Study On The Mechanism Of Action Of Compound Angelica Injection On Ischemic Stroke Based On Network Pharmacology

Ischemic stroke(IS)is a neurovascular disease that results in softening and necrosis of the brain tissue due to ischemic hypoxia.The incidence,relapse and mortality rates of stroke are high around the world.The pathophysiological mechanism of IS is complex;various cellular,molecular and signaling pathways are involved in the process of stroke.The intricate process of stroke includes bioenergy failure,imbalance of ion homeostasis,apoptosis,excitotoxicity,oxidative stress,immune reaction,inflammation,etc.At present,the main treatments of IS is intravenous thrombolysis and mechanical thrombectomy,but due to the limitation of treatment time window and stroke subtypes,it cannot serve most patients clinically.Compound Angelica Injection(CAI)is a compound Chinese medicine injection composed of Danggui,Chuanxiong and Honghua.It has the effects of huoxue,huayu and tongluo.It is widely used in clinical practice for various diseases with blood stasis,such as Dysmenorrhea,Amenorrhea,Fall injury and Rheumatic arthralgia.Network pharmacology is based on the theory of systems biology,which applies highthroughput omics data analysis and processing,computer graphics and virtual computing,network database information retrieval etc to the network analysis of biological systems.Funrich is a software that uses high-throughput analysis to describe genomes,transcriptome,proteome and metabolome.It has powerful enrichment analysis function.The phenotypic enrichment analysis can reduce the cost of drug R&D and improve the benefit/risk ratio of drug R&D,which is of great significance to expand the clinical indications of traditional Chinese medicine compound.In this study,we will be based on network pharmacology to explore the mechanism of CAI’s intervention of IS,and conduct preliminary experimental demonstrations.Objective:The active components,key targets and biological mechanisms of CAI intervention in IS were studied by network pharmacology method,so as to provide theoretical and experimental basis for CAI treatment of IS.The protective effect of CAI on MCAO rats and its related mechanism were studied by preliminary experimental experiments.Methods:1.The disease targets of CAI indications(Dysmenorrhea,Amenorrhea,Fall injury,Rheumatic arthralgia)and IS were retrieved from GeneCards,OMIM and TCMIP databases.To explore whether CAI has potential value in intervening IS,the clinical indications and comparative results were constructed by Funrich software.The active components of CAI were retrieved from CNKI,and the targets of CAI active components were obtained from Swiss Target Prediction.The targets of CAI active components and the targets of IS disease were analyzed,and the co-expression targets with the highest correlation were obtained.GO and KEGG enrichment analysis and molecular docking were performed to further explore the potential mechanism of CAI’s intervention in IS.2.The SD rats were randomly divided into control group(normal saline 3 mL/kg ip.qd.)and MCAO group,which were modeling with MCAO on the right side.Based on mNSS,the MCAO group were randomly divided into model group(normal saline 3 mL/kg ip.qd.),Edaravone group(edaravone injection 3 mL/kg ip.qd.)and CAI group(CAI 10 mL/kg ip.qd.),all of them were treated for 7 d.Finally,mNSS was used to observe the changes of nerve function.Triphenyltetrazolium chloride(TTC)staining was used to detect the change of the volume ratio of ischemic area.Hematoxylin-eosin(HE)staining was used to observe the pathomorphologic changes of brain tissue.The expressions of IL-17A,IL-17RA,TRAF6 and CXCR2 were detected by Western blot.Enzyme linked immune sorbent assay(Elisa)was used to detect the expression levels of CXCL1 in serum.The expression of IL-17RA and CXCR2 in ischemic hemispheric was detected by immunohistochemistry.Results:1.The indications for CAI overlapped with the clinical phenotypes of IS,mainly concentrated in abnormal neurological function and abnormal central nervous system.2.There were 6 members of CAI active ingredient,ferulic acid,hydroxysafflor yellow A,chlorogenic acid,cryptochlorogenic acid,neochlorogenic acid and caffeic acid,thus the active ingredient targets were 260,and a total of 165 targets intersected with IS.A total of 27 key intersected targets were obtained through MCODE analysis.3.Bioinformatics analysis of key intersection targets was established,and BP showed that the intervention of CAI in IS were related to cell apoptosis,cell energy supply and tissue inflammation.MF showed that the intervention of CAI in IS were related to information transmission,energy metabolism,nerve cell function and survival,etc.CC showed that the intervention of CAI in IS were related to information transduction,protein synthesis and material transport.KEGG enrichment analysis showed that CAI intervention was closely related to 20 signaling pathways,including TNF signaling pathway,IL-17 signaling pathway,HIF-1 signaling pathway,PI3K-Akt signaling pathway,Toll-like receptor signaling pathway,and JAK-STAT signaling pathway,and the "component-target-pathway,network diagram was constructed.Molecular docking experiments showed that ferulic acid,neochlorogenic acid,cryptochlorogenic acid and caffeic acid had the best binding effect with MMP9,hydroxyl safflower yellow A had the best binding effect with MTOR,and chlorogenic acid had the best binding effect with MAPK8.4.mNSS results showed that compared with model group,the neurological function in CAI group was significantly improved(P<0.01),TTC staining results showed that compared with model group,the cerebral infarct volume of CAI group was significantly decreased(P<0.01).The results of HE staining showed that the pathological morphology of brain tissue was improved in CAI group compared with model group.5.Western blot results showed that compared with the model group,the expression of IL17A and IL-17RA in CAI group was significantly decreased(P<0.05,P<0.01),while the expression of TRAF6 was not significantly different(P>0.05).Immunohistochemical results showed that the expression of IL-17RA in brain tissue in CAI group was significantly decreased(P<0.01).Elisa results showed that the expression of CXCL1 in CAI group was significantly decreased compared with model group(P<0.01).Immunohistochemistry and Western blot results showed that the expression of CXCR2 in brain tissue of rats in CAI group was significantly decreased compared with model group(P<0.05).Conclusion:CAI is a potential medicine of IS,which can intervene the progression of IS through multiple targets and pathways,including anti-apoptosis,anti-inflammation and protecting the integrity of the blood-brain barrier.CAI has a neuroprotective effect which may be related to regulating the IL-17 signaling pathway.