mice.Urodynamic evaluations were performed to assess the bladder function of EAC mice.Results:1.The EAC model exhibited bladder tissue edema,inflammatory cell infiltration,and bladder epithelial damage,similar to the pathological manifestations of IC/BPS.Urodynamics also demonstrated a significant increase in micturition frequency(MF)and a decrease in inter-contraction interval(ICI),indicating that EAC mice can effectively simulate the phenotypic and pathophysiological changes of IC/BPS.2.CXCL13 and CXCR5 were significantly up-regulated in IC/BPS and EAC(p<0.05).The JNK and NF-κB signal path downstream of CXCL13/CXCR5 axis is activated in IC/BPS and EAC.Compared with the control group,the score of bladder tissue in EAC group was significantly higher(p<0.05),and the expressions of inflammatory factors(IL-6,TNF-α,IL-1β)and apoptosis-related proteins(Bax,Caspase-3,Caspase-8)were significantly increased(p<0.05),JNK and NF-κB signal pathways were activated,MF was significantly increased(p<0.05),and ICI was significantly shortened(p<0.05).Compared with the EAC group,the bladder tissue score of EAC+TAK-779 group was significantly lower,and the expression of inflammatory factors(IL-6,TNF-α,IL-1β)and apoptosis-related proteins(Bax,Caspase-3,Caspase-8)decreased significantly,JNK and NF-κB signal pathway activation was blocked,MF was significantly reduced(p<0.05),ICI was significantly prolonged(p<0.05),and bladder function was significantly recovered.3.GSE11783 data of IC/BPS patients and sequence analysis of EAC animal proteomics revealed that S100A8 and S100A9 were key targets of abnormal immune-inflammatory reactions in IC/BPS and EAC.Immunohistochemistry and Western blotting demonstrated high expressions of S100A8 and S100A9 in IC/BPS patients and EAC animals,particularly in the bladder mucosa and muscle layer(p<0.05).Furthermore,analysis of GSE11783 data and EAC animal proteomics sequencing indicated increased infiltration of macrophages,T cells,and mast cells in IC/BPS patients and EAC animals(p<0.05).Single-cell sequencing analysis and immunofluorescence analysis suggested that S100A8 and S100A9 in IC/BPS and EAC bladder tissue may primarily originate from macrophages.Moreover,single-cell sequencing analysis discovered significantly increased number and intensity of communication between EAC cells,with macrophages and T cells exhibiting the most significant increase(p<0.05).Bioinformatics analysis indicated that S100A8 and S100A9-related TLR/My D88,NF-κB,and p38 inflammatory signaling pathways were activated in the bladder of IC/BPS and EAC patients.4.Compared to the control group,the EAC group displayed a significantly increased bladder tissue score(p<0.05),elevated expression of inflammatory factors(IL-6,TNF-α,IL-1β)and apoptosis-related proteins(Bax,Caspase-1,Caspase-3,Caspase-8),decreased expression of bladder epithelial marker proteins UPK2 and UPK3A(p<0.05),and increased infiltration of mast cells,macrophages(F4/80),and T cells(CD4)(p<0.05).TLR4/My D88/NF-κB and TLR4/My D88/p38 signaling pathways were activated,MF significantly increased(p<0.05),and ICI significantly shortened(p<0.05).After administering Paquinimod to EAC mice and knocking out S100A9 in EAC mice,the activation of TLR4/My D88/NF-κB and TLR4/My D88/p38 signaling pathways were blocked.The bladder tissue score in the EAC group significantly decreased(p<0.05),expression of inflammatory factors(IL-6,TNF-α,IL-1β)and apoptosis-related proteins(Bax,Caspase-1,Caspase-3,Caspase-8)significantly reduced(p<0.05),and expression of bladder epithelial marker proteins UPK2 and UPK3 A significantly increased(p<0.05).Additionally,infiltration of mast cells,macrophages(F4/80),and T cells(CD4)significantly decreased(p<0.05),MF significantly decreased(p<0.05),and ICI significantly prolonged(p<0.05).Conclusions1.CXCL13/CXCR5 axis activation is involved in the pathophysiology of IC/BPS and EAC.The activation of NF-κB and JNK signaling pathways downstream of the CXCL13/CXCR5 axis involved in the pathophysiology of IC/BPS and EAC,which was blocked by TAK-779 effectively,thereby ameliorating cystitis injury and bladder function in EAC mice.2.Proteomic sequencing,single-cell sequencing,bioinformatics analysis,and knockout animal studies confirm that S100A8 and S100A9 are critical proinflammatory and pathogenic molecules in IC/BPS,and their activation exacerbates the inflammatory response and tissue damage in the IC/BPS bladder.S100A8 and S100A9 in the bladder of IC/BPS and EAC may primarily originate from macrophages.The number and intensity of communication between macrophages and T cells in the EAC bladder are increased,suggesting that macrophage and T cell interactions may play a role in the pathophysiology of IC/BPS.3.S100A8 and S100A9 can activate TLR4/My D88/NF-κB and TLR4/My D88/p38 signaling pathways,leading to cystitis injury and bladder function impairment in EAC mice.Paquinimod administration and S100A9 knockout can significantly block the activation of TLR4/My D88/NF-κB and TLR4/My D88/p38 signaling pathways,improving the cystitis injury and bladder function in EAC mice.