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

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.