Study On The Material Basis And Mechanism Of Zhachong Shisanwei Pills In Preventing Chronic Cerebral Ischemia

Objective: Chronic cerebral ischemia is a common underlying pathophysiological mechanism leading to dementia and other neurodegenerative diseases.Zhachong Shisanwei Pills(ZC13)is a classic prescription of Mongolian medicine,which has a significant effect on the treatment of cardiovascular and cerebrovascular diseases.However,the complex ingredients,unknown drug substances and unclear mechanism of action severely limit its development and application.This article takes ZC13 as the research object.We evaluated its therapeutic effect on chronic cerebral ischemia by establishing animal models,and utilized the spectrum effect screening method to determine its bioactive substances,then Explored its possible mechanism of action,so as to provide a certain reference for the regulation of ZC13’s clinical application.Methods: 1.Animal models of chronic cerebral ischemia in rats with permanently ligated bilateral common carotid arteries were established.28 days after administration of ZC13,the experimental rat’s cerebral blood flow was detected with cerebral blood flow meter and laser Doppler imaging system.The kits were used to detected the content of MPO,MCP-1,TNF-α and the activity of SOD in rat’s serum.2.UPLC/Q-TOF-MS technology combined with literature reports were used to analyze and identify the chemical composition of ZC13.The five principles,TPSA and number of rotation keys were regard as a standard to predict the absorbable components.String11.0 and databases of Pharmmapper,Uniprot,KEGG was adopted to predict and analyze the Network pharmacology on the targets and pathways of absorbable components in ZC13.3.UPLC/Q-TOF-MS combined with an NF-κB dual fluorescence reporter gene system and NO content detection were utilized to identify the anti-inflammatory bioactive substances in ZC-13.Network pharmacology and bioinformatics methods were used to predict the main targets and pathways of these anti-inflammatory active ingredients.Finally,we verified the anti-inflammatory pathway of PI3K-AKT-NF-κB with Costunolide.Results: 1.The results showed that compared with the model group,the cerebral blood flow of the rats in ZC13 administration group was significantly increased.In rat’s serum,the expression levels of TNF-α,MCP-1 and MPO were decreased,and the activity of SOD was improved.2.38 chemical components of ZC13 were identified by UPLC/Q-TOF-MS,from which 23 components were predicted to be absorbed by the body such as gallic acid,6-Hydroxy-2-(2-phenylethyl)chromone,Liquiritigenin,Costunolide,Dehydrocostus lactone,Mysristicin and so on.The results of network pharmacological analysis show that the above components may play an anti-inflammatory effect by regulating 27 targets such as CDK6,GSTA3,PDPK1,MMP3,INSR,KDR,GSK3β,EGFR,MMP9 and so on,thereby regulating Signaling pathways such as PI3K-AKT,fluid shear stress and atherosclerosis,IL-17,MAPK and T cell receptor.3.Screening results showed that in ZC13,four kinds of components related to NF-κB inhibition were identified: gallic acid,ellagic acid,liquiritin apioside,glycyrrhizic acid,and four kinds of components related to NO release inhibition were found: gallic acid,liquiritigenin,costunolide,and dehydrocostus lactone.The above components exert anti-inflammatory activities mainly through the regulation of PDK1,MAPK14,GSK3β and other anti-inflammatory-related targets,and further adjust the PI3K-AKT,MAPK,m TOR pathways.Among them,costunolide can inhibit AKT phosphorylation and NF-κB nuclear transfer.Conclusion: ZC13 has a certain improvement effect on chronic cerebral ischemia in rat model,the function may related to it’s components of Gallic acid,ellagic acid,Liquiritin apioside,Glycyrrhizic acid,Liquiritigenin,costunolideand Dehydrocostus lactone which have an anti-inflammatory effect by regulating PI3K-AKT,MAPK,m TOR and other signaling pathways.