基于PACAP-cAMP信号通路探讨电针治疗骶上脊髓损伤后神经源性膀胱的效应机制

Neurogenic bladder is a severe complication of spinal cord injury (SCI), the main cause of death in late patients with paraplegia. More than 80% of SCI patients are injured above the sacral segment, which lead to bladder dysfunction mainly as detrusor hyperreflexia，urinary bladder storage problems, and urinary incontinence.Inhibiting secondary synaptic and neuronal apoptosis after spinal cord injury, and promoting the bladder detrusor relaxation can effectively improve the neurogenic bladder after spinal cord injury above sacral segment. PACAP-cAMP signal pathway plays an important role in inhibiting synaptic and neuronal apoptosis and promoting the bladder detrusor relaxation.The project takes PACAP-cAMP signal pathway as a starting point, rat model with urinary incontinence caused by spinal cord injury above sacral segment as subjects,uses electroacupuncture as treatment, uses the urine flow dynamics, light microscopy, immunohistochemistry, RT-PCR, Western-blot and other means of detection, by observing the changes of urodynamics and detrusor tissue morphology, the changes of local tissue morphology and apoptosis related protein of spinal cord injury，the changes of PACAP, cAMP content in the cytoplasm, the changes of phosphorylation of regulatary protein and gene expression in the downstream of the PACAP-cAMP signaling pathways , to explore the possible effect mechanism of electroacupuncture on neurogenic bladder after spinal cord injury above sacral segment.

神经源性膀胱是脊髓损伤(SCI)后严重的合并症，是截瘫患者晚期的主要病死原因。SCI患者80%以上为骶上脊髓损伤，其所导致的膀胱功能障碍主要表现为逼尿肌反射亢进，膀胱储尿困难、尿失禁。抑制脊髓损伤后继发神经元凋亡，促进膀胱逼尿肌舒张可有效改善骶上脊髓损伤后神经源性膀胱的功能，PACAP-cAMP信号通路在抑制神经元凋亡及促进膀胱逼尿肌舒张方面均有重要作用。本项目以此为切入点，以骶上脊髓损伤所致的尿失禁大鼠模型为受试对象，采用电针治疗，运用尿流动力学、光镜、免疫组化、RT-PCR、Western-blot等多种检测手段，观察膀胱尿流动力学及逼尿肌组织形态，脊髓损伤局部组织形态及凋亡相关蛋白含量，胞浆内PACAP、cAMP含量，PACAP-cAMP信号通路下游环节中各调节蛋白磷酸化状态及相关基因表达量等的变化，探讨电针治疗骶上脊髓损伤后神经源性膀胱可能的效应机制。

骶上脊髓损伤（supersacral spinal cord injury,SSCI）引起脑桥排尿中枢和骶段脊髓之间的联系中断，正常的抑制机制被破坏，将导致逼尿肌反射亢进，由此形成神经源性膀胱(neurogenic bladder,NB)是脊髓损伤患者的主要并发症之一，导致膀胱和尿道功能障碍，进而产生一系列下尿路症状。由于其病情复杂、难以控制，病变持续，致残率高，严重损害患者身心健康，降低生存质量，已成为医学界亟待解决的重点与难点。本项目在前期研究的基础上，通过观察电针“大椎”、“次髎”、“中极”、“三阴交”对SSCI后NB大鼠膀胱尿流动力学改变和髓内凋亡因子及膀胱逼尿肌的影响，进一步探讨电针干预SSCI后NB功能改善的可能机理。 研究表明：电针可明显增加骶上脊髓损伤后神经源性膀胱模型大鼠膀胱最大容量，降低膀胱内压，提高膀胱的顺应性，促进膀胱逼尿肌舒张，并抑制逼尿肌的病理损害，从而改善膀胱的功能；电针可通过激活PACAP-cAMP-PKA信号通路，上调骶上脊髓损伤后神经源性膀胱模型大鼠逼尿肌内PACAP、cAMP的含量，增加骶上脊髓损伤后神经源性膀胱模型大鼠逼尿肌内PKA的表达，提高逼尿肌内MLCK、 MLCP的磷酸化，使MLC去磷酸化，抑制逼尿肌的收缩过程，从而发挥促进膀胱逼尿肌舒张，重建膀胱功能的效应；电针可通过激活PACAP-cAMP-ERK信号通路，降低髓内细胞凋亡率，减少髓内细胞凋亡，达到改善膀胱神经支配，缓解逼尿肌无抑制性收缩。本项目以PACAP-cAMP信号通路为切入点，通过观察电针对骶上脊髓损伤后神经源性膀胱模型大鼠尿流动力学、逼尿肌组织及脊髓组织形态学，膀胱逼尿肌 PACAP-cAMP-PKA 信号通路及其下游收缩元件磷酸化的影响，脊髓组织PACAP-cAMP-ERK信号通路及凋亡因子的影响，在功能、形态、分子等多个层面探讨了电针治疗骶上脊髓损伤后神经源性膀胱的效应及其生物学机制，为针灸治疗本病提供了一定的实验和理论依据。