钩藤碱治疗骶上脊髓损伤致逼尿肌反射亢进大鼠的实验研究

摘要

目的：探讨钩藤碱对骶上脊髓损伤（ SSCI ）致神经源性膀胱大鼠逼尿肌反射亢进的治疗作用及其机制。方法将10周龄健康雌性SD大鼠30只（ SPF级）按照随机数字表法分为对照组、模型组、治疗组，每组10只。对照组不做任何手术处理，模型组和治疗组采用脊髓横断法制备神经源性膀胱模型。对照组和模型组腹腔注射0.9％氯化钠溶液，治疗组注射钩藤碱（5 mg／kg ）。对照组10只大鼠全部存活，模型组8只造模成功，治疗组8只造模成功。4周后，大鼠膀胱行尿动力学检测，离体逼尿肌条电生理学指标检测，膀胱病理切片HE染色观察，蛋白质印迹法检测c-kit蛋白的表达，激光共聚焦显微镜下观察Cajal间质细胞（ ICC）数量变化，实时荧光PCR检测ICC中T型钙通道蛋白亚型α1G基因表达水平。结果与模型组相比，治疗组膀胱最大容量增大（q＝8.251， P＜0.05），膀胱漏尿点压（q＝3.764， P＜0.05）和膀胱充盈压均减小（q＝5.687， P＜0.05），逼尿肌收缩频率降低（q＝9.661， P＜0.05），最小张力增大（q＝5.217， P＜0.05）， c -kit 蛋白表达降低（q＝13.688， P＜0.05）， ICC 数量减少（q＝7.060， P＜0.05），α1G基因表达量降低（q＝8.762， P＜0.05），差异均有统计学意义。病理切片下观察到治疗组逼尿肌无断裂，肌间隙增宽减少。结论钩藤碱通过减少神经源性膀胱逼尿肌ICC数量，抑制ICC T型钙通道蛋白亚型α1G基因的表达来抑制ICC起搏兴奋活性，从而减轻逼尿肌的过度亢进，使逼尿肌兴奋性趋于正常。%Objective To investigate the effects of phynchophylline on detrusor overactivity induced by sacral spinal cord injury ( SSCI) in rats and the underlying mechanism .Methods Using random number table method , we divided 30 10-week female healthy SD rats into control group , model group and treatment group , with 10 rats in each group.No surgical intervention was made in control group , neurogenic bladder models were made in model group and treatment group by spinal cord transaction.Control group and model group were administrated with 0.9%sodium chloride solution by intraperitoneal infection . Treatment group was administrated with phynchophylline (5 mg/kg).10 rats in control group were all alive, successful model building was made in 8 rats in model group and 8 rats in treatment group . Four weeks later , urine dynamics testing was undertaken with the bladder of rats , in vitro test of the electrophysiological indicators of detrusor was conducted , the pathological sections of bladder were observed by HE staining method , c-kit protein expression was tested by western blotting method , the change in the number of ICC was observed under laser scanning confocal microscope , and the expression level of α1G gene of subunit of T-type calcium channels in ICC was detected by real -time PCR.Results Compared with the model group , the treatment group had larger maximum capacity of bladder (q=8.251, P<0.05), lower bladder leak point pressure (q=3.764, P<0.05), lower bladder filling pressure (q =5.687, P <0.05), lower detrusor contraction frequency (q =9.661, P <0.05), higher minimum tension (q=5.217, P<0.05), lower expression level of c -kit protein (q=13.688, P<0.05), lower number of ICC (q =7.060, P <0.05) and lower α1G gene expression (q =8.762, P <0.05).Conclusion Rhynchophylline can reduce the number of ICC cells in neurogenic detrusor and inhibit the pacemaker activity of ICC cells by inhibiting the expression of α1G gene of subunit of T -type calcium channels in ICC , thereby reducing excessive detrusor hyperactivity and make the excitability of detrusor become normal .