| Objective: Acute spinal cord injury(ASCI)refers to acute spinal cord structural changes and neurological dysfunction caused by multiple factors,especially research reports that emphasize the importance of inflammatory processes in mediating tissue damage after ASCI.more and more.We also screened formononetin(FMN),a monomer component of flavonoids,through the TCMSP Chinese Medicine Database.FMN has been widely reported to have many pharmacological properties,such as antioxidant,cardioprotective and neuroprotective activities,vascular relaxants,Anticancer,anti-inflammatory and antiviral activities,etc.Spinal cord injury will have pathological features such as formation of oxidative free radicals,inflammation,and apoptosis.Therefore,it is speculated that FMN may have a therapeutic effect on ASCI,but its specific regulatory mechanism is still uncertainty.This research is to investigate the specific effect of FMN on ASCI.The mechanism provides new targets and ideas for the treatment of ASCI and the recovery of neurological function.Method:This research will take FMN as the research object and predict its mechanism of action in spinal cord injury through network pharmacology.The Swiss Target Prediction database was used to find potential targets of FMN,and then the target proteins corresponding to FMN were screened out,and the targets with duplicate,non-human origin and no standard gene name were removed,and finally 103 targets were collected.In the Gene Ontology Resource database,the research species was limited to "Homo sapiens",and the research disease was "Spinal cord injury",and finally 115 genes related to spinal cord injury were obtained.Subsequently,the obtained FMN-target genes and spinal cord injury-related genes were passed through the online tool Venny 2.1,and 4 overlapping targets were obtained,accounting for 1.8% of the collection,among which the gene EGFR attracted our attention.The study found that the epithelial factor growth receptor EGFR binds to its receptor,which can cause the activation of microglia and the mitosis-activated protein kinase(p38 MAPK) cascade.It has been reported that phosphorylated p-p38 plays a key role in activating microglia in spinal cord injury and can increase the release of microglial pro-inflammatory factors,such as interleukin-1 β(IL-1 β),TNF α,etc.Therefore,inhibition of the EGFR/MAPK signaling pathway has been shown to reduce microglial inflammatory responses and associated secondary injuries after SCI.Then,we used the rat spinal cord injury model and IL-1β-induced primary glial cells and HMC3 human microglia to verify whether the monomeric component FMN in Zhenbao Pills inhibits microglial inflammation through EGFR/MAPK signaling response,thereby reducing the degree of spinal cord tissue damage.Results:1.Elisa detects the expression of inflammatory factors TNFα and IL-6 in the cell supernatant: Compared with the Control group,the expressions of TNFα and IL-6 in the primary glial cells and HMC3 human microglia in the IL-1 β group were significantly increased,while the expressions of TNFα and IL-6 in primary glial cells and HMC3 human microglia in IL-1 β +FMN group were significantly decreased,and the difference was statistically significant(P<0.05);2.WB detection of EGFR,p-EGFR,p38,p-p38 expression:Compared with the Control group,the expression of p-EGFR and p-p38 in the primary glial cells and HMC3 human microglia in the IL-1 β group was significantly higher The expression of p-EGFR and p-p38 in primary glial cells and HMC3 human microglia decreased in IL-1 β +si-EGFR group,and the difference was statistically significant(P<0.05).The expression of inflammatory factors TNFα and IL-6 in the serum showed that the expressions of TNFα and IL-6 in primary glial cells and HMC3 human microglia were significantly decreased in IL-1 β +si-EGFR group;3.WB detection of EGFR,p-EGFR,p38,p-p38expression: p-EGFR and p-p38 were up-regulated in IL-1β group,but negatively regulated in FMN group,and the difference was statistically significant(P < 0.05);4.BBB score(Rats in each group underwent motor function tests on days 1,4,7,and 14 after SCI,on a scale of 0to 21.): Compared with the Sham group,the BBB score of the SCI group decreased significantly at each time point,but the opposite was true in the SCI+FMN(20,40 mg/kg)group,and the difference was statistically significant(P<0.05);The tissue Iab1(cytoplasmic localization)and p-EGFR(cellular membrane localization)immunofluorescence double staining : Iba1 labeled microglia,compared with the Sham group,the microglia in the SCI group were activated,and the expression of p-EGFR was increased,and the co-localization was obvious.The FMN group could inhibit this phenomenon to different degrees,and the difference was statistically significant(P<0.05).);Conclusion:1.FMN can reduce the inflammatory response of microglia stimulated by IL-1β;2.Inhibiting the expression of EGFR can reduce the inflammatory response of microglia in spinal cord injury;3.FMN can reduce the inflammatory response of microglia by targeting the inhibition of EGFR/MAPK signaling pathway.Glial cell inflammatory response;4.It was verified in animals that FMN can alleviate the degree of spinal cord injury by down-regulating the expression of EGFR;... |
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