背景：间质性膀胱炎/膀胱疼痛综合征（Interstitial Cystitis/Bladder Pain Syndrome, IC/BPS）是一种慢性非细菌性膀胱炎症。患者中重度长期疼痛严重影响生活质量，现有治疗方式、方法均未能达到理想治疗效果。IC/BPS的发病机制目前尚无定论，炎症因子在IC/BPS的发生发展中起着重要作用，但缺乏以此为靶点的治疗药物。

目的：探索IC/BPS患者在膀胱水扩张下发生溃疡和点状出血的可能机制，并筛选出的潜在的炎症治疗靶点。基于炎症治疗靶点，针对性地使用PDE4抑制剂，探索其在大鼠IC/BPS中的治疗作用并通过多组学明确其治疗机制。最终为临床上IC/BPS一线用药的开发提供理论基础和研发方向。

方法：首先，收集人源膀胱黏膜组织样本，进行单细胞测序。对数据进行Seurat单细胞数据处理，Cellchat细胞通信分析和scMetabolism代谢分析，通过TCGA数据库对关键差异基因进行分析，并结合GEO公开数据集，对单细胞分析结果进行验证。随后，根据分析结果选用新型PDE4抑制剂，在IC/BPS大鼠模型中试验其治疗效果。采用硫酸鱼精蛋白联合脂多糖隔天一次膀胱灌注的方式构建IC/BPS大鼠模型。通过HE染色，甲苯胺蓝染色分析大鼠膀胱组织学改变；通过尿液组胺水平分析大鼠膀胱症状严重程度；通过尿斑实验和代谢笼实验分析大鼠的膀胱容量和尿频、尿急等下尿路症状；通过强迫游泳实验，悬尾实验和糖水偏好实验分析大鼠的抑郁表现；通过酶联免疫吸附试验和蛋白免疫印记分析大鼠膀胱和血清中IL-1β、IL-6和TNF-α炎症因子的表达和新生血管丰富程度；通过实时荧光定量聚合酶链式反应分析PDE4B基因的转录水平；并结合转录组学、代谢组学和蛋白组学，从基因表达到代谢过程，再到蛋白质功能，全面解读PDE4抑制剂在IC/BPS治疗中的生物学特性。最后，使用SV-HUC-1细胞系，分析新型PDE4抑制剂对SV-HUC-1细胞活力的影响，通过酶联免疫吸附试验对IL-1β、IL-6和TNF-α炎症因子进行检测、通过TUNEL染色和流式细胞术对凋亡进行检测；使用划痕试验对细胞迁移能力进行分析；通过酶联免疫吸附试验分析cAMP/PKA/CREB信号通路及其下游的IL-17家族在PDE4抑制剂发挥治疗作用中的意义。

结果：本研究通过单细胞测序分析，进一步证实了IC/BPS是一种与炎症和自身免疫相关性极高的慢性疾病。本研究发现具有炎症特征的高表达S100P基因的上皮细胞参与了Hunner's溃疡的形成，而中性粒细胞浸润和胶原分解代谢等可能是介导溃疡的主要因素。NHIC更高比例的抑制性免疫细胞在一定程度上控制了炎症，从而没有引起溃疡的发生。SEMA3D，S100P，COL4A1等参与的大量新生血管生成和Ⅳ型胶原减少导致的血管壁脆弱，形成了IC/BPS膀胱水扩张下更容易出现小球样出血的特征。IC/BPS更高比例的神经元细胞和广泛炎症浸润则加重了膀胱区域疼痛的表现。IC/BPS的关键差异基因LAYN在肿瘤的发生发展中起到重要作用，可能是IC/BPS患者增加肿瘤风险的因素。重要的是，本研究提出炎症可作为直接针对病理生理机制的IC/BPS治疗靶点，为使用抗炎药物（如PDE4抑制剂）治疗IC/BPS提供了理论支持。接下来，本研究同时针对TNF-α和IL-6这两个靶点，使用PDE4抑制剂在动物模型中探究其对IC/BPS的治疗作用。本研究使用了一种全新的PDE4抑制剂，并通过膀胱灌注的方式给药。本研究发现，新型PDE4抑制剂可以减轻IC/BPS大鼠的膀胱组织水肿（模型组水肿程度评分2.17±0.75，治疗组水肿程度评分1.17±0.75，P=0.047），减少肥大细胞数量（模型组每视野肥大细胞数量7.67±2.16，治疗组每视野肥大细胞数量3.50±2.88，P=0.009），缓解大鼠膀胱疼痛、感觉过敏（P<0.0001）。尿斑实验显示模型组尿斑个数5.17±1.47，治疗组尿斑个数2.00±0.63，差异有统计学意义（P<0.001）；模型组最大尿斑面积15.93±2.94mm²，治疗组最大尿斑面积31.36±7.77mm²，差异有统计学意义（P=0.002），单位时间内模型组和治疗组总尿量（P=0.203）和角落尿斑面积（P=0.252）无显著差异。同时治疗组的抑郁行为表现也较IC/BPS模型组有所缓解（P<0.05）。新型PDE4抑制剂显著降低了IC/BPS大鼠膀胱组织和血清中的IL-1β、IL-6和TNF-α炎症因子的高表达（P<0.05），也降低了大鼠尿液中组胺水平（模型组4.88±1.29，治疗组2.65±0.49，P=0.001）。PDE4抑制剂还降低了PDE4B的转录水平（P<0.0001），并逆转了IC/BPS大鼠的新生血管生成。随后，运用转录组学、代谢组学和蛋白组学方法，全面解读了PDE4抑制剂在大鼠IC/BPS中发挥治疗作用的机制。本研究发现炎症通路是PS/LPS构建IC/BPS模型的核心通路，并且与人的IC/BPS存在相似的信号传导通路和差异代谢通路。代谢产物分析显示PDE4抑制剂可能直接或间接通过影响花生四烯酸代谢通路来发挥抗炎作用。蛋白组学则提示PDE4抑制剂可能通过修复黏膜，对IC/BPS的膀胱刺激症状起到缓解作用。TNF信号通路、NF-κB信号通路、IL-17信号通路是IC/BPS病理生理过程中的重要通路，同时也是PDE4抑制剂发挥作用的共同途径。PDE4抑制剂可通过降低IL-17信号通路等的活性，从而缓解IC/BPS。最后，本研究通过CCK8方法在SV-HUC-1细胞中筛选出了最适药物浓度，PDE4抑制剂在SV-HUC-1细胞中显著降低了IL-1β、IL-6和TNF-α炎症因子表达（P<0.05）。PDE4抑制剂通过cAMP/PKA/CREB通路对炎症起到抑制作用并呈剂量依赖性。此外，PDE4抑制剂还能减轻PS/LPS诱导的细胞凋亡，并通过调节IL-17信号通路发挥治疗作用。基于人转录组水平的数据的结果显示IL-17A、IL17F、IL-17RA、IL-17RB、IL17RC在IC/BPS患者中的表达均上调（P<0.05），这提示IL-17家族在IC/BPS的发生和治疗中具有重要价值。

结论：IC/BPS是一种与炎症和自身免疫相关性极高的慢性疾病，SEMA3D，S100P，COL4A1和LAYN等基因在其表现和发展中起到重要作用。炎症可作为直接针对病理生理机制的IC/BPS治疗靶点。动物实验证明PDE4抑制剂可以在IC/BPS中发挥治疗效果，其机制涉及cAMP/PKA/CREB信号通路及其调控的IL-17炎症信号通路等。本研究为临床上IC/BPS用药缺乏问题提供解决的理论基础和潜在的治疗选择。

Background: Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic non-bacterial inflammation of the bladder. Patients suffer from long-term severe pain that seriously affects their quality of life. Existing treatment methods have failed to achieve ideal therapeutic effects. The pathogenesis of IC/BPS is currently unclear, and inflammatory factors may play an important role in the occurrence and development of IC/BPS, but there is a lack of drugs for targeted treatment of this mechanism.

Objective: To explore the possible mechanisms of ulceration and bleeding in the bladder under hydrodistention in IC/BPS patients, and to identify potential inflammatory therapeutic targets. Based on these targets, selectively utilize PDE4 inhibitors to investigate their therapeutic effects in rat IC/BPS models and elucidate their treatment mechanisms through multi-omics approaches. Ultimately, provide a theoretical basis and research direction for the development of first-line medications for IC/BPS in clinical practice.

Methods: Initially, human bladder mucosal tissue samples were collected for single-cell sequencing. The data was processed using Seurat for single-cell data analysis, Cellchat for cell communication analysis, and scMetabolism for metabolism analysis. Key differential genes were analyzed using the TCGA database, and the single-cell analysis results were validated using publicly available datasets from GEO datasets. Subsequently, a novel PDE4 inhibitor was selected based on the analysis results and tested for its therapeutic effects in an IC/BPS rat model. The IC/BPS rat model was established by intravesical instillation of fish protein polysaccharide and lipopolysaccharide every other day. HE staining and toluidine blue staining were performed to analyze histological changes in rat bladder tissue. Urinary histamine levels were analyzed to assess the severity of bladder symptoms. The urinary spot test and metabolic cage experiment were conducted to analyze bladder capacity, urinary frequency, urgency, and other lower urinary tract symptoms in rats. The forced swimming test, tail suspension test, and sucrose preference test were performed to evaluate depressive behaviors in rats. ELISA and Western blot were utilized to analyze the expression of IL-1β, IL-6, and TNF-α inflammatory factors and neovascularization in rat bladder tissue and serum. Real-time fluorescence quantitative polymerase chain reaction was employed to analyze the transcription levels of the PDE4B gene. By integrating transcriptomics, metabolomics, and proteomics analyses, this study comprehensively elucidated the biological characteristics of PDE4 inhibitors in the treatment of IC/BPS, from gene expression to metabolic processes, and finally to protein functionality. Furthermore, the impact of novel PDE4 inhibitors on the viability of SV-HUC-1 cells was assessed, and ELISA were performed to detect the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α. Apoptosis was evaluated using TUNEL staining and flow cytometry. The migratory ability of cells was analyzed using a scratch assay. Additionally, the significance of the cAMP/PKA/CREB signaling pathway and downstream IL-17 family in the therapeutic effects of PDE4 inhibitors was investigated through ELISA.

Results: This study confirmed through single-cell sequencing analysis that IC/BPS is a chronic condition highly correlated with inflammation and autoimmune factors. The formation of Hunner's ulcers involves the destruction of cells and mucosal barriers caused by inflammation. This study identified epithelial cells with high expression of the inflammatory marker gene S100P as participants in Hunner's ulcer formation, while neutrophil infiltration and collagen degradation metabolism may serve as primary mediators of ulceration. A higher proportion of inhibitory immune cells in NHIC partially controlled inflammation, thus preventing ulcer occurrence. Extensive neovascularization involving SEMA3D, S100P, COL4A1, and others, coupled with reduced Type IV collagen, led to weakened vessel walls, characteristic of bladder wall fragility in IC/BPS, predisposing to bleeding. Increased proportions of neuronal cells and widespread inflammation exacerbate bladder area pain manifestations. The key differential gene LAYN plays an important role in the occurrence and development of tumors, which may be a contributing factor to increased cancer risk in IC/BPS patients. Importantly, this section proposes inflammation as a direct therapeutic target for IC/BPS pathophysiology, providing theoretical support for the use of anti-inflammatory agents such as PDE4 inhibitors in the treatment of interstitial cystitis. Next, this study explored the therapeutic effects of a PDE4 inhibitor targeting both TNF-α and IL-6 on IC/BPS in an animal model. This study used a novel PDE4 inhibitor administered via bladder infusion and observed that the novel PDE4 inhibitor alleviated bladder tissue inflammation and edema in IC/BPS rats (P=0.047), reduced the number of mast cells (P=0.009), and relieved bladder pain in rats (P<0.0001). The micturition test showed significant improvements in lower urinary tract symptoms such as frequency (P<0.001) and urgency (P=0.003) in the treatment group. Moreover, the treatment group exhibited alleviated depressive behavior compared to the IC/BPS model group (P<0.05). The novel PDE4 inhibitor significantly reduced the high expression of inflammatory factors, including IL-1β, IL-6, and TNF-α, in bladder tissue and serum of IC/BPS rats (P<0.05). It also decreased histamine levels in rat urine (P=0.001). The PDE4 inhibitor downregulated the transcription levels of PDE4B (P<0.0001) and reversed neovascularization in IC/BPS rats.Subsequently, this study employed transcriptomics, metabolomics, and proteomics methods to comprehensively elucidate the mechanisms underlying the therapeutic effects of PDE4 inhibitors in IC/BPS rats. Our study revealed that the inflammatory pathway is the core pathway in constructing the PS/LPS-induced IC/BPS model in rats, which shares similar signaling transduction pathways and differential metabolic pathways with human IC/BPS. Metabolite analysis demonstrated that PDE4 inhibitors may exert anti-inflammatory effects by directly or indirectly influencing the arachidonic acid metabolic pathway. Proteomics findings suggested that PDE4 inhibitors may alleviate bladder irritative symptoms in IC/BPS by repairing the mucosa. The TNF signaling pathway, NF-κB signaling pathway, and IL-17 signaling pathway were identified as important pathways in the pathophysiological processes of IC/BPS and also as common pathways through which PDE4 inhibitors exert their effects. PDE4 inhibitors can alleviate IC/BPS by reducing the activity of IL-17 signaling pathway, among others. Lastly, in this study, the optimal drug concentration was determined through CCK-8 assay in SV-HUC-1 cells. Significant reductions in the expression of IL-1β, IL-6, and TNF-α inflammatory factors were observed in SV-HUC-1 cells treated with PDE4 inhibitors (P<0.05). The inhibitory effects of PDE4 inhibitors on inflammation were mediated through the cAMP/PKA/CREB pathway and exhibited a dose-dependent response. Moreover, PDE4 inhibitors alleviated LPS-induced cell apoptosis and exerted therapeutic effects by modulating the IL-17 signaling pathway. Analysis of human transcriptomic data revealed upregulation of IL-17A, IL17F, IL-17RA, IL-17RB, and IL17RC expression in IC/BPS patients (P<0.05), suggesting the crucial value of the IL-17 family in the occurrence and treatment of IC/BPS.

Conclusion: IC/BPS is a chronic disease highly associated with inflammation and autoimmune factors. Genes such as SEMA3D, S100P, COL4A1, and LAYN play important roles in its manifestation and development. Inflammation can be considered a direct therapeutic target against the pathological mechanisms of IC/BPS. Animal studies have demonstrated the remarkable therapeutic efficacy of PDE4 inhibitors in IC/BPS, involving the modulation of the cAMP/PKA/CREB signaling pathway and the IL-17 inflammatory signaling pathway. This research provides a theoretical foundation and a direction for the development of frontline medications for IC/BPS in clinical practice.