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

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.