Rehabilitation Medicine Department, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China.
School of Rehabilitation Medicine, Capital Medical University, Beijing, China.
CNS Neurosci Ther. 2024 Sep;30(9):e70045. doi: 10.1111/cns.70045.
We aimed to explore whether the combination of CLP290 and bumetanide maximally improves neuropathic pain following spinal cord injury (SCI) and its possible molecular mechanism.
Rats were randomly divided into five groups: Sham, SCI + vehicle, SCI + CLP290, SCI + bumetanide, and SCI + combination (CLP290 + bumetanide). Drug administration commenced on the 7th day post-injury (7 dpi) and continued for 14 days. All rats underwent behavioral assessments for 56 days to comprehensively evaluate the effects of interventions on mechanical pain, thermal pain, cold pain, motor function, and other relevant parameters. Electrophysiological assessments, immunoblotting, and immunofluorescence detection were performed at different timepoints post-injury, with a specific focus on the expression and changes of KCC2 and NKCC1 proteins in the lumbar enlargement of the spinal cord.
CLP290 and bumetanide alleviated SCI-associated hypersensitivity and locomotor function, with the combination providing enhanced recovery. The combined treatment group exhibited the most significant improvement in restoring Rate-Dependent Depression (RDD) levels. In the combined treatment group and the two individual drug administration groups, the upregulation of potassium chloride cotransporter 2 (K-Clcotransporter 2, KCC2) expression and downregulation of sodium potassium chloride cotransporter 1 (Na-K-Clcotransporter 1, NKCC1) expression in the lumbar enlargement area resulted in a significant increase in the KCC2/NKCC1 ratio compared to the SCI + vehicle group, with the most pronounced improvement seen in the combined treatment group. Compared to the SCI + vehicle group, the SCI + bumetanide group showed no significant paw withdrawal thermal latency (PWTL) improvement at 21 and 35 dpi, but a notable enhancement at 56 dpi. In contrast, the SCI + CLP290 group significantly improved PWTL at 21 days, with non-significant changes at 35 and 56 days. At 21 dpi, KCC2 expression was marginally higher in monotherapy groups versus SCI + vehicle, but not significantly. At 56 dpi, only the SCI + bumetanide group showed a significant difference in KCC2 expression compared to the control group.
Combined application of CLP290 and bumetanide effectively increases the ratio of KCC2/NKCC1, restores RDD levels, enhances GABA receptor-mediated inhibitory function in the spinal cord, and relieves neuropathic pain in SCI; Bumetanide significantly improves neuropathic pain in the long term, whereas CLP290 demonstrates a notable short-term effect.
本研究旨在探讨 CLP290 联合布美他尼是否能最大限度地改善脊髓损伤（SCI）后的神经病理性疼痛，并探讨其可能的分子机制。
将大鼠随机分为五组：假手术组（Sham）、SCI+载体组、SCI+CLP290 组、SCI+布美他尼组和 SCI+联合治疗组（CLP290+布美他尼组）。药物治疗从损伤后第 7 天（7 dpi）开始，持续 14 天。所有大鼠在 56 天内进行行为评估，全面评估干预措施对机械痛、热痛、冷痛、运动功能和其他相关参数的影响。在不同时间点进行电生理学评估、免疫印迹和免疫荧光检测，重点关注脊髓腰膨大处 KCC2 和 NKCC1 蛋白的表达和变化。
CLP290 和布美他尼减轻了 SCI 引起的感觉过敏和运动功能障碍，联合治疗组的恢复效果更为显著。联合治疗组的恢复率依赖性抑制（Rate-Dependent Depression，RDD）水平最为显著。在联合治疗组和两种单药治疗组中，腰膨大区钾氯离子共转运蛋白 2（K-Clcotransporter 2，KCC2）表达上调和钠离子钾氯离子共转运蛋白 1（Sodium-Potassium-Chloride Cotransporter 1，NKCC1）表达下调导致 KCC2/NKCC1 比值显著升高，其中联合治疗组最为明显。与 SCI+载体组相比，SCI+布美他尼组在 21 和 35 dpi 时的热痛缩足潜伏期（Paw Withdrawal Thermal Latency，PWTL）无明显改善，但在 56 dpi 时显著改善。相比之下，SCI+CLP290 组在 21 dpi 时显著改善 PWTL，而在 35 和 56 dpi 时无明显变化。在 21 dpi 时，两种单药治疗组的 KCC2 表达均高于 SCI+载体组，但差异无统计学意义。在 56 dpi 时，只有 SCI+布美他尼组的 KCC2 表达与对照组相比有显著差异。
CLP290 联合布美他尼可有效增加 KCC2/NKCC1 比值，恢复 RDD 水平，增强脊髓 GABA 受体介导的抑制功能，缓解 SCI 后的神经病理性疼痛；布美他尼能显著改善 SCI 后的慢性神经病理性疼痛，而 CLP290 则具有显著的短期疗效。
