电针通过抑制PDK1/Akt/HCN4通路改善大鼠神经源性尿潴留

Electroacupuncture inhibits PDK1/Akt/HCN4 pathway to improve neurogenic urinary retention in rats

目的:以3-磷酸肌醇依赖性蛋白激酶1(PDK1)/蛋白激酶B(Akt)/超极化激活环核苷酸门控离子通道4(HCN4)通路为切入点,探究电针治疗大鼠神经源性尿潴留的分子机制。方法:实验选取SD雌性大鼠,采用改良Hassan Shaker脊髓全断法建立逼尿肌无力型尿潴留大鼠模型,并按随机数字表法随机分为模型组、电针组、PDK1抑制剂组、HCN4阻断剂组、电针+HCN4阻断剂组,每组20只,另取20只大鼠作为假手术组。电针组、电针+HCN4阻断剂组取大鼠“中极”“中髎”电针治疗20 min,1次/d,PDK1抑制剂组隔天1次腹腔注射OSU-03012(20 mg/kg),HCN4阻断剂组隔天1次腹腔注射伊伐布雷定(10 mg/kg),以上治疗均持续10 d。多通道生理记录仪检测大鼠尿流动力学指标;肌条实验检测逼尿肌兴奋性;HE染色观察膀胱形态学变化;免疫荧光双染法检测膀胱组织HCN4与酪氨酸蛋白激酶KIT(C-Kit)阳性表达;Western blot法检测膀胱组织PDK1/Akt/HCN4通路蛋白及热休克蛋白27(HSP27)表达。结果:与假手术组相比,模型组大鼠出现排尿功能障碍,漏尿点压力、离体逼尿肌自发收缩频率、膀胱组织C-Kit荧光强度、HCN4+/C-Kit+共表达、HCN4及磷酸化(p)-HSP27/HSP27蛋白表达降低(P<0.05),膀胱最大容量及顺应性、离体逼尿肌收缩时最小张力、膀胱组织PDK1及p-Akt/Akt蛋白表达升高(P<0.05);与模型组相比,电针组、PDK1抑制剂组大鼠以上指标均逆转(P<0.05);与电针组相比,电针+HCN4阻断剂组和HCN4阻断剂组大鼠排尿功能障碍严重,漏尿点压力、自发收缩频率、C-Kit荧光强度、HCN4+/C-Kit+共表达、HCN4及p-HSP27/HSP27蛋白表达降低(P<0.05),膀胱最大容量及顺应性、离体逼尿肌收缩时最小张力、膀胱组织PDK1及p-Akt/Akt蛋白表达升高(P<0.05)。HE染色示模型组大鼠膀胱上皮组织脱落、层次紊乱,膀胱壁细胞空泡化、黏膜层及肌层中性粒细胞浸润等病理损伤明显,电针组及PDK1抑制剂组有所减轻,HCN4阻断剂组较模型组加重,电针+HCN4阻断剂组上述病理损伤较电针组严重。结论:电针刺激“中髎”“中极”可能通过抑制PDK1/Akt通路活化,促进HCN4介导的逼尿肌兴奋性收缩、排尿电信号活化,从而改善神经源性尿潴留大鼠排尿功能障碍。

Objective To observe the therapeutic effect of electroacupuncture(EA)on neurogenic urinary re⁃tention rats,so as to explore the underlying mechanism of EA in treating neurogenic urinary retention by focusing on 3-phosphoinositide-dependent protein kinase 1(PDK1)/protein kinase B(Akt)/hyperpolarization activated cyclic nucleotide-gated cation channel 4(HCN4)pathway.Methods Female SD rats were randomly divided into sham operation,model,EA,PDK1 inhibitor,HCN4 blocker and EA+HCN4 blocker groups,with 20 rats in each group.The model of sacral spinal cord injury was established by modified Hassan Shaker spinal cord transection method.EA(2 Hz/15 Hz,0.5 mA)was applied to“Zhongji”(CV3)and“Zhongliao”(BL33)for 20 min,once daily for 10 days.Rats of the PDK1 inhibitor group received intraperitoneal injection of OSU-03012(20 mg/kg),and rats of the HCN4 blocker group received intraperitoneal injection of ivabradine(10 mg/kg),both once every other day for 10 days.The urody⁃namic indexes of rats were detected by multi-channel physiological recorder;muscle strip test was used to detect detru⁃sor excitability;the morphological changes of bladder were observed by HE staining.Immunofluorescence double stain⁃ing was used to detect the co-expression of HCN4 and C-Kit,a specific marker of interstitial cells of Cajal in bladder.Western blot was used to detect the expression of PDK1/Akt/HCN4 pathway proteins in bladder tissue and heat shock protein 27(HSP27),a protein related to bladder contraction function.Results Compared with the sham operation group,the rats in the model group showed urinary dysfunction,decreased leak point pressure,isolated detrusor spon⁃taneous contraction frequency,fluorescence intensity of C-Kit positive cells,HCN4+/C-Kit+co-expression,HCN4 and p-HSP27/HSP27 protein expression in bladder tissue(P<0.05),and increased maximum bladder capacity and comp-liance,minimum tension during contraction of isolated detrusor,PDK1 and p-Akt/Akt protein expression in bladder tissue(P<0.05).Meanwhile,the above index were all reversed after EA and PDK1 inhibitor intervention(P<0.05).In compari⁃son with the EA group,the rats had severe urinary dysfunction,the urine leakage point pressure,spontaneous contrac⁃tion frequency,fluorescence intensity of C-Kit positive cells,the co-expression of HCN4+/C-Kit+,and the protein expres⁃sion of HCN4 and p-HSP27/HSP27 were decreased(P<0.05),the maximum bladder capacity and compliance,the minimum tension during contraction of isolated detrusor,and the protein expression of PDK1 and p-Akt/Akt in bladder tissue were increased(P<0.05)in both HCN4 blocker and EA+HCN4 blocker groups.HE staining showed exfoliated bladder epithelium and disordered layers,vacuolization of bladder wall cells,with infiltration of neutrophils in mucosal and muscular layers in the model group,which were relatively milder in the EA and PDK1 inhibitor groups,but worse in the HCN4 blocker and EA+HCN4 blocker groups.Conclusion EA can improve the urinary dysfunction in rats with neurogenic urinary retention,which may be related to its effect in inhibiting the activation of PDK1/Akt pathway,promo-ting HCN4-mediated detrusor excitatory contraction and urinary electrical signal activation.