如意珍宝丸干预卒中后中枢痛小鼠痛觉过敏及脊髓中枢敏化的药效特点及神经炎症机制

Pharmacodynamic Characteristics and Neuroinflammatory Mechanisms of Ruyi Zhenbaowan in Treating Nociceptive Hypersensitivity and Central Sensitisation of Spinal Cord in Mouse Model of Central Post-stroke Pain

摘要:

目的 明确如意珍宝丸（RYZBW）干预卒中后中枢痛（CPSP）小鼠痛觉过敏及脊髓中枢敏化的药效特点及神经炎症机制。方法 SPF级雄性8周龄ICR小鼠，分为假手术组（Sham）、模型组（CPSP）、如意珍宝丸低（RYZBW-L）、中（RYZBW-M）、高（RYZBW-H）剂量组和普瑞巴林组（PGB）。在第1天进行丘脑腹后外侧核Ⅳ型胶原酶注射以构建CPSP模型。第14~17天进行灌胃给药，其中Sham及CPSP给予等体积生理盐水，RYZBW-L/M/H组给予0.303、0.607、1.214 g·kg^-1如意珍宝丸，PGB给予0.046 g·kg^-1普瑞巴林；分别于第0、3、4、7、10、14、17天进行机械痛敏检测，第18天取脊髓L₅段采用免疫炎症芯片检测炎症因子、酶联免疫吸附测定法（ELISA）检测CXC型趋化因子配体16（CXCL16）、免疫组化法检测降钙素基因相关肽（cGRP）、荧光双标分析CXCL16、树突状细胞标记物CD11c、巨噬细胞标记物CD68、小胶质细胞标记物TMEM119、内皮细胞标记物CD31、CXCR6及T细胞标记物CD3的表达情况。结果 与Sham组比较，CPSP组在第3~17天的机械痛阈值显著低于Sham组，具有稳定的痛觉过敏症状。第7天PGB组的机械痛阈值显著高于CPSP组，具有明显镇痛效果（P<0.01）。第10~17天，RYZBW-H组的机械痛阈值明显高于CPSP组，展现出稳定的镇痛效果（P<0.05）。第17天，如意珍宝丸的镇痛效果呈量效相关（R²=0.303 7）；第4~17天，RYZBW-H组机械痛阈值与时间正相关（R²=0.111 5），上述结果提示RYZBW的镇痛呈时效相关。第17天CPSP小鼠脊髓背角内中枢敏化标志物cGRP表达量相比于Sham组明显增加（P<0.05），RYZBW以剂量依赖的方式将其下调（R²=0.500 8），提示如意珍宝丸显著抑制CPSP导致的脊髓中枢敏化。第17天的脊髓炎症芯片结果显示，与CPSP组比较，RYZBW-H组抑制CXCL16表达（P<0.01）。基于独立重复样品的ELISA结果表明，如意珍宝丸对CXCL16的蛋白脊髓表达的抑制呈剂量依赖性（R²=0.250 4）；免疫荧光双标结果显示，与Sham组比较，CPSP组CD11c阳性的树突细胞内CXCL16表达量增加，CD68阳性细胞数量增加（P<0.05），与CPSP组比较，RYZBW将其下调：CD31阳性的内皮细胞、CD68阳性的巨噬细胞、TMEM119阳性的小胶质细胞内，CXCL16表达量均增加，TMEM119阳性的小胶质细胞细数量、胞体面积均显著增加（P<0.05）；CD3阳性的T细胞数量（P<0.05）及其中CXCR6表达量增加。如意珍宝丸以剂量依赖性抑制内皮细胞、巨噬细胞的激活，减少小胶质细胞、T细胞浸润（R²=0.691 4，R²=0.551 5，R²=0.653 2，R²=0.180 6，R²=0.287 5，R²=0.298 6，R²=0.511 6）。结论 RYZBW可有效缓解CPSP小鼠卒中后中枢痛痛觉过敏及脊髓中枢敏化，其机制与调控CXCL16-CXCR6配体-受体、抑制小胶质细胞及巨噬细胞浸润及激活、树突状细胞、内皮细胞及T细胞活化有关。

Abstract:

Objective To clarify the pharmacodynamic characteristics and neuroinflammatory mechanisms of Ruyi Zhenbaowan （RYZBW） in treating nociceptive hypersensitivity and central sensitisation of spinal cord in the mouse model of central post-stroke pain （CPSP）.Method SPF-grade male ICR mice of 8 weeks old were assigned into the sham operation （Sham）， model （CPSP）， low-， medium-， and high-dose （0.303， 0.607 1.214 g·kg^-1） RYZBW （RYZBW-L， RYZBW-M， and RYZBW-H， respectively）， and pregabalin （PGB， 0.046 g·kg^-1， positive control） groups. The rat model of CPSP was established by injection of type Ⅳ collagenase into the ventral posterior lateral nucleus of the thalamus on day 1. Rats were administrated with corresponding drugs or normal saline （Sham and CPSP groups） by gavage from day 14 to day 17. The mechanical pain sensitivity test was performed on days 0， 3， 4， 7， 10， 14， 17. On day 18， the L₅ segment of spinal cord was collected for the detection of inflammatory cytokines by immunoinflammatory microarray， CXC chemokine ligand 16 （CXCL16） by enzyme-linked immunosorbent assay， and calcitonin gene-related peptide （cGRP） by immunohistochemistry. In addition， fluorescence dual-labeling was employed to determine the expression levels of CXCL16， the dendritic cell marker CD11c， the macrophage marker CD68， the microglia marker TMEM119， the endothelial cell markers CD31 and CXCR6， and the T cell marker CD3.Result Compared with the Sham group， the mechanical pain threshold of the CPSP group was significantly lower than that of the Sham group from day 3 to day 17， with stable hyperalgesia symptoms. On the 7^th day， the mechanical pain threshold of the PGB group was significantly higher than that of the CPSP group， with significant analgesic effect （P<0.01）. On days 10-17, the mechanical pain threshold of the RYZBW-H group was significantly higher than that of the CPSP group， showing a stable analgesic effect （P<0.05）. On the 17^th day， the analgesic effect of RYZBW was dose-effect correlated （R²=0.303 7）. From day 4 to day 17， the mechanical pain threshold of RYZBW-H group was positively correlated with time （R²=0.111 5）. The above results suggested that the analgesia of RYZBW was time-dependent. On the 17 th day， the expression of central sensitization marker cGRP in the spinal dorsal horn of CPSP mice was significantly increased compared with the Sham group （P<0.05）， and RYZBW down-regulated it in a dose-dependent manner （R²=0.500 8）， suggesting that RYZBW significantly inhibited the central sensitization of the spinal cord caused by CPSP. The results of spinal cord inflammation chip on the 17^th day showed that compared with CPSP group， RYZBW-H group inhibited CXCL16 expression （P<0.01）.The results of ELISA based on independent repeated samples showed that RYZBW inhibited the expression of CXCL16 protein in spinal cord in a dose-dependent manner （R²=0.250 4）. The results of immunofluorescence double labeling showed that compared with Sham group, the expression of CXCL16 in CD11c positive dendritic cells in CPSP group increased， and the number of CD68 positive cells increased （P<0.05）. Compared with CPSP group， RYZBW down-regulated it: the expression of CXCL16 in CD31 positive endothelial cells， CD68 positive macrophages and TMEM119 positive microglia increased， and the number and cell body area of TMEM119 positive microglia increased significantly （P<0.05）. The number of CD3 positive T cells （P<0.05） and the expression of CXCR6 in CD3 positive T cells were increased. RYZBW inhibited the activation of endothelial cells and macrophages in a dose-dependent manner， and reduced the infiltration of microglia and T cells （R²=0.691 4， R²=0.551 5， R²=0.653 2， R²=0.180 6， R²=0.287 5， R²=0.298 6，R²=0.511 6）.Conclusion RYZBW can effectively alleviate nociceptive hypersensitivity and central sensitisation of the spinal cord in CPSP mice by regulating CXCL16-CXCR-6， inhibiting the infiltration and activation of microglia and macrophages， and the activation of dendritic cells， endothelial cells， and T cells.