Objective:To explore the potential molecular mechanism of Erigeron breviscapus in the prevention and treatment of ischemic stroke through network pharmaco-molecular docking.Methods:The Chinese Herbal Medicine Systematic Pharmacology Platform(TCMSP)database provided active ingredients and potential targets of Erigeron breviscapus.Ischemic stroke-related targets were searched through the Online Mendelian Inheritance in Man(OMIM)database,the bioinformatics and chemoinformatics(DrugBank)database and human gene comprehensive database(GeneCards).The targets criteria were de-weighted by the Protein Data Bank(Uniprot)and imported into the Venny online platform to obtain the intersecting targets of both.The intersecting targets were visualized by STRING database and Cytoscape 3.7.1 software for protein-protein interaction(PPI),followed by the enrichment analysis of intersection targets for gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.AutoDock Vina1.5.6 software was used to verify the molecular docking of key active ingredients and core targets and realize the vi-sualization of docking results.Results:Eleven active ingredients and 176 targets were obtained.There were 690 targets ischemic stroke-related targets and 69 intersection targets.Through PPI network,10 core genes were screened according to the degree value,including tumor necrosis factor(TNF),interleukin-6(IL-6),serine/threonine protein kinase 1(AKT1),interleukin-1β(IL-1β)and vascular endothelial growth factor A(VEGFA).KEGG enrichment included the advanced glycation end products-receptor(AGE-RAGE)signaling pathway,interleukin-17(IL-17)signaling pathway,tumor necrosis factor(TNF)signaling pathway,etc.The top 3 active ingredients and the top 5 target proteins were selected according to the degree value,and the molecular docking results demonstrated a considerable binding ability.Conclusion:Erigeron breviscapus in the treatment of ischemic stroke may work through multiple active ingredients,such as quercetin,kakaferol,and luteolin,which act on TNF,IL-6,AKT1,IL-1β,and VEGFA,and through a varie-ty of signaling pathways such as IL-17,TNF,etc.showing the characteristics of multi-components,multi-targets,and multi-pathways.

Objective:To investigate the effect of panax notoginseng saponins(PNS)on the expression of tumor necrosis factor-α(TNF-α)in lipopolysaccharide(LPS)-induced activated BV2 microglia through p38 mitogen-activa-ted protein kinase(p38 MAPK)pathway.Methods:BV2 microglia were divided into control group,LPS activated group and LPS+panax notoginseng saponins intervention group(LPS+PNS).The CCK-8 method was used to detect the viability of BV2 microglia and determine the optimal drug intervention concentration.Western Blot and immunofluo-rescence were used to detect the expression of p38 MAPK and TNF-α and the phosphorylation level of p38 MAPK(p-p38 MAPK)in BV2 microglia.Results:Compared with the blank control group,there was no significant difference in the cell viability of BV2 microglia,and finally 100 mg/L was selected as the drug intervention concentration.Western Blot and immunofluorescence results indicated that after LPS activation,the expression of TNF-α and the phosphoryla-tion level of p38 MAPK in BV2 microglia were significantly increased(P＜0.05).After PNS intervention,compared with LPS-activated group,the expression of TNF-α and the phosphorylation level of p38 MAPK were significantly decreased(P＜0.05).After treatment with p38 MAPK pathway inhibitor(SB203580),there was no significant differ-ence in the expression levels of p-p38 MAPK and TNF-α in PNS combined with SB203580 group(LPS+PNS+I)com-pared with LPS+PNS group(P＞0.05).In addition,the changes of p38 MAPK in each group were not statistically sig-nificant(P＞0.05).Conclusion:PNS may inhibit the expression of inflammatory factor TNF-α secreted by activated BV2 microglia through p38 MAPK pathway.

Objective:To explore the impact of scutellarin on the levels of phosphatidylinositol 3-kinase(P13K)and phosphorylated P13K(p-PI3K)in activated microglia.Methods:A rat cerebral ischemia model was made by middle cerebral artery occlusion(MCAO)method.SD rats were randomly divided into three groups:sham operation group(Sham),cerebral ischemia group(MCAO),and cerebral ischemia with scutellarin group(MCAO+S).A model of ischemia-hypoxia injury of BV2 microglia was established by glucose-oxygen deprivation(OGD)and randomly assigned as control group(Control),OGD group and OGD+scutellarin group(OGD+S).Changes in PI3K and p-PI3K expres-sion in microglia were assessed using double immunofluorescence staining and Western Blot.Results:Immunofluores-cence staining and Western Blot analyses revealed a significant increase in p-PI3K levels in activated microglia both in vivo and in vitro(P＜0.05).Furthermore,treatment with scutellarin led to a further elevation in p-PI3K(P＜0.05).However,there were no significant alterations observed in PI3 K expression among the groups(P＞0.05).Conclusion:Scutellarin may play a positive role in the treatment of cerebral ischemia by up-regulating the phosphorylation level of P13K in activated microglia.

Objective:To investigate the effects of cannabidiol(CBD)on the NOD-like receptor protein 3(NLRP3)inflammasome in the brains of rats with multiple cerebral concussions(MCC).Methods:Rats were subjec-ted to the MCC model and divided into Sham,MCC,vehicle(MCC+TW),CBD-L(10 mg/kg),and CBD-H(40 mg/kg)groups.Immunofluorescence double staining was used to observe changes in NLRP3 and microglial cells in the brain,and Western Blot was performed to detect the expression changes of the NLRP3 inflammasome.Results:A sig-nificant increase in lectin-positive microglial cells of the cortex with enlarged cell bodies and elevated immunofluores-cence intensity of NLRP3 in the activated microglial cells was revealed by immunofluorescence double staining following MCC(P＜0.05).The immunofluorescence intensity of NLRP3 in the activated microglial cells was downregulated by the administration of CBD,with a more pronounced effect observed in the CBD-H group compared to the CBD-L group(P＜0.05).The expression of NLRP3,caspase-1,and apoptosis-associated speck-like protein(ASC)in the cortex,hippocampus,and basal ganglia of rats following MCC was significantly increased,as shown by Western Blot analysis(P＜0.05),and cortical areas are more elevated.The expression of these proteins in different brain regions was reduced by CBD-10 and CBD-40 intervention(P＜0.05).Conclusion:Cannabidiol can reduce the inflammatory response of multiple cerebral concussions rats through NLRP3 inflammasome and protect nerve tissue.

Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage(HIBD)in neonatal rats.Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group,HIBD group and gastrodin treatment group.Western blotting was used to detect the expressions of TNF-α,IL-1β,IL-10 and TGF-β1 in the corpus callosum of the rats.The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis.The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation(OGD)were detected with Western blotting.The effects of LY294002(a specific inhibitor of the PI3K/AKT pathway)and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR.Results In the neonatal rats with HIBD,gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum.Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K.Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD.In BV-2 cells with OGD,gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions,and this effect was strongly attenuated by LY294002 treatment.Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage(HIBD)in neonatal rats.Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group,HIBD group and gastrodin treatment group.Western blotting was used to detect the expressions of TNF-α,IL-1β,IL-10 and TGF-β1 in the corpus callosum of the rats.The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis.The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation(OGD)were detected with Western blotting.The effects of LY294002(a specific inhibitor of the PI3K/AKT pathway)and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR.Results In the neonatal rats with HIBD,gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum.Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K.Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD.In BV-2 cells with OGD,gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions,and this effect was strongly attenuated by LY294002 treatment.Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.