氯胺酮致膀胱平滑肌损伤及其分子机制研究

Ketamine is a well-known noncompetitive antagonist for N-methyl-D-aspartate receptor (NMDAR) and is being increasingly used as recreation drug in China due to its hallucinatory effects. Most of ketamine abusers reported the presence of lower urinary tract symptoms, such as severe dysuria, painful hematuria, urinary frequency, urgency, and urge urinary incontinence. Clinical examination often indicates a contracted bladder with chronic inflammation, ulceration, or necrotic mucosa, but with sterile pyuria, giving rise to the term ketamine cystitis. Although the number of patients diagnosed with ketamine cystitis is increasing, there is no single definitive treatment reported to date, because the exact pathophysiological mechanism of ketamine on the urinary bladder is unknown. we found that ketamine could dose-dependently and directly impair bladder smooth muscle (BSM), we concluded that smooth muscle is an important target for ketamine-induced pathology. In this study, ketamine-induced cystitis mouse model and bladder smooth muscle NMDAR homozygous knockout mouse (M-NR1-/-) will be generated. By using a combination of cystometrogram, voiding spot assay (VSA), myography and other pathological experiments, we will demonstrate the role of NDMAR in bladder physiological function and the effects of ketamine on regulating bladder smooth muscle properties by inhibition of NMDAR. And using calcium imaging, we will explore the mechanisms of ketamine in inhibition of calcium influx and will discover ketamine inhibition of calcium influx whether by blocking NMDAR. More importantly, we will unravel the downstream signaling of NDMAR and demonstrate that the molecular mechanism of ketamine-associated pathologies in bladder smooth muscle. This study will provide insight into the pathogenesis of ketamine cystitis and other related pathology and offer a great potential treatment approach for ketamine-induced pathologies.

氯胺酮作为非竞争性N-甲基-D天门冬氨酸酸受体（N-methyl-D-aspartate receptor, NMDAR）拮抗剂，是我国最常被滥用的毒品，多数滥用者出现尿困难、尿频、尿急、尿痛，被称为氯胺酮相关性膀胱炎。该病严重影响患者生活质量，由于其致病机制不清楚，目前无有效治疗方案。我们前期研究发现氯胺酮对人源膀胱平滑肌原代细胞具有直接毒性，能阻断细胞外钙离子内流，抑制膀胱平滑肌收缩，提示膀胱平滑肌受损可能是氯胺酮相关性膀胱炎发生的重要因素。本项目建立了氯胺酮相关性膀胱炎小鼠模型，膀胱平滑肌NMDAR特异敲除小鼠模型，将通过分子细胞生物学结合生理病理学实验方法，明确氯胺酮通过抑制膀胱平滑肌NMDAR对膀胱生理功能的影响，探明氯胺酮阻断平滑肌细胞外钙离子内流机制及NMDAR下游信号通路，阐明氯胺酮致膀胱平滑肌损伤分子机制。本研究对氯胺酮相关性膀胱炎治疗靶点选择及治疗方案探索具有重要意义。

在我国，氯胺酮是最常被滥用的毒品，30%的滥用者出现严重的下尿路症状，由于其致病机制尚不清楚，目前没有有效的治疗药物。本研究通过建立氯胺酮相关性膀胱炎小鼠模型，发现氯胺酮相关性膀胱炎小鼠膀胱平滑肌具有明显的病理生理功能改变，包括: 膀胱壁具有明显损伤，上皮细胞脱落，膀胱黏膜屏障变薄，肌肉层间隙增大，结缔组织显著增加，并有明显的炎性细胞和肥大细胞的浸润，肌肉层表现出明显的病理变化。排尿行为模式研究和尿动力学实验显示模型小鼠排尿频率增加、膀胱容量降低和膀胱顺应性下降，表现出明显的膀胱功能损伤。离体肌条张力实验发现小鼠模型膀胱平滑肌在电刺激、胆碱信号刺激、嘌呤信刺激号以及去极化刺激下收缩力显著降低。蛋白印迹和免疫荧光检测发现M2和P2X1蛋白表达显著降低，而ITGB1及其相关信号蛋白均显著增加。由于NMDAR是氯胺酮重要的作用靶点，能被氯胺酮结合并抑制，本研究通过建立平滑肌特异NMDAR敲除小鼠模型，明确了NMDAR缺失在正常生理和病理条件下对膀胱病理生理功能的影响，发现了膀胱平滑肌中NMDAR的缺失对正常生理条件和病理条件下的小鼠膀胱形态、结构以及生理功能没有影响，揭示了MK801能够显著改善病理条件下的膀胱形态、结构和生理功能，但平滑肌中的NMDAR不是MK801作用治疗靶点。我们进一步发现了氯胺酮是L型钙离子通道Cav1.2的新型抑制剂，能通过拮抗Cav1.2影响膀胱平滑肌的收缩。最后，在已有的氯胺酮相关性膀胱炎发生理论和机制基础上结合我们自己的研究，对氯胺酮相关性膀胱炎中表皮受损机制、膀胱固有层炎症和纤维化发生机制、血管损伤机制、神经纤维病变机制、膀胱平滑肌受损机制以及它们对膀胱功能的影响进行了总结和阐明。综上，本项目的研究扩展了氯胺酮相关性膀胱炎发生理论，明确了膀胱平滑肌受损是氯胺酮相关性膀胱炎发生的重要因素，确定了MK801治疗作用和平滑肌中的NMDAR的作用，发现了氯胺酮新的作用靶点Cav1.2, 最后对氯胺酮相关性膀胱炎中各组织损伤机制以及它们对膀胱功能的影响进行了总结分析。