Study On The Mechanism Of Tongqiao Huoxue Decoction In Treating Ischemic Stroke Based On Network Pharmacology And Molecular Docking Technology

Objective:1.The active components,key targets and biological mechanism of Tongqiao Huoxue decoction in the treatment of ischemic stroke were studied by the method of network pharmacology,so as to provide theoretical and experimental basis for Tongqiao Huoxue decoction in the treatment of ischemic stroke.2.Based on the results of network pharmacology,the mechanism of Tongqiao Huoxue decoction regulating MAPK signal pathway and apoptosis in the treatment of ischemic stroke was demonstrated by animal experiments.Methods:1.Network pharmacological analysis: the active components and action targets of Tongqiao Huoxue decoction were searched by TCMSP database,HERB database,Pub Chem and Swiss Target Prediction,and the disease targets of ischemic stroke were obtained by OMIM,Genecards,Pharm GKB,TTD and Drug Bank databases.The drug components and disease targets were matched,the drug-component-disease-target interaction network was constructed by Cytoscape software,and the protein interaction network was constructed by String database.Combined with Metascape database,the enrichment analysis of GO and KEGG was carried out,and the molecular docking between the key active components and core targets was verified by Auto Dock software..2.Animal experiment: 50 healthy SD rats of SPF grade were selected as the research objects,and 40 of them were occluded by thread occlusion to establish MCAO model.24 hours later,Zea-Longa neurological deficit score was used to evaluate the modeling effect.The rats were randomly divided into model group and high,middle and low dose Tongqiao Huoxue decoction groups with 10 rats in each group.Another 10 rats were used as sham operation group,without vascular ligation or thread occlusion,and the rest was treated in the same way as MCAO.Rats in sham operation group and model group were perfused with distilled water of 1ml/100 g volume,while rats in high,middle and low dose groups of Tongqiao Huoxue decoction were fed with Tongqiao Huoxue decoction of 0.72ml/100 g,0.36ml/100 g and0.18ml/100 g combined with distilled water of 0.28ml/100 g,0.64ml/100 g and0.82ml/100 g volume,respectively.Rats in each group were intragastrically fed once a day for 7 consecutive days.The changes of neurological function of rats in each group were evaluated by Zea-Longa score after 24 hours,4 days and 7 days.At the end of the last intragastric administration,the rats were killed,and the brain tissue was dissected for TTC staining,and the volume changes of the infarcted area were measured.The expression of p38MAPK/p-p38 MAPK,Caspase-3 and Bax/Bcl-2 in the brain tissue of rats in each group was detected by Westernblot method.Results:1.Network pharmacology: 77 key components of Tongqiao Huoxue decoction in the treatment of ischemic stroke were screened,including quercetin,N-demethylnuciferine,Morin,kaempferol,testosterone,etc.,with 13 core targets,including MAPK3,SRC,MAPK1,RELA,JUN,TP53,MYC,ESR1,HSP90AA1,MAPK14,AKT1,FOS,STAT1.The results of molecular docking showed that quercetin,N-demethylnuciferine,Morin,kaempferol and testosterone had good binding ability with MAPK3,SRC,MAPK1,RELA,JUN,TP53,MYC,ESR1,HSP90AA1,MAPK14,AKT1,FOS and STAT1.The results of GO enrichment analysis showed that biological processes were related to MAPK cascade regulation,apoptosis signal pathway regulation,neuron death regulation and so on.According to the enrichment analysis of KEGG pathway,209 signal pathways were obtained,including MAPK signal pathway,TNF signal pathway,Alzheimerdisease signal pathway,IL-17 signal pathway and HIF-1 signal pathway.Among them,there are the most enriched targets in the MAPK signal pathway.According to the analysis of the results of network pharmacology,the main mechanism of Tongqiao Huoxue decoction in the treatment of ischemic stroke may be related to the regulation of MAPK signal pathway mediated apoptosis.This study will carry on the follow-up preliminary experimental demonstration based on this.2.Animal experiments:(1)Neurological deficit score: the neurological deficit score of rats in each group was 0 at 24 hours,4 days and 7 days after modeling,and the rats could move normally and the neurological function was not damaged.Compared with the sham operation group,the score in the model group was significantly higher than that in the sham operation group(P < 0.01).After 4 days of modeling,there was no significant difference between Tongqiao Huoxue decoction group and model group(P > 0.05).After 7 days of modeling,compared with the model group,the level in the Tongqiao Huoxue decoction group was significantly higher than that in the model group(P < 0.05),and the difference was significant in the high dose group(P < 0.01).(2)Percentage change of cerebral infarction volume: after TTC staining,there was no infarction focus in the sham operation group,and the pale infarction focus in the MCAO model group was more obvious than that in the sham operation group.Compared with the sham operation group,the cerebral infarction volume in the model group increased significantly(P < 0.01),and compared with the model group,the cerebral infarction volume in the Tongqiao Huoxue decoction group decreased significantly(P < 0.05),and there was significant difference between the middle and high dose groups(P< 0.01).(3)Changes of MAPK signal pathway and apoptosis-related protein expression: the results of Westernblot experiment showed that compared with the sham operation group,the expression level of p38MAPK/p-p38 MAPK protein in the model group increased,while the expression level of Bcl-2 protein increased and the expression level of Caspase-3 and Bax decreased in each dose group of Tongqiao Huoxue decoction.Conclusion(s):1.Network pharmacological analysis shows that the main mechanism of Tongqiao Huoxue decoction in the treatment of ischemic stroke may be related to the regulation of MAPK signal pathway mediated apoptosis.2.Based on the results of network pharmacological analysis,the rat model of ischemic stroke was established.the results of animal experiments showed that Tongqiao Huoxue decoction could improve the neurological defect,reduce the volume of cerebral infarction and protect brain tissue,which may be related to its regulation of MAPK signal pathway and inhibition of neuronal apoptosis.