益元灸对骶髓损伤后神经源性膀胱大鼠尿流动力学及膀胱组织TRPV4/ATP/P2X5信号通路的影响

Effect of Yiyuan moxibustion on urodynamics and TRPV4/ATP/P2X5 signal pathway of bladder tissue in rats with neurogenic bladder after sacral cord injury

目的:观察益元灸对骶髓损伤后逼尿肌无反射型神经源性膀胱(NB)大鼠尿流动力学的作用,探讨益元灸调控膀胱组织中瞬时受体电位香草酸4(TRPV4)、三磷酸腺苷(ATP)、酪氨酸蛋白激酶KIT(C-Kit)和ATP受体P2X5的表达以促进NB大鼠排尿功能恢复的机制。方法:80只雌性SD大鼠,随机选取12只作为假手术组,其余大鼠用骶髓横断法制备膀胱逼尿肌无反射型NB模型,造模成功的大鼠随机分为模型组、益元灸组、益元灸+抑制剂组、抑制剂组,每组12只。术后第15天开始治疗,益元灸组在手法排尿基础上给予益元灸“神阙”“关元”治疗,抑制剂组在手法排尿基础上给予HC067047膀胱灌注(1 mL,1μmol/L,30 min),益元灸+抑制剂组在手法排尿基础上给予益元灸+HC067047膀胱灌注,模型组除手法排尿外不予其他治疗。末次干预结束后采用尿流动力学评价大鼠膀胱功能,HE染色法观察膀胱组织形态,比色法检测膀胱组织中ATP含量,免疫荧光法观察膀胱组织中C-Kit及P2X5的阳性表达率,Western blot法检测膀胱组织中TRPV4、C-Kit和P2X5蛋白表达水平。结果:与假手术组比较,模型组大鼠最大膀胱容量、膀胱顺应性均升高(P<0.01),漏尿点压力、膀胱组织中ATP含量、C-Kit和P2X5的阳性表达率及TRPV4、C-Kit、P2X5蛋白表达水平均降低(P<0.01)。与模型组比较,益元灸组大鼠最大膀胱容量、膀胱顺应性均降低(P<0.01),漏尿点压力、膀胱组织中ATP含量、C-Kit和P2X5的阳性表达率及TRPV4、C-Kit、P2X5蛋白表达水平均升高(P<0.01,P<0.05);抑制剂组大鼠最大膀胱容量、膀胱顺应性均升高(P<0.01),漏尿点压力、膀胱组织中ATP含量、C-Kit和P2X5的阳性表达率及TRPV4、C-Kit、P2X5蛋白表达水平均降低(P<0.05,P<0.01)。与益元灸+抑制剂组比较,益元灸组大鼠最大膀胱容量、膀胱顺应性均降低(P<0.01),漏尿点压力、膀胱组织中ATP含量、C-Kit和P2X5的阳性表达率及TRPV4、C-Kit、P2X5蛋白表达水平均升高(P<0.01,P<0.05);抑制剂组大鼠最大膀胱容量、膀胱顺应性均升高(P<0.01),漏尿点压力、膀胱组织中ATP含量、C-Kit和P2X5的阳性表达率及TRPV4、C-Kit、P2X5蛋白表达水平均降低(P<0.01,P<0.05)。结论:益元灸对膀胱逼尿肌无反射型NB大鼠尿流动力学有明显改善作用,其作用机制可能是激活膀胱组织中的TRPV4通道,促进膀胱上皮细胞释放ATP,从而增加膀胱Cajal间质细胞数量及其嘌呤能受体P2X5表达,提高逼尿肌收缩能力,改善膀胱功能。

ObjectiveTo observe the effect of Yiyuan m oxibustion on urodynam ics and the expressions of transient receptor potential vanilloid 4(TRPV4),adenosine triphosphate(ATP),tyrosine protein kinase KIT(C-Kit)and adenosine triphosphate receptor P2X5 in bladder tissue of rats with detrusor reflex-free neurogenic bladder(NB)after sacral cord injury(SCI),so as to explore its m echanism in prom oting the recovery of urination function of NB rats.MethodsFem ale SD rats were random ly divided into sham operation,m odel,Yiyuan m oxibustion,Yiyuan m oxibus⁃tion+inhibitor(com bination)and inhibitor groups,with 12 rats in each group.The m odel of detruser reflex-free NB after sacral SCI was established by m odified Hassan Shaker spinal cord transection m ethod.The behavioral score of Basso Beasttie Bresnahan(BBB)and urodynam ic indexes were used to evaluate the m odel of rats after operation.Fifteen daysafter m odeling,Yiyuan m oxibustion was applied to“Shenque”(CV8)and“Guanyuan”(CV4)for 20 m in,once daily for 14 days.Rats of the inhibitor and com bination groups were given intravesical instillation of HC067047(1 m L,1μm ol/L,30 m in).After the interventions,urodynam ics was used to evaluate the bladder function of rats.HE staining was used to observe the m orphology of bladder tissue.ATP content in bladder tissue was detected by colorim etric m ethod.The positive expression rates of C-Kit and their receptor P2X5 in bladder tissue were observed by im m unofluo⁃rescence double labeling m ethod,and TRPV4,C-Kit,and P2X5 protein expression levels in bladder tissue were de⁃tected by W estern blot.ResultsCom pared with the sham operation group,the m axim um bladder capacity and blad⁃der com pliance of rats in the m odel group were increased(P<0.01),the leak point pressure,ATP content,the possi⁃tive expression rates of C-Kit and P2X5,and the protein expression levels of TRPV4,C-Kit,P2X5 in bladder tissue were decreased(P<0.01).In com parison with the m odel and com bination groups,the Yiyuan m oxibustion group showed a decrease in m axim um bladder capacity and bladder com pliance(P<0.01),an increase in leakage point pres⁃sure,ATP content,the possitive expression rates of C-Kit and P2X5,and TRPV4,C-Kit,and P2X5 protein expression levels(P<0.01,P<0.05);However,these indicators showed opposite trends in the inhibitor group(P<0.01,P<0.05).ConclusionYiyuan m oxibustion can im prove the urodynam ics and bladder function in rats with bladder detrusor non⁃reflective after SCI,which m ay be related to its effect in activating the TRPV4 channel in bladder tissue,prom oting the release of ATP from bladder epithelium,thus increasing the expression of bladder Cajal interstitial cells and their puri⁃nergic P2X5 receptors.