Formononetin Inhibits Microglial Inflammatory Response And Contributes To Spinal Cord Injury Repair By Targeting The EGFR/MAPK Pathway

Objective: Zhenbao Pill contains many Chinese herbal medicinal ingredients and has been proven to have therapeutic effects on the repair of spinal cord injury(SCI).This study attempts to investigate the role of formononetin(FMN),an ingredient of Zhenbao Pill,in regulating neuroinflammation after SCI and the underlying mechanism.Methods:By using the Pub Chem software,the 2D chemical structure of FMN was downloaded.The Swiss Target Prediction was used and the predictive result showed 103 targets of FMN.The Gene Ontology Resource database focuses on the association between disease phenotypes and their causative genes,from which we obtained 115 genes related to SCI.By taking the intersection,4 genes,including TNF,PTPRS,EGFR,and Hsp90 were gained.Based on the previous reports,EGFR is involved in regulating the development of SCI.Thus,the EGFR was selected for further experiments.Primary microglia isolated from the spinal cord of newborn rats and human microglial clone 3(HMC3)cells were stimulated with IL-1β followed by FMN incubation.The cell viability and inflammatory cytokine levels were detected.The target of FMN was predicted and screened using databases.By silencing or overexpression of epidermal growth factor receptor(EGFR),the anti-neuroinflammatory effect of FMN was assessed in vitro.In vivo,FMN was intraperitoneally injected into rats after SCI followed by the neurological function and histopathology examination.Results:1.The isolated microglia were in high purity(99.22%),It can be used for subsequent experiments.and the different concentrations of FMN incubation had no toxic effects on primary microglia and HMC3 cells.Elisa was used to detect the expression of TNFα and IL-6in the cell supernatant,FMN reduced the inflammatory cytokine levels(TNF-α and IL-6)in a concentration-dependent manner,The difference was statistically significant(P <0.05).2.EGFR silencing or FMN incubation decreased p-EGFR and p-p38 levels and down-regulated inflammatory cytokine levels in IL-1β-stimulated cells or supernatants.In addition,EGFR silencing induced a significant decrease in EGFR phosphorylation and p38 phosphorylation in primary microglia and HMC3 cells treated with IL-1β,The difference was statistically significant(P <0.05).Meanwhile,in the cell supernatant,EGFR silencing decreased the concentrations of TNF-α and IL-6,The difference was statistically significant(P <0.05).3.the effects of FMN on microglial inflammation were reversed by EGFR overexpression:EGFR was overexpressed in primary microglia and HMC3 cells and then stimulated with IL-1β.The results showed that FMN treatment significantly reduced the levels of p-EGFR and p-p38 in IL-1β-treated cells,whereas EGFR overexpression enhanced these levels.The concentration of TNF-α and IL-6 in the cell supernatant may be reduced after FMN treatment,whereas EGFR overexpression significantly increased these levels,The difference was statistically significant(P <0.05).4.In vivo,FMN treatment improved the neuromotor function,repaired tissue injury,and inhibited EGFR/p38 MAPK phosphorylation,The difference was statistically significant:As the concentration of FMN increased,the BBB score and tilt Angle of SCI rats were significantly higher than those of SCI rats and sham-operated rats(P < 0.05).SCI surgery and FMN injection had no toxic effect on rats,and the difference was not statistically significant(P > 0.05).The serum levels of TNF-α and IL-6 in SCI group were increased,and FMN was down-regulated in a dose-dependent manner,and the difference was statistically significant(P < 0.05).In addition,the increased p-EGFR and p-p38 protein levels in the spinal cord of SCI rats were gradually decreased after the cumulative FMN treatment,and the difference was statistically significant(P < 0.05).Conculsions:Formononetin inhibits microglial inflammatory response and contributes to SCI repair via the EGFR/p38 MAPK signaling pathway:(1)FMN had no toxic effect on microglia.(2)EGFR silencing or FMN incubation reduced the phosphorylation of EGFR/p38 MAPK and inhibited microglial inflammation;(3)Overexpression of EGFR abolished the anti-neuroinflammatory effect of FMN.(4)In vivo experiments showed that FMN could inhibit the phosphorylation of EGFR/p38 MAPK and inflammatory response in SCI rats,and improve spinal cord tissue injury and functional recovery.