逼尿肌细胞缝隙连接蛋白43表达及半通道开放与间质性膀胱炎发病机制关系的研究

Painful bladder syndrome/interstitial cystitis (PBS/IC) is a chronic bladder inflammatory disease that is characterized by suprapubic and pelvic pain and voiding symptoms such as urinary urgency, and frequency. Recently, the incidence of PBS/IC has increased obviously in China, and the quality of life of the patients is very poor. To date, the pathological basis for PBS/IC is still poorly understood.. Gap junctions (GJs) are clusters of transmembrane channels that permit the direct exchange of ions, secondary messengers, and small signaling molecules among neighboring cells. Each GJ channel is composed of two hemichannels that reside in the plasma membrane of two closely apposed cells. The proteins that form the gap junctions are called connexin (Cx). In bladder smooth muscle cells, Cx43 is the main functional GJ protein. The Cx-formed GJs are thought to play an important role in the control of a variety of cellular functions. Besides intercellular GJ channels, there are the nonjunctional Cx hemichannels on the membrane, which have been thought to be implicated in various pathological situations. Based on our previous studies, we speculate that Cx43-formed GJs and hemichannel may participate in the BPS/IC pathogenesis and serve as the critical regulatory structure.. In this study, we will establish the mice models of interstitial cystitis induced by cyclophosphamide (CYP) through intraperitoneal injection. During the experiments in vitro, bladder smooth muscle cells will be stimulated by acrolein (ACR), the intracorporal metabolite of CYP. We will investigate the mRNA level and protein expression of Cx43 under the stimulation of ACR, and also test the distribution of Cx43 on the membrane by immunofluorescent staining and GJs' function by scrape loading dye transfer assay. The electrophysiological characteristics of GJs channels will be examined by patch clamp techniques. In the next step, we will use various specific inhibitors and siRNAs to test the involvement of key enzymes and transcriptional factors in the regulation of Cx43 expression, such as NF-κB, iNOS and COX-2. Additionally, the status of hemichannels and exchange of small molecules will be examined by high performance liquid chromatography. . In the experiments in vivo, we will compare the alteration of Cx43 expression and small molecule concentration in urine after intraperitoneal injection of CYP, in the offsprings of heterozygous Cx43 knockout mice, including Cx43+/+ and Cx43+/- (Cx43-/- mice will die after birth). Application with intravesical instillation with fluorescent marker calcein-AM, hemichannel opening will be investigated with two-photon confocal excitation microscopy. Other function experiments include pain score for mice behavior and strip contraction. . Collectively, we will provide a potentially important molecular mechanism for PBS/IC pathogenesis, and targeting Cx43-formed GJs and signal pathways concerned may hold promise for PBS/IC.

膀胱疼痛综合症/间质性膀胱炎（PBS/IC）的核心临床表现是疼痛，患者生活质量差，近年发病率上升明显。PBS/IC发病机制不明确，是泌尿外科研究中的热点和难点。细胞缝隙连接（GJ )及半通道是保持组织和细胞内稳态的核心调节结构，连接蛋白43（Cx43）是调节逼尿肌功能的主要连接蛋白，我们推测其在PBS/IC发病过程中具有重要作用。我们建立环磷酰胺（CYP）诱导的小鼠膀胱炎模型，体外细胞实验应用CYP代谢产物丙烯醛（ACR），证实其调节Cx43蛋白及mRNA水平和相关的信号传导通路，明确其引起逼尿肌细胞半通道开放的状态与机制；根据体外实验结果，体内条件下证实转基因Cx43半表达小鼠和调节Cx43表达的信号通路抑制剂对CYP诱导膀胱炎的影响，比较不同小鼠间及应用抑制剂前后小鼠痛学评分和逼尿肌肌条收缩能力的改变，探索体内干预的可能。研究成果有助阐明PBS/IC的发病机制，为临床治疗提供理论依据。

膀胱疼痛综合症/间质性膀胱炎（PBS/IC）的核心临床表现是疼痛，患者生活质量差，近年发病率上升明显。PBS/IC发病机制不明确，是泌尿外科研究中的热点和难点。细胞缝隙连接（GJ )及半通道是保持组织和细胞内稳态的核心调节结构，连接蛋白43（Cx43）是调节逼尿肌功能的主要连接蛋白，我们的研究旨证实其在PBS/IC发病过程中具有重要作用。我们建立环磷酰胺（CYP）诱导的小鼠膀胱炎模型，体外细胞实验应用CYP代谢产物丙烯醛（ACR），证实其调节Cx43蛋白及mRNA水平和相关的信号传导通路（NF kappaB-iNOS-NO，以及COX2-PGI2， cAMP信号通路），明确其引起逼尿肌细胞半通道开放的状态与机制（GSH和ATP）；根据体外实验结果，体内条件下证实转基因Cx43半表达小鼠和调节Cx43表达的信号通路抑制剂对CYP诱导膀胱炎的影响，比较了不同小鼠间及应用抑制剂前后小尿动力学指标的改变，探索了体内干预的可能。研究成果有助阐明PBS/IC的发病机制，为临床治疗提供理论依据。