电针治疗对完全性骶上脊髓损伤后神经源性膀胱大鼠尿流动力学和脊髓组织中CyclinD1、Ngn1的影响

Effects of electroacupuncture on urodynamics and expression of CyclinD1,NGN1 in spinal cord tissue in rats with neurogenic bladder after complete suprasacral spinal cord injury

目的观察电针治疗完全性骶上脊髓损伤(SSCI)后神经源性膀胱(逼尿肌反射亢进)大鼠的效果,探讨电针基于Wnt/β-catenin信号通路调控脊髓组织中CyclinD1、Ngn1 mRNA及蛋白的表达及其促进模型大鼠排尿功能恢复的作用机制。方法SD雌性SPF级大鼠60只,随机分为假手术组12只和模型组48只。模型组大鼠手术致完全性SSCI,采用BBB评分和尿流动力学对大鼠模型进行评价。术后2周,将模型复制成功的大鼠再次随机分为模型对照组、电针组和电针对照组,每组12只。电针组在大椎穴、次髎穴(次髎穴隔日左右更替)用电针治疗;电针对照组亦于相同时间在大椎穴、次髎穴远脊柱侧(次髎穴用法同上)1 cm处为针刺点,进行电针治疗。电针参数采用疏密波(疏波10 Hz/9 s,密波50 Hz/5 s),留针20 min,强度以针刺处出现规律性抽动为宜,1次/d,连续1周。末次电针治疗后各组大鼠行尿流动力学检测,采用RT-PCR、Western blotting和免疫组织化学法检测大鼠脊髓组织中Cyclin D1、Ngn1 mRNA及蛋白表达。结果①尿流动力学:与假手术组比较,其他3组大鼠膀胱基础压力和膀胱最大压力升高,膀胱最大容量和膀胱顺应性降低;模型对照组和电针对照组大鼠膀胱漏尿点压力升高(P<0.05)。与模型对照组比较,电针组大鼠膀胱基础压力、膀胱最大压力和膀胱漏尿点压力降低,膀胱最大容量和膀胱顺应性升高(P<0.05)。与电针组比较,电针对照组大鼠膀胱基础压力、膀胱最大压力和膀胱漏尿点压力升高,膀胱最大容量和膀胱顺应性降低(P<0.05)。②Cyclin D1 mRNA及蛋白:与假手术组比较,其他3组Cyclin D1 mRNA及蛋白表达升高(P<0.05);与模型对照组比较,电针组Cyclin D1 mRNA及蛋白表达升高(P<0.05);与电针组比较,电针对照组Cyclin D1 mRNA及蛋白表达降低(P<0.05)。③Ngn1蛋白:与假手术组比较,模型对照组和电针对照组Ngn1蛋白表达降低(P<0.05);与模型对照组比较,电针组Ngn1蛋白表达升高(P<0.05);与电针组比较,电针对照组Ngn1蛋白表达降低(P<0.05)。④Ngn1 mRNA:与假手术组比较,其他3组Ngn1 mRNA表达下降(P<0.05);与模型对照组比较,电针组Ngn1 mRNA表达升高(P<0.05);与电针组比较,电针对照组Ngn1 mRNA表达降低(P<0.05)。结论电针大椎穴、次髎穴对完全性SSCI后神经源性膀胱(逼尿肌反射亢进)大鼠尿流动力学具有明显改善作用,其作用机制可能是通过Wnt/β-catenin信号通路调控Cyclin D1、Ngn1 mRNA及蛋白表达,促进脊髓内源性神经干细胞的增殖和活化而实现。

Objective To observe the effect of electroacupuncture on rats with neurogenic bladder(detrusor hyperreflex)after complete supraorbital spinal cord injury(SSCI),and to investigate the mechanism of electroacupuncture based on Wnt/β-catenin signaling pathway regulating the expression of Cyclin D1 and Ngn1 mRNA and protein in spinal cord tissue.Methods Sixty female SPF SD rats were randomly divided into sham operation group(12 rats)and model group(48 rats).The model group rats with complete SSCI induced by surgery was evaluated by BBB score and urodynamics.On the two weeks after surgery,the rats of model replication successful were randomly divided into model control group,electroacupuncture group,and electroacupuncture control group,with 12 rats in per group.The electroacupuncture group was treated with electroacupuncture at'Dazhui'point and'Ciliao'point('Ciliao'point replacement around the next day);the electroacupuncture control group was treated with electroacupuncture at the 1cm acupuncture point next to the'Dazhui'point and'Ciliao'point.The electroacupuncture used Dilatational wave(10 Hz/9 s,50 Hz/5 s)for 20 min,and the intensity was suitable for regular contraction and twitching at the acupuncture point,once a day for a week in a row.After the last electroacupuncture intervention,the urodynamic test was performed in each group,and the expression levels of Cyclin D1,Ngn1 mRNA,and protein in the spinal cord tissues of rats were detected by Western blotting,RT-PCR,and IHC.Results Urodynamic testing:Compared with the sham operation group,the bladder base pressure and maximum pressure were increased in the other groups,and the bladder maximum capacity and compliance were decreased.The pressure of the bladder leak point in the model control group and electroacupuncture control group were increased(P<0.05).Compared with the model control group,the bladder base pressure,maximum pressure,and leak point pressure were decreased in the electroacupuncture group,the bladder maximum capacity and compliance were increased(P<0.05).Compared with the electroacupuncture group,the bladder basic pressure,maximum pressure,and leak point pressure were increased in the electroacupuncture control group,while the bladder maximum capacity and compliance were decreased(P<0.05).Cyclin D1 mRNA and protein expression:Compared with the sham operation group,the expression levels of Cyclin D1 mRNA and protein in the other groups were increased(P<0.05).Compared with the model control group,the expression levels of Cyclin D1 mRNA and protein in the electroacupuncture group were increased(P<0.05).Compared with the electroacupuncture group,the expression levels of Cyclin D1 mRNA and protein in the electroacupuncture control group were decreased(P<0.05).Ngn1 protein expression:Compared with the sham operation group,the expression levels of Ngn1 protein in the model control groups and electroacupuncture control group were decreased(P<0.05).Compared with the model control group,the expression levels of Ngn1 protein in the electroacupuncture group were increased(P<0.05).Compared with the electroacupuncture group,the expression levels of Ngn1 protein in the electroacupuncture control group were decreased(P<0.05).Ngn1 mRNA expression:Compared with the sham operation group,the expression levels of Ngn1 mRNA in the other groups were decreased(P<0.05).Compared with the model control group,the expression levels of Ngn1 mRNA in the electroacupuncture group were increased(P<0.05).Compared with the electroacupuncture group,the expression levels of Ngn1 mRNA in the electroacupuncture control group were decreased(P<0.05).Conclusion Electroacupuncture on'Dazhui'point and'Ciliao'point has a significant effect on improving urodynamics of rats with neurogenic bladder(detrusor hyperreflex)after completes SSCI,and its mechanism is achieved by regulating the Cyclin D1 and Ngn1 mRNA and protein expression in Wnt/β-catenin signal pathway,promote the proliferation and activation of ENSCs in the spinal cord.