Based On Network Pharmacology To Study The Mechanism Of Refined Qingkailing Injection Against Ischemic Stroke

Stroke is the main cause of death and disability.Because of its high morbidity,high disability rate and high mortality rate,it has become one of the common problems faced by the public health fields in various countries.Qingkailing injection is an important traditional Chinese medicine injection for the treatment of ischemic stroke.Based on Qingkailing injection,the team developed the baicalin and geniposide through the screening and optimization of the prescription.A group of traditional Chinese medicines consisting of bile acid and hyodeoxycholic acid is used to refine Qingkailing injection.In order to gain an in-depth understanding of the pharmacological mechanisms of refined Qingkailing injection against ischemic stroke at the system level,we designed this study using network pharmacology and pharmacological experiments.This study firstly used network pharmacology to integrate multiple online databases to predict the target information of four components of refined Qingkailing injection,construct a refined Qingkailing injection component-target interaction network,and analyze the target using the cytoscape software module.Topological parameters in the network,analysis of target enrichment in the tissue using RichFun software,and analysis of biological processes and molecular functions involved in the target using the David online database.Secondly,the online database was used to retrieve the target of ischemic stroke and the Venn diagram was used to obtain the target of refined Qingkailing against ischemic stroke.The AlzData database was used to analyze the high expression of these targets in different cells of the brain,and to establish a four-component refining Qingkailing injection-anti-ischemic stroke target-high expression cell type interaction network.The David database was used to enrich the biological expression of neurons,endothelial cells,astrocytes and microglia,respectively,to explain the regulation of refined Qingkailing injection on different cell types..Third,in order to study the potential biological mechanism of refined Qingkailing injection against ischemic stroke at the level of protein-protein interaction,we used Cytoscape software to construct refined Qingkailing injection target and anti-ischemic brain,respectively.The target of stroke is the two proteinprotein interaction networks of seeds,and the two are intersected and merged and the plug-in is used to screen the network to obtain the core protein-protein interaction network.KEGG enrichment analysis was performed on the core protein-protein interaction network,and molecular docking software was used to molecularly dock the four components with important proteins on the signaling pathway.In order to verify some of the results of network pharmacology,we used the suture method to prepare rat cerebral ischemia-reperfusion model,and studied the refinement of Qingkailing injection on the neurological function score,cerebral infarction volume and blood-brain barrier of the model rats.The effect of rate,brain water content,etc.In addition,we also studied the effects of refined Qingkailing injection on apoptosis and the effects of apoptosis-related proteins,PI3K/Akt signaling pathway-related proteins,inflammatory factors,and NLRP3 signaling pathway-related proteins.We also used laser confocal 3D reconstruction techniques to study the effects of refined Qingkailing injection on the volume and surface area of astrocytes and microglia.The results of the study showed that 312 pharmacological studies yielded 312 targets for ischemic stroke and 430 targets for refined Qingkailing injection.The refined Qingkailing injection target is significantly enriched in human umbilical vein endothelial cells,blood vessels,cerebellum,nervous system and other tissues and cells,and may be involved in hypoxia response,inflammatory reaction,negative regulation of cell death and calcium channel activity.The learning process indicates the potential therapeutic effect of refined Qingkailing injection on cerebrovascular disease.The target of refined Qingkailing injection was combined with the target of ischemic stroke to obtain 47 overlapping targets,which have different high expression in different cells in the brain: neuron cells(31),star Glial cells(14),endothelial cells(13),microglia(11),less synaptic glial cells(11)and oligodendrocyte precursor cells(11).Analysis of the biological processes involved in the targeting of these targets in neurons,endothelial cells,astrocytes,and microglia revealed that refined Qingkailing injection affects neuronal learning or memory,chemical synaptic transmission,and peroxidation.Hydrogen reaction,hypoxia response,calcium ion transmembrane transport,negative regulation of apoptosis,glutamate receptor signaling pathway,leukocyte-cell adhesion and other biological processes affect endothelial cell response to hypoxia,Positive response to VEGF stimulation,angiogenesis,positive regulation of proliferation,positive regulation of cell migration involved in germination angiogenesis,chemokine production,inflammatory response,negative regulation of apoptosis process,etc.,affecting astrocytes Hypoxia response,negative regulation of reactive oxygen metabolism,positive regulation of angiogenesis,positive regulation of ERK1 and ERK2 cascades,positive regulation of VEGFR signaling pathway,positive regulation of chemokine production,ionotropic glutamate receptors Signal pathways,positive regulation of B cell proliferation,negative regulation of apoptosis,and other biological processes that affect the positive regulation of microglial chemokine production,and the introduction of NF-kB into the nucleus Regulation,regulation of cytokine secretion,I-kappa B phosphorylation,positive regulation of nitric oxide synthase biosynthesis,production of cytokines,MyD88-dependent Toll-like receptor signaling pathway,inflammatory response,immune response,etc.process.Topological screening of the core protein-protein interaction network yielded a protein-protein interaction network containing 189 nodes and 5754 edges.The KEGG signaling pathway analysis of nodes in the network found that the top 15 signaling pathways associated with ischemic stroke include TLR,TNF,Wnt,NF-kappa B,and apoptosis.NOD-like receptors,TGF-beta,FoxO,Hippo,MAPK,HIF-1,neurotrophic factors,estrogen and PI3K/Akt signaling pathways.Here we focus on the PI3K/Akt signaling pathway and important molecules of the NLRP3 inflammatory body pathway such as AKT,PTEN,TLR4,NF?B,NLRP3 and CASSASE1,and found that the four components of refined Qingkailing injection have a certain binding rate with these proteins.In the experimental part,refined Qingkailing injection can significantly reduce the neurological damage of rats,inhibit the infarct volume of the brain and alleviate the damage of the blood-brain barrier,all of which have a dose-dependent effect,indicating that the refined Qingkailing injection has potential for ischemic stroke.Therapeutic effect.Refined Qingkailing injection can also inhibit the apoptosis of brain cells after ischemic stroke,increase the ratio of anti-apoptotic protein BCL2 and pro-apoptotic protein BAX,and activate the phosphorylation levels of AKT and PI3 K.Refined Qingkailing Injection can also significantly inhibit the volume and surface area of astrocytes and microglia activated by ischemic stroke,and inhibit astrocyte and microglial marker proteins GFAP and IBA-1.The expression promotes the expression of the neuronal marker protein NEUN.We also studied the effect of refined Qingkailing injection on the inflammatory response of ischemic stroke.It was found that refined Qingkailing injection can significantly increase the expression levels of Lgals1,Timp1,Il10,Prlr and Kitlg,and significantly reduce Il1 b,Adipoq.Expression levels of Cxcl2,Ccl2,Icam1,Trem1,F11 r,Gas1,Nrp1,Tnfrsf12 a,Cxcl3,Flt3 lg,Tnf and Il6.These differentially expressed proteins are involved in TNF,cytokine-cytokine receptor interaction,malaria African trypanosomiasis,NOD-like receptors,Legionnaires' disease,NF-?B,Toll-like receptors,Jak-STAT,chemokines and other signaling pathways.Since NLRP3 is an important component of the Nod-like receptor signaling pathway,we next investigated the effect of refined Qingkailing injection on NLRP3 inflammatory signaling pathway.We found that refined Qingkailing injection can significantly down-regulate the levels of NLRP3 and CLIL1 B,and also down-regulate the levels of CL-Caspase1 and ASC protein.In summary,this study used a combination of network pharmacology and animal experiments to visualize the interaction between Qingkailing injection components,component targets,disease targets,and distributed cell types through network pharmacology visualization.Relationships to study the multi-target characteristics of refined Qingkailing injection and related important signaling pathways in biological networks.The experimental results also show that refined Qingkailing injection has clear pharmacological activity against ischemic stroke,and its mechanism may be reflected in multiple levels of regulation,including antiapoptosis,inhibition of astrocytes and small glue.Activation of cytoplasmic cells,inhibition of inflammatory responses,activation of the PI3K/Akt signaling pathway,and inhibition of the NLRP3 signaling pathway.