如意珍宝丸干预出血性脑卒中小鼠继发性脑损伤及中枢痛的作用及免疫炎症机制

Effect and Immunoinflammatory Mechanism of Ruyi Zhenbaowan in Treatment of Secondary Brain Injury and Central Pain in Hemorrhagic Stroke Mice

摘要:

目的 评价如意珍宝丸（RYZBW）对出血性脑卒中小鼠继发性脑损伤及中枢痛的干预效果，并从神经炎症角度探索其修复神经-血管单元的药理机制。方法 以Ⅳ型胶原酶丘脑腹后外侧核区显微注射的方式构建小鼠卒中后中枢痛（CPSP）模型，构建当天记为D1并分为假手术组（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（PGB）组。模型构建后第4天（D4），进行2次/d的灌胃给药。Sham及CPSP给予等体积生理盐水，RYZBW-L、RYZBW-M、RYZBW-H组给予1.214、1.821、2.428 g·kg^-1如意珍宝丸，PGB组给予0.046 g·kg^-1普瑞巴林；分别于建立模型前1 d（D0）、建立模型后第3天（D3）及D4第1次灌胃给药后检测机械痛敏，D1及D4第2次灌胃给药后评价神经损伤。D4取外周血进行血常规检测，取丘脑进行免疫炎症芯片分析。在独立样品中，以免疫荧光双标染色及酶联免疫吸附测定法（ELISA）、蛋白免疫印迹法（Western blot）对相关免疫炎症因子-受体-细胞定位进行定量分析。结果 与Sham组比较，CPSP小鼠具有显著的继发性神经损伤，卒中后中枢痛行为（P<0.05，P<0.01），外周血红细胞分布宽度（RDW）明显增高（P<0.05），血红蛋白浓度（HGB）明显下降（P<0.05）。炎症芯片结果显示，与Sham组比较，CPSP组CC趋化因子配体2（CCL2）明显增加（P<0.05），而CX3C趋化因子配体1（CX3CL1）明显降低（P<0.05），上述结果在ELISA及免疫荧光统计中被证实。Western blot结果显示，与Sham组比较，CPSP组CX3CL1受体CX3CR1的蛋白表达量显著降低（P<0.01）。免疫荧光双标染色结果显示，与Sham组比较，CPSP组Ly6C⁺CX3CR1⁺标记的非典型单核细胞数量无显著变化，CX3CR1^-Ly6C⁺标记的典型单核细胞的数量显著增加（P<0.01），CX3CR1在小胶质细胞内表达显著增加（P<0.01）。与CPSP组比较，RYZBW剂量依赖性地改善神经损伤（R²=0.367 9）及中枢痛症状（R²=0.501 9）；RYZBW-H明显改善外周血RDW及HGB（P<0.05）；免疫炎症芯片及ELISA结果显示，RYZBW-H显著抑制CCL2表达（P<0.01）并提高CX3CL1表达（P<0.05）。Western blot实验结果显示RYZBW-L/H组明显提高CX3CR1蛋白表达（P<0.05）。免疫荧光双标染色结果显示，与CPSP组比较，RYZBW剂量依赖性地升高CX3CR1总体表达量（R²=0.619 6），抑制小胶质细胞上CX3CR1表达，降低小胶质细胞数量（R²=0.494 5）及胞体面积（R²=0.571 7）。同时，RYZBW剂量依赖性地增加CX3CR1⁺Ly6C⁺（R²=0.635 3）的非典型核细胞浸润，并有效抑制Ly6C⁺CX3CR1^-（R²=0.483 6）的典型单核细胞浸润。结论 RYZBW可有效缓解CPSP小鼠继发性损伤及中枢痛，其机制与调控CX3CL1-CX3CR1轴、抑制小胶质细胞浸润及激活、促进血管修复性的非典型单核细胞浸润并抑制炎症吞噬性的典型单核细胞浸润有关。

Abstract:

Objective To evaluate the intervention effect of Ruyi Zhenbaowan （RYZBW） on secondary brain injury and central pain in mice with hemorrhagic stroke and to explore its pharmacological mechanism of repairing the neurovascular unit from the perspective of neuroinflammation.Method A mouse model of central post-stroke pain （CPSP） was established by microinjecting type Ⅳ collagenase into the ventroposterior thalamic nucleus. The day of model establishment was recorded as D1， and the mice were divided into Sham operation group （Sham）， model group （CPSP）， low （RYZBW-L）， medium （RYZBW-M）， and high （RYZBW-H） dose groups of RYZBW， and positive drug pregabalin （PGB） group. On the 4th day （D4） after model establishment， gavage administration was performed twice daily. The Sham and CPSP groups received an equal volume of normal saline， while the RYZBW-L， RYZBW-M， and RYZBW-H groups received RYZBW at 1.214， 1.821， 2.428 g·kg^-1， respectively， and the PGB group received PGB at 0.046 g·kg^-1. Mechanical hyperalgesia was assessed before model establishment （D0）， on the 3rd day （D3）， and after the first gavage on D4. Nerve damage was evaluated after the second gavage on D1 and D4. On D4， peripheral blood was collected for routine blood tests， and the thalamus was collected for immune-inflammation microarray analysis. In independent samples， quantitative analysis was performed on the localization of immune-inflammatory factors， receptors， and cells via immunofluorescence staining， enzyme-linked immunosorbent assay （ELISA）， and Western blot analysis.Result Compared with the Sham group， CPSP mice showed significant secondary nerve injury， central pain after stroke （P<0.05，P<0.01）， increased red blood cell distribution width （RDW） in peripheral blood （P<0.05）， and decreased hemoglobin （HGB） concentration （P<0.05）. Immune-inflammation microarray analysis showed that CC chemokine ligand 2 （CCL2） in the CPSP thalamus was significantly increased compared to the Sham group （P<0.01）， while CX3C chemokine ligand 1 （CX3CL1） was significantly decreased （P<0.05）. These results were confirmed by ELISA and immunofluorescence staining. Western blot analysis indicated that the protein expression of CX3CR1， the receptor for CX3CL1， was significantly decreased in the CPSP group compared to the Sham group （P<0.01）. Immunofluorescence staining revealed that the number of Ly6C⁺CX3CR1⁺ non-classical monocytes in the CPSP group did not change significantly， while the number of classical monocytes （CX3CR1^-Ly6C⁺） significantly increased （P<0.01）. The expression of CX3CR1 in microglia was significantly increased in the CPSP group （P<0.01）. Compared with the CPSP group， RYZBW improved neurological deficits （R²=0.367 9） and central pain symptoms （R²=0.501 9） in a dose-dependent manner. RYZBW-H significantly improved peripheral blood RDW and HGB （P<0.05）. Immune-inflammation microarray analysis and ELISA results showed that RYZBW-H significantly inhibited CCL2 expression （P<0.01） and increased CX3CL1 expression （P<0.05）. Western blot results indicated that the protein expression of CX3CR1 in the RYZBW-L and RYZBW-H groups was significantly increased （P<0.05）. Immunofluorescence staining demonstrated that RYZBW increased the overall expression of CX3CR1 in a dose-dependent manner （R²=0.619 6）， inhibited the expression of CX3CR1 on microglia， and decreased both the number （R²=0.494 5） and soma area （R²=0.571 7） of microglia compared with the CPSP group. Additionally， RYZBW increased the infiltration of CX3CR1⁺Ly6C⁺ non-classical monocytes in a dose-dependent manner （R²=0.635 3） and effectively inhibited the infiltration of Ly6C⁺CX3CR1^- classical monocytes （R²=0.483 6）.Conclusion RYZBW can effectively alleviate secondary injury and central pain in CPSP mice， and its mechanism involves regulating the CX3CL1-CX3CR1 ligand-receptor interaction， inhibiting microglial infiltration and activation， promoting non-classical monocyte infiltration for vascular repair， and suppressing the infiltration of classical monocytes for inflammatory phagocytosis.