HCN4通道在OAB膀胱感觉信号通路中的作用及其调控机制研究

Overactive bladder (OAB) is a prevalent and chronic medical condition that greatly impacts an individual's QOL, while the multifactorial pathophysiology of this condition and intrinsic compensatory mechanisms confound the evaluation of treatment response. Dysfunction of bladder afferent signaling has been proposed to contribute to the pathogenesis of OAB. Ion channel disorders may lead to the dysfunction of afferent signaling and induce OAB symptom through affecting afferent cell membrane potential and excitability. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been shown to play key roles in bladder afferent signaling. Surpported from the former National Natural Science Foundation, our pilot study revealed the upregulation of expression of HCN4 channels in OAB rat bladder interstitial cells (ICC) and dorsal root ganglion (DRG) neurons. And these cells play key roles in the bladder afferent signaling. However, the mechanisms underlying HCN4 channels in the dysfunction of bladder afferent signaling remain unclear. In this project, multiple experimental techniques, including morphology, molecular biology, whole cell patch clamp and afferent nerve electrophysiology will be employed. And we aim to investigate the functions and modulation mechanisms of HCN4 channels in afferent signaling in OAB, through investigation of the bio-physiologic feature, regulation effects and plasticity changes of HCN4 channels in bladder ICC and DRG neurons in OAB rats. Furthermore, this study might provide experimental basis for understanding the etiology and exploring more effective novel drug targets for OAB treatment.

膀胱过度活动症（OAB）严重影响患者生活，是临床上发病机制不清且又最缺乏有效治疗手段的一类疾病。膀胱感觉信号的异常是引发OAB的重要因素。离子通道的病变可能通过影响感觉细胞膜电位及兴奋性引起膀胱感觉信号异常并引发OAB症状。超极化激活环核苷酸门控阳离子通道(HCN)与膀胱感觉信号通路密切相关。在前一项国科金青年项目资助下，我们发现OAB大鼠膀胱感觉信号传递通路中的关键细胞膀胱间质细胞及脊髓背根节神经元中HCN4通道表达明显上调。但HCN4通道如何参与OAB膀胱异常感觉信号传递尚不清楚。基于此，我们拟采用形态学、分子生物学、全细胞膜片钳及膀胱感觉神经电生理等技术，观察OAB大鼠膀胱间质细胞及背根节神经元内HCN4通道的生物物理特性、调制机制及其可塑性变化，明确HCN4通道在OAB膀胱异常感觉信号传递的作用及调控机制，为进一步了解OAB的发病新机制，寻找新的OAB药物作用靶点提供实验基础。

膀胱感觉信号的异常是引发OAB的重要因素。超极化激活环核苷酸门控阳离子通道(HCN)与膀胱感觉信号通路密切相关。我们对正常人膀胱ICC内HCN通道不同亚型表达进行研究，结果显示正常人膀胱ICC内有HCN1,2,3及4亚型的表达，其中HCN4表达量最高。进一步研究发现OAB大鼠膀胱感觉信号传递通路中的关键细胞膀胱间质细胞及脊髓背根节神经元中HCN4通道表达明显上调。同时，采用形态学、分子生物学、全细胞膜片钳及膀胱感觉神经电生理等技术，观察OAB大鼠膀胱间质细胞及背根节神经元内HCN4通道的生物物理特性、调制机制及其可塑性变化，1.膀胱尿动力学检测结果提示：OAB组膀胱逼尿肌自发性收缩频率随时间明显升高，且8周组明显高于4周及6周组。2. Western blot检测显示，HCN4通道蛋白在对照组及OAB组均有表达，与对照组相比，OAB组的膀胱粘膜层及肌层中HCN4蛋白表达量随OAB发生时间均显著增高(P<0．01)，在OAB-8周组达到最高。3. 支配膀胱的DRG组织中HCN各亚型较正常对照组大鼠明显升高，且HCN4通道蛋白随OAB发生时间显著增高(P<0．01)，OAB-8周组表达量最高；4.OAB模型组大鼠DRG神经元Ih电流阳性细胞率较正常对照组明显升高；5.OAB模型组大鼠DRG神经元Ih电流密度较正常对照组明显升高，ZD7288可完全抑制Ih电流的发生。HCN4通道在大鼠膀胱粘膜层、肌层以及膀胱对应脊髓DRG中均有表达，且OAB组较对照组显著增多，并随OAB发生时间而升高。提示HCN4通道可能参与了OAB膀胱功能的可塑性变化。DRG中HCN通道蛋白表达增强时，可导致膀胱感觉神经末梢和胞体在接近静息膜电位水平附近时产生自发放电，同时受刺激后产生高频簇放电，形成异常膀胱感觉信号，从而引发OAB症状。本项目为明确HCN4通道在OAB膀胱异常感觉信号传递的作用及调控机制，寻找新的OAB药物作用靶点提供实验基础。