miR-199a-3p调控c-kit/ERK通路在电针关元穴治疗神经源性尿潴留大鼠的作用机制

目的：探讨miR-199a-3p调控c-kit/细胞外信号调节激酶（ERK）通路在电针关元穴治疗神经源性尿潴留中的作用机制。方法: 采用改良脊髓横断法建立神经源性尿潴留大鼠模型60只,通过随机数表法平均分为5组：模型组、电针组、miR-199a-3p antagomir（抑制剂）组、miR-199a-3p antagomir阴性对照组、电针+miR-199a-3p antagomir组,另选取SD大鼠12只,设为假手术组,使用药物分组干预处理后,检测大鼠尿动力学,比较各组膀胱最大容量、膀胱基础压、漏尿点压力;肌条实验检测大鼠逼尿肌兴奋性,比较各组肌条自发性收缩频率;HE染色检测各组大鼠膀胱组织病理形态改变;试剂盒测定各组大鼠血清炎性细胞因子环氧化酶（COX-2）、白细胞介素（IL-18）、诱导型一氧化氮合酶（iNOS）水平;实时荧光定量（qRT-PCR）实验检测各组大鼠膀胱组织miR-199a-3p与c-kit mRNA表达水平;蛋白免疫印迹法检测各组大鼠膀胱组织c-kit/ERK通路蛋白表达水平。结果：与假手术组相比,模型组大鼠膀胱组织呈现严重病理损伤改变,膀胱最大容量、膀胱基础压、漏尿点压力、血清COX-2、IL-18及iNOS水平显著升高（P＜0.05）,肌条自发性收缩频率、膀胱组织miR-199a-3p与c-kit mRNA表达水平、膀胱组织c-kit/ERK通路蛋白p-ERK/ERK、c-kit表达水平显著降低（P＜0.05）。与模型组、电针+miR-199a-3p antagomir组分别相比,电针组大鼠膀胱组织病理损伤均减轻,膀胱最大容量、膀胱基础压、漏尿点压力、血清COX-2、IL-18及iNOS水平均降低（P＜0.05）,肌条自发性收缩频率、膀胱组织miR-199a-3p与c-kit mRNA表达水平、膀胱组织c-kit/ERK通路蛋白p-ERK/ERK、c-kit表达水平均升高（P＜0.05）;miR-199a-3p antagomir组大鼠膀胱组织病理损伤均加重,膀胱最大容量、膀胱基础压、漏尿点压力、血清COX-2、IL-18及iNOS水平均升高（P＜0.05）,肌条自发性收缩频率、膀胱组织miR-199a-3p与c-kit mRNA表达水平、膀胱组织c-kit/ERK通路蛋白p-ERK/ERK、c-kit表达水平均降低（P＜0.05）。与模型组相比,miR-199a-3p antagomir阴性对照组大鼠各指标水平无显著差异（P＞0.05）。结论：电针关元穴可通过上调miR-199a-3p表达而抑制c-kit/ERK通路激活,减轻神经源性尿潴留大鼠膀胱组织炎症损伤,增强逼尿肌兴奋性,修复其膀胱功能,改善大鼠尿潴留症状。

The mechanism of miR-199a-3p regulating c-kit/ERK pathway in the treatment of neurogenic urinary retention with electroacupuncture at Guanyuan acupoint rats

AIM: To investigate the mechanism of miR-199a-3p regulating c-kit/extracellular signal-regulated kinase (ERK) pathway in the treatment of neurogenic urinary retention with electroacupuncture at Guanyuan acupoint. METHODS: Sixty model rats of neurogenic urinary retention were established by modified spinal cord transection method, and they were divided into five groups by random number table method: model group, electroacupuncture group, miR-199a-3p antagomir (inhibitor) group, miR-199a-3p antagomir negative control group and electroacupuncture + miR-199a-3p antagomir group, and another 12 SD rats were selected as sham operation group. After group intervention with drugs, the urodynamics of the rats were tested, and the maximum bladder capacity, basal bladder pressure, and urine leakage point pressure were compared in each group; muscle strip experiment was used to detect the excitability of rat detrusor muscles and compare the spontaneous contraction frequency of muscle strips in each group; HE staining was used to detect the pathological and morphological changes of the bladder tissues of rats in each group; the kit was used to measure the serum levels of inflammatory cytokine cyclooxygenase (COX-2), interleukin (IL-18) and inducible nitric oxide synthase (iNOS) of rats in each group; real-time fluorescence quantitative (qRT-PCR) experiment was used to detect the expression levels of miR-199a-3p and c-kit mRNA in the bladder tissues of rats in each group; western blotting was used to detect the expression levels of c-kit/ERK pathway proteins in the bladder tissues of rats in each group. RESULTS: Compared with the sham operation group, the bladder tissue of the model group showed severe pathological damage, the maximum bladder capacity, basal bladder pressure, urine leakage point pressure, serum COX-2, IL-18 and iNOS levels were significantly increased (P<0.05), the spontaneous contraction frequency of muscle strips, the expression levels of miR-199a-3p and c-kit mRNA in bladder tissue, and the expression levels of c-kit/ERK pathway proteins p-ERK/ERK and c-kit in bladder tissue were significantly reduced (P<0.05). Compared with the model group and the electroacupuncture + miR-199a-3p antagomir group, the pathological damage of the bladder tissue of rats in the electroacupuncture group was reduced, the maximum bladder capacity, basal bladder pressure, urine leakage point pressure, serum COX-2, IL-18 and iNOS levels were reduced (P<0.05), the spontaneous contraction frequency of muscle strips, the expression levels of miR-199a-3p and c-kit mRNA in bladder tissue, and the expression levels of c-kit/ERK pathway proteins p-ERK/ERK and c-kit in bladder tissue were increased (P<0.05); the pathological damage of the bladder tissue of rats in the miR-199a-3p antagomir group was aggravated, the maximum bladder capacity, basal bladder pressure, urine leakage point pressure, serum COX-2, IL-18 and iNOS levels were increased (P<0.05), the spontaneous contraction frequency of muscle strips, the expression levels of miR-199a-3p and c-kit mRNA in bladder tissue, and the expression levels of c-kit/ERK pathway proteins p-ERK/ERK and c-kit in bladder tissue were reduced (P<0.05). Compared with the model group, there was no significant difference in the levels of indicators in the miR-199a-3p antagomir negative control group (P>0.05). CONCLUSION: Electroacupuncture at Guanyuan acupoint can inhibit the activation of c-kit/ERK pathway by up-regulating the expression of miR-199a-3p, reduce the inflammatory damage of bladder tissue in neurogenic urinary retention rats, enhance the excitability of detrusor, repair the bladder function, and improve the symptoms of urinary retention in rats.