中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (5): 912-921.doi: 10.4103/1673-5374.268904

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

钠-钾-氯转运蛋白1在慢性压迫性损伤大鼠背根神经节神经元中的表达及作用

  

  • 出版日期:2020-05-15 发布日期:2020-06-01

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Chao-Yang Tan1, 2, 3, Yan-Ping Wang2, 4, Yuan-Yuan Han1, 5, Bi-Han Lu1, Wei Ji1, Li-Cang Zhu6, Yang Wang1, 7, Wen-Yan Shi1, 7, Li-Ya Shan1, 7, Liang Zhang1, 7, Ke-Tao Ma1, 7, Li Li1, 2, 7, Jun-Qiang Si1, 7, 8, 9#br#   

  1. 1 Department of Physiology, College of Medicine, Shihezi University, Shihezi, Xinjiang Uygur Autonomous Region, China
    2 Department of Physiology, Medical College of Jiaxing University, Jiaxing, Zhejiang Province, China
    3 Department of Health, Karamay Army Division, Chinese People’s Liberation Army, Karamay, Xinjiang Uygur Autonomous Region, China
    4 Department of Nursing, Medical College of Jiaxing University, Jiaxing, Zhejiang Province, China
    5 Department of Clinical Medicine, Karamay College of Xinjiang Medical University, Karamay, Xinjiang Uygur Autonomous Region, China
    6 Department of Neurosurgery, First Affiliated Hospital, College of Medicine, Shihezi University, Shihezi, Xinjiang Uygur Autonomous
    Region, China
    7 The key Laboratory of Xinjiang Endemic and Ethnic Diseases, College of Medicine, Shihezi University, Shihezi, Xinjiang Uygur Autonomous Region, China
    8 Department of Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei Province, China
    9 Department of Physiology, School of Basic Medical Sciences, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
  • Online:2020-05-15 Published:2020-06-01
  • Contact: Jun-Qiang Si, PhD,sijunqiang@shzu.edu.cn; Li Li, PhD, lily7588@163.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 30160026 (to JQS); the High Level Talent Research Project of Shihezi University of China, No. RCSX201705 (to YW).

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摘要：

为了探讨持续性痛觉过敏大鼠背根神经节神经元上钠-钾-氯转运蛋白1(NKCC1)和钾-氯共转运蛋白2 (KCC2)的变化与外周疼痛的传递相关，实验将大鼠随机分为4组，对照组正常饲养，假手术组大鼠仅暴露坐骨神经，CCI组大鼠结扎坐骨神经建立慢性压迫性损伤(CCI)模型，CCI+布美他尼组大鼠在CCI造模的基础上给予布美他尼干预，在测量热退缩潜伏期的实验中通过静脉注射的方式给予布美他尼 (15 mg/kg)，在膜片钳实验时，将布美他尼(10 μg/μL)与急性分离的背根神经节神经元(造模后第14天)共孵育1 h或鞘内注射布美他尼(5 μg/μL)。(1)采用Hargreaves实验检测大鼠热痛觉过敏的变化发现，CCI建模后第7，14和21天，大鼠热退缩潜伏期显著降低；在给予布美他尼干预后，由CCI建模造成的热退缩潜伏期的降低明显被抑制；(2)采用免疫组织化学染色和蛋白质印迹分析发现，建模后第7，14和21天，CCI组大鼠背根神经节神经元NKCC1的免疫反应及蛋白表达显著增加；但未观察到KCC2的免疫反应和蛋白表达；(3)采用氯离子荧光探针技术评估CCI模型大鼠背根神经节神经元中Cl-浓度的变化发现，建模后第7，14天，CCI组术侧的背根神经节神经元相对光密度值显著降低；(4)采用全细胞膜片钳技术检测发现，造模第14天，CCI组背根神经节神经元的静息电位和动作电位频率增加，动作电位的阈值和基强度降低；并且，在给予布美他尼干预后，上述结果指标均显著被抑制；(5)上述结果证实，CCI能诱导NKCC1的过表达，进而增加背根神经节神经元中的Cl-浓度，从而增强背根神经节神经元的兴奋性，最终出现痛觉过敏和异常性疼痛，采用布美他尼干预可达到镇痛的效果。实验方案已于2017-02-22经中国石河子大学医学院第一附属医院动物伦理委员会批准(A2017-169-01)。

orcid: 0000-0001-6704-2115 (Jun-Qiang Si) 

         0000-0001-8591-0676 (Li Li)

关键词: NKCC1, KCC2, 背根神经节, 慢性压迫性损伤, 神经病理性疼痛, 全细胞膜片钳, 初级传入去极化, 背根反射, 布美他尼, 痛觉过敏

Abstract: Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 μg/μL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl– concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl– concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

Key words: bumetanide, chronic constriction injury, dorsal root ganglion, dorsal root reflex, hyperalgesia, KCC2, nerve regeneration, neuropathic pain, NKCC1, primary afferent depolarization, whole-cell patch clamp