泌尿道“专性/兼性厌氧菌”失衡介导丁酸盐减少引起上皮功能障碍在膀胱过度活动症中的作用机制研究

Abnormalities of afferent sensory nerve signaling mediated by the urothelial dysfunction is thought to be a main pathogenesis of overactive bladder (OAB). Previous researches of OAB were focus on bladder but ignored the effects of urinary tract microenvironment. In our previous work, the aberrant urinary microbiome with increased facultative anaerobes and decreased obligate anaerobes were observed distinctly in OAB patients, and we also demonstrated that butyrate concentrations in urine of OAB group were significantly decreased compared to controls. It has been known that reduction of butyrate may result in insufficient activation of PPAR-γ, inducing impaired epithelial barrier. Therefore, we propose a hypothesis that decreased butyrate mediated by aberrant urinary microbiome may cause insufficient activation of PPAR-γ and sequentially impair bladder epithelial barrier. Then infiltration of harmful substances may lead to abnormalities of afferent sensory nerve signaling and finally trigger the initiation of OAB. This project is aimed specifically at the connections between urinary microbiome and host. At human, animal, and cell levels, we plan to further study the concrete mechanism how urinary microbiome decreases butyrate and consequently impairs bladder epithelial barrier. We will perform a series of experiments in vivo and vitro, including high-throughput sequencing for the analysis of microbiome composition and metabolic function, isolating specific strains for transplantation and co-culture, supplementing with butyrate and regulating PPAR-γ and PKC signaling pathway. We hope to provide experimental and theoretical basis for clarifying the pathogenesis of OAB and finding novel therapeutic targets ultimately.

膀胱传入神经信号异常是膀胱过度活动症（OAB）的主要发病机制之一，而膀胱上皮功能受损是介导异常传入神经信号形成的重要原因。既往研究聚焦于膀胱本身，未关注到泌尿道微环境的影响。我们发现OAB患者泌尿道存在“专性厌氧菌减少/兼性厌氧菌增多”的菌群紊乱模式及丁酸盐含量显著降低。已知丁酸盐可通过激动核受体PPAR-γ，增强上皮屏障和线粒体β氧化功能。因此提出假设：泌尿道菌群紊乱介导丁酸盐减少和PPAR-γ激动不足，造成膀胱上皮功能障碍和β氧化受损，局部微环境发生改变，从而诱发OAB。本项目拟从菌群与宿主相互作用的角度入手，在人体、动物、细胞三个层次，通过高通量测序进行菌群结构和代谢功能谱分析、分离特异菌株进行菌株移植和共培养、补充丁酸盐、调控PPAR-γ等体内外实验，进一步研究菌群紊乱介导丁酸盐减少和膀胱上皮屏障受损的具体机制，从微生态视角为阐明OAB发病机制和寻找新治疗靶标提供依据。

发现了OAB患者的泌尿道菌群紊乱模式，初步探究了泌尿道微生态与OAB发生发展之间的关系。在前期研究的基础上扩大OAB患者样本量，按病情程度分为轻度组与中重度组，通过对其中段尿行16S rDNA高通量测序发现，二组OAB患者在门水平最常见的是厚壁菌门，其次为变形杆菌、放线杆菌和拟杆菌；Chao1指数和Ace指数(反映细菌群落丰富度)在中/重度组中显著升高，Shannon指数与Simpson指数（反映细菌总体多样性）在轻度组显著降低，Pielou指数（反应细菌均匀度）无显著差异，表明OAB症状的严重程度与泌尿道菌群丰富度有关，而与其均衡性无关。同时，OABSS评分与上述尿液菌群的多样性指标均有显著相关性。另外通过导尿方式收集了55名OAB患者和18名对照的尿液样本并行NGS检测，发现JC病毒是尿液中最常见的病毒，并且病毒感染组的OABSS评分、OAB-V8评分、O’Leary-Sant评分、PUF量表评分均显著高于非病毒感染组，提示病毒感染可能是导致OAB病情进展的可能因素。通过进一步分析发现， OAB病毒感染组组的华氏葡萄球菌、人葡萄球菌和表皮葡萄球菌的相对丰度显著增加。我们发现的JC病毒主要为7B亚型和7A亚型，既往已有报道OAB与JC病毒的感染相关，且其亚型主要集中分布于欧洲的1型和1B型，本团队的研究符合JC病毒的流行分布，且扩充了致OAB的JC病毒亚型。米拉贝隆作为β3受体激动剂药物已用于OAB的常规治疗，但对于部分OAB患者其疗效甚微，本团队通过对64例成年女性OAB患者的尿样进行16S rRNA基因测序，记录米拉贝隆治疗前后患者的膀胱过度活动症症状评分(OABSS)，并将患者分为有效组和无效组，分析了尿液菌群中某些属的相对丰度与OABSS的关系；对亚组分析后发现泌尿道菌群中产丁酸盐菌丰度较低组中，OABSS评分在米拉贝隆治疗后显著降低，而在丰度较高组中，米拉贝隆治疗前后OABSS评分无明显变化，说明丁酸盐在米拉贝隆缓解OAB的疗效中起着反面角色；最后根据OAB患者尿液菌群中乳杆菌属的相对丰度建立了米拉贝隆对于OAB的疗效预测模型。