氯离子转运体在神经病理性疼痛大鼠背根神经节上表达的研究

摘要

Objective:The present study aimed to investigate the expression of sodium-potassium-chloride co-transorter 1 (NKCC1) and potassium-chloride co-transporter 2 (KCC2) in rat dorsal root ganglion (DRG) neurons after chronic constriction injury (CCI) of the sciatic nerve and their possible role in the development of neuropathic pain.Methods:In this study, we employed the CCI model to induce neuropathic pain in SD rats and tested the thermal withdrawal latency (TWL) using hot plate test. Immunohistochemistry andWestern Blot analysis were utilized to identify tissue specific expression of NKCC1 and KCC2 in L4~ L6 DRG neurons of SD rats at 7, 14, 21 days of postoperative, respectively. The change of Cl- concentration in DRG neurons of CCI model was assessed using MQAE (N-Ethoxycarbonylmethyl -6-methoxyquinolinium bromide) chloride fluorescence probe.Results: (1) Hot plate test showed a much shorter TWL in posterior limb of operation side of CCI model group in comparison to that of control group (P< 0.01,n=10), and the heat hyperalgesia persisted for 21 days after surgery in CCI rat. Moreover, TWL in posterior limb of operation side had no significant difference between sham operation group and control group. (2) Immunohistochemistry revealed that the protein expression of NKCC1 broadly distributed in DRG neurons, KCC2 immunostainings were not observed in DRGs. (3) the protein expression of NKCC1 significantly increased in 2 categories of DRG neurons, the small and and the medium-sized neurons, on 14 and 21 day after CCI (P < 0.01,n= 1000). (4) Compared with that in control group, the expression of NKCC1 in DRG of operation side of CCI model was enhanced at the 14th and 21th day after operation (P < 0.01,n = 6). (5) A significant increase in Cl-concentration was observed in DRG of operation side of CCI model at day 7 and 14 after operation (P< 0.05). Conclusion:CCI-induced overexpression of NKCC1 may lead to the increase of Cl- concentration in DRG neurons. Cl- efflux may induce primary afferent depolarization (PAD) in the primary sensory nerves and dorsal root reflexes (DRRs) in primary sensorium, and promote the production of hyperalgesia and allodynia.%目的：为了探讨氯离子转运体的表达及其功能在神经病理性疼痛的产生和维持中的作用。方法：建立大鼠坐骨神经压迫模型(chronic constriction injury, CCI)；检测大鼠热刺激缩足反射潜伏期(thermal withdrawal latency, TWL)；应用免疫组化、Western Blot技术检测钠-钾-氯共转运体1(sodium-potassium-chloride co-transorter 1, NKCC1)和钾-氯共转运体2(potassium-chloride co-transorter 2, KCC2)在CCI模型背根神经节(dorsal root ganglion, DRG)神经元上表达的改变；应用氯离子荧光探针MQAE（N-Ethoxycarbonylmethyl -6-methoxyquinolinium bromide）检测DRG神经元氯离子浓度的变化。结果：（1）CCI组大鼠TWL较正常对照组明显缩短（P<0.01，n=10），至术后21天测试结束仍较明显（P<0.05，n=10）。假手术组与正常组差异无统计学意义。（2）正常情况下， NKCC1微弱表达于DRG大、中、小型神经元，KCC2未见表达。CCI模型术后第7天、14天、21天，NKCC1表达增加，KCC2未见表达。（3）CCI组大鼠术后第7天DRG、NKCC1阳性细胞计数、中型神经元显著增加（P<0.01，n=1000）；术后第14天，中、小型神经元增加（P<0.01，n=1000）；术后第21天，中型神经元增加（P<0.01，n=1000）。（4）CCI术后14、21天DRG神经元NKCC1蛋白表达显著增加。（5）CCI术后7、14天DRG神经元中氯离子浓度明显升高（P<0.05， n=1000）。结论：神经病理性疼痛发生时，NKCC1在DRG神经元表达异常增加，引起细胞内Cl-浓度升高，继而可能参与了介导初极感觉外周末梢去极化(primary afferent depolarization, PAD)或/和诱发初极感觉中枢末梢端的背根反射(dorsal root reflexes,DRRs)引发痛觉过敏和异常疼痛产生。