ClC-3氯通道调控脊髓小胶质细胞表型转变在A型肉毒毒素治疗神经病理性疼痛中的机制研究

Botulinum toxin type A (BoNT/A) has been used to treat a number of pain conditions including neuropathic pain, but its internal mechanism remains largely unknown. ClC-3 is capable of regulating the activation of glial cells and the release of inflammatory factors via NF- kappa B pathway and plays an important role in mechanism of neuropathic pain. Our previous study demonstrated that BoNT/A can be transported retrograde from peripheral to dorsal horn and dorsal root ganglion, which inhibits the activation of microglia and the release of tumor necrosis factor- α. Our preliminary experiments have indicated that BoNT/A is capable of regulating the expression and function of ClC-3 chloride channel, promoting the M1 / M2 phenotypic transition of microglia, and modulating the excitability of neurons. Therefore, we assume that ClC-3 may play an important role in the analgesic mechanism of BoNT/A by regulating the activation of microglia and phenotypic transition. In this study, a spared sciatic nerve injury model (SNI) was used to observe the effect of BoNT/A on the expression and function of ClC-3.The effect of ClC-3 on M1 / M2 phenotypic transition of microglia via TLR4 / NF- κ B pathway is also investigated. Furthermore, the regulation of ClC-3 by M2 type microglia is also examined. The present research is expected to reveal the significance of phenotypic transition of microglia mediated by ClC-3 in the treatment of neuropathic pain by BoNT/A, thus provide the important theoretical basis for further application of BoNT/A on treatment of various pain

A型肉毒毒素（BoNT/A）对病理性疼痛疗效显著，但机制不明。氯离子通道3（ClC-3）能够通过NF-κB通路调控胶质细胞的激活和炎症因子释放，在病理性疼痛中意义重要。我们前期研究表明，BoNT/A可抑制脊髓背角和背根神经节中小胶质细胞的激活及TNFα的释放。预实验发现，BoNT/A可影响ClC-3的表达和功能，介导小胶质细胞M1/M2表型转变，缓解病理性疼痛。因此我们设想，ClC-3可能通过调控小胶质细胞激活和表型转变，在BoNT/A的镇痛机制中发挥重要作用。本研究拟在疼痛动物模型观察BoNT/A对ClC-3的调节，研究ClC-3通过TLR4/NF-κB通路对小胶质细胞M1/M2表型转变的调控机制，并进一步研究M2型小胶质细胞激活后对ClC-3的反向调控作用，旨在阐明ClC-3调控小胶质细胞表型转变在BoNT/A治疗神经病理性疼痛中的重要意义，为进一步应用BoNT/A治疗疼痛提供新的思路

神经病理性疼痛严重影响患者生活质量，目前尚无满意的治疗方案。A型肉毒毒素（BoNT/A）对临床多种病理性疼痛有较好疗效，但机制不明。氯离子通道3（ClC-3）能够通过NF-κB通路调控胶质细胞激活和炎症因子释放，在病理性疼痛中意义重要。我们前期研究表明，BoNT/A可抑制脊髓背角和背根神经节（DRG）中小胶质细胞激活及TNFα的释放；预实验发现，BoNT/A可影响ClC-3的表达和功能，介导小胶质细胞M1/M2表型转变，缓解疼痛。本项目以神经损伤病理性疼痛动物为研究对象，首先明确BoNT/A的镇痛作用，进而研究BoNT/A可能调节ClC-3，通过TLR4/NF-κB通路介导小胶质细胞M1/M2表型转变发挥镇痛作用的机制，并进一步探究M2型小胶质细胞激活对ClC-3的反向调控作用。.研究结果发现：（1）BoNT/A后可显著减轻外周神经损伤模型SNI和L5 VRT引起的神经病理性疼痛；BoNT/A下调SNI术后DRG中钠通道Nav1.3表达，减小功能性TTX-S钠电流；BoNT/A减小了L5 VRT大鼠DRG中TTX-S和TTX-R钠电流，降低了神经元的兴奋性；（2）ClC-3表达分布于脊髓背角和DRG中与感觉传导相关的神经元上，同时在脊髓前角神经元上也可见分布；SNI术后不同时间点（第3、7、14天）脊髓背角和DRG中ClC-3表达显著下调；BoNT/A干预后可使SNI术后原本下调的ClC-3表达发生上调；（3）SNI术后脊髓背角和DRG中小胶质细胞激活增多，DRG中M1型小胶质细胞激活增多，M2型小胶质细胞激活减少；BoNT/A干预后不同时间点（第3、7、14天）小胶质细胞的激活减少，且可使SNI术后上调的M1型小胶质细胞激活减少，下调的M2型小胶质细胞激活增多，而氯通道阻断剂Tamoxifen可以翻转BoNT/A的作用；（4）BoNT/A可使SNI术后脊髓背角和DRG中表达上调的TLR4和p-NF-κB表达发生显著下调；阻断ClC-3和TLR4后，BoNT/A的镇痛效应下降；（5）SNI大鼠鞘内注射抑炎因子rrIL-10增大了ClC-3氯电流。.本研究结果表明BoNT/A可能调节ClC-3通过TLR4/NF-κB通路介导小胶质细胞表型转变产生镇痛的中枢作用机制，为病理性疼痛的治疗提供了重要靶点，也为进一步应用BoNT/A治疗疼痛提供新的思路和理论依据。