A Network Pharmacology Approach To Investigate The Mechanism Of Shuxuening Injection In The Treatment Of Ischemic Stroke

Objective:To explore molecular mechanisms of Shuxuening Injection(SXNI)in the treatment of Ischemic stroke(IS)by the Network pharmacology and animal experiment.Methods: ?.Network pharmacology1.The main active components of GBE were discerned through the TCMSP,TCMID,Batman-TCM databases.DL ?0.18 and OB ?30% were set as the thresholds to screen the active compounds.we screened the targets related to the active compounds using the TCMSP,STITCH,CNKI and PubMed databases,and obtained the Gene name and Gene ID of the targets from the Uniprot database,active compounds and targets are imported into the software of Cytoscape 3.7.1 to construct the “compound-target” network.2.The targets related to IS were obtained using Genecards,OMIM,TTD,and Disgenet databases.After removing the duplicate genes,the overlapping genes of targets related to IS and active compounds were collected as the potential targets.3.GO and KEGG pathway analyses were performed to analyze the potential of targets via the DAVID 6.8.4.Based on the above analysis,a “Compound-Target-Pathway” network were built via Cytoscape software.Moreover,the median of topological indexes were calculated,and the core targets and core compounds were obtained.5.Core targets and core compounds were obtained from the above analysis for molecular docking.To ensure the reliability of molecular docking,a protein crystal structure with a resolution of less than 2.8? was selected to establish the molecular docking model.the RMSD was calculated via Maestro 11.9.A docking is considered reliable if the RMSD of all ligand atoms is less than 2.0.AutoDock Vina were used to analyze the binding properties of the ligands for each protein.And sorted the docking scores and calculated the mean values,screening out the gene target of afiniy<-7.The three genes with the lowest affinity score were selected as the core target.Maestro 11.9 was used to show the specific docking situation in 3D graphics and analyze the hydrogen bond energy,Pi-pi bond and other interactions in the key amino acid residues.?.Animal experiment1.The MCAO model of rats were established.Modified Neurological severity score(mNSS)were evaluated.2.Infarct volume was calculated by TTC staining in rats.3.HE staining observed the morphology of neurons in the hippocampus of rats.4.RT-qPCR were used to measure the mRNA expression levels of the key gen es.5.Western blotting were used to measure the protein expression levels of the key genes.Results: ?.Network pharmacology1.Based on TCMSP,TCMID and Batman-TCM databases,After screening was performed for OB ?30% and DL ?0.18,17 active compounds were obtained,and 112 targets of the active compounds were searched.The “compound-target” network were constructed by the Cytoscape software.2.A total of 1537 IS-related genes were obtained via the Genecards,OMIO,TTD and Disgenet databases.After merging IS-related targets and active compound targets,52 overlapping targets were recognized as the potential targets.3.52 candidate targets were put into the DAVID 6.8 database for annotation GO enrichment analysis and KEGG pathway enrichment analysis.Based on the “count” values,a total of 27 pathways were obtained,and the top 10 pathways are shown as the core pathways.4.The“Compound-Target-Pathway” network was established using Cytoscape soft ware.The median of three topological indexes were calculated.28 core targets and 9core compounds were obtained.5.Based on the above analysis,28 core targets and 9 core compounds were selected for molecular docking.And sorted the docking scores and calculated the mean values for 28 genes,15 genes with the average affinity scores of less than 7 were selected.The lowest scores of the three genes were PTGS2,NOS3,CASP3 and they were selected as the key genes,Then find the compounds with the lowest affinity corresponding to genes(Kaempferol,luteolin,quercetin).And the combination of the compounds and genes were shown in a 3D graph by Maestro 11.9 software.?.Animal experiment1.After 24 h,48h and 72 h of reperfusion,the mNSS were evaluated.The results showed that there was no neurological deficit in Sham group.Compared with Sham group,mNSS scores of SXNI group and I/R group were improved(P < 0.05).Compared with I/R group,mNSS score of SXNI group was reduced(P< 0.05).With the prolongation of the treatment,the decreasing trend was increased.2.TTC staining showed that the cerebral infarct volume of the SXNI group was reduced(P<0.05)compared with that of the I/R group.The results showed that SXNI had a protective effect on the cerebral infarct volume of the I/R group,and reduced the cerebral infarct volume.3.HE staining: In the Sham group,the hippocampal neurons appeared to be healthy,in terms of an orderly arrangement,and the nuclei were in the center of the cell,and the cell membranes were intact.In the I/R group,there were significant areas of necrosis in the brain tissue,and inflammatory cells had infiltrated the hippocampus and microglial cells had proliferated.In the SXNI group,some neurons were swollen and ruptured,and a small number of inflammatory cells had infiltrated the hippocampus.4.The results of RT-qPCR showed that PTGS2 and CASP3 mRNA expression was down-regulated in the SXNI group(P<0.05).The mRNA expression of NOS3 was up-regulated in the SXNI group compared with the I/R group(P<0.05).5.Western blotting indicated that PTGS2 and CASP3 protein was down-regulated in the SXNI group compared with the I/R group(P<0.05),the protein expression of NOS3 in the SXNI group was up-regulated compared with the I/R group(P<0.05).Conclusion:The results of animal experiments in vivo showed that SXNI treat IS by multiple targets,multiple compounds and multiple pathways.Its mechanisms are related to the inhibition of inflammation,the regulation of oxidative stress level,and the reduction of neuron apoptosis in brain tissue.