基于脊髓星型胶质细胞JNK/CXLCL1信号通路探讨独活香豆素治疗神经病理性痛的分子作用机制

Neuropathic pain (NP) is a devastating neurological disease that seriously affects patients’ quality of life. Because of Academic System that think neuron as the center is not sufficient, astrocyte that is capable of regulating neuronal has been considered as a promising target against neuropathic pain. Radix angelicae pubescentis is proved to be effective and low side effect to pain，and has been used clinically for curing pain for a long history. Our present study showed that coumarins from Radix angelicae pubescentis (CRAP) attenuated the development of mechanical and thermal hypersensitivity, and reduced the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 of dorsal root ganglion in a spared nerve injury model of neuropathic pain. It indicated that CRAP could regulate astrocytes, which play the role of immune cells in neuropathic pain. Pre-Experiment showed that astrocytic activation in spinal cord was suppressed by CRAP in a spared nerve ligation model of neuropathic pain. JNK/CXCL1 pathway plays an important role in astrocytic activation. Therefore, we proposed that CRAP regulated astrocytic activation by JNK/CXCL1 pathway, resulting reduction of neuronal excitability and analgesic effect. In order to certify this view, whole-cell patch-clamp technique, western blotting, quantitative PCR, gene transfection and immunofluorescence were applied to clarify which molecule was involved in alleviating neuropathic pain by CRAP. This project provides theoretical basis for the clinical application of CRAP.

神经病理性痛(Neuropathic pain，NP)严重影响患者的生活质量。星型胶质细胞对神经元的调节作用，使其成为前景广阔的镇痛靶点。独活镇痛历史悠久，疗效确切，副作用小。研究证明：独活香豆素(CRAP)能够降低坐骨神经部分损伤模型大鼠的机械痛敏和热痛敏，抑制炎症因子TNF-α，IL-1β和IL-6的表达，提示CRAP调节星型胶质细胞这一疼痛免疫细胞的潜在作用。预实验表明：CRAP能够抑制坐骨神经部分结扎大鼠脊髓星型胶质细胞激活。JNK/CXCL1对星型胶质细胞激活至关重要。因此，本课题组提出“CRAP通过JNK/CXCL1调节脊髓星型胶质细胞激活，降低神经元兴奋性而发挥镇痛作用”的科学假说。本项目拟采用膜片钳技术、western blotting、实时定量荧光PCR、基因转染、免疫荧光等多种分子生物学技术，阐明CRAP治疗NP的分子作用机制，为CRAP的临床应用提供理论依据。

星形胶质细胞-神经元相互作用在维持神经病理性疼痛中起重要作用。靶向星形胶质细胞抑制剂被认为是治疗神经病理性疼痛的有效方法。CRAP是独活的主要成分，是一种具有悠久历史的消炎止痛的中药。CRAP能否通过星形胶质细胞减轻神经病理性疼痛，目前尚不清楚。采用CCI手术建立小鼠神经病理性疼痛模型。CCI小鼠用CRAP（5、10或20 mg/kg/天）治疗14天。采用机械性痛觉过敏和热痛觉过敏两种方法，观察其治疗效果。在机制研究中，观察了星形胶质细胞的活化，星形胶质细胞中p-JNK和CXCL1蛋白的表达，mEPSPs和eEPSP电位的变化，神经元中CXCR2、GluA1、GluN2B、p-ERK、p-CREB和c-fos的水平。此外，还应用星形胶质细胞和神经元共培养技术研究了CRAP对星形胶质细胞-神经元相互作用和信号传递的直接影响。应用脂多糖、CXCL1中和抗体、JNK抑制剂SP600125、JNK-siRNA、CXCL1重组蛋白和CXCR2抑制剂SB225002证实CRAP对神经病理性疼痛的作用。CRAP治疗能明显减轻CCI小鼠的机械性痛觉过敏和热痛觉过敏。JNK参与CCI诱导的痛敏反应，JNK通路参与CRAP诱导的脊髓CXCL1下调。CRAP降低的CXCR2表达与CXCL1呈剂量依赖关系。然而，CRAP对CXCL1和CXCR2没有直接影响。经CRAP处理后，脊髓I-II层神经元mEPSPs频率和eEPSPs波幅降低。下游信号分子pGluA1、pGluN2B、p-ERK、p-CREB和c-fos在CRAP治疗的神经痛小鼠中也自动减少。CRAP通过抑制JNK依赖的CXCL1/CXCR2信号通路对小鼠神经病理性疼痛具有保护作用，可作为一种潜在的减轻神经病理性疼痛的药物治疗。