To Explore The Mechanism Of "Gualou-xiebai" On Phlegm And Blood Stasis-related Cardiovascular Pharmacology And Metabonomics Diseases Based On Network

Objective:This study aimed to investigate the mechanism of“Trichosanthis Fructus-Allii Macrostemonis Bulbus"(GX)on phlegm and blood stasis syndrome rats combining the methods of metabolomics and network pharmacology.Method:HPLC-Q-TOF-MS method and literatures were used to collect the active ingredients of GX.The SwissTargetPrediction platform,Similarity ensemble approach(SEA),Therapeutic Target Database(TTD)databases were used to predict potential targets of GX and were integrated with the predicted targets for the treatment of phlegam and blood stasis syndrome.A protein-protein interaction network model was constructed by using String database and Cytoscape 3.8.0 software;the DAVID 6.8 platform was used for Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Then we constructed network of drug components-targets-pathways by Cytoscape 3.8.0 software and used to dock the molecules of GX with the key disease targets by PyMOL software.The prediction targets were validated in high-fat diet combined with ice-water bath and adrenaline(0.8 g·kg-1)hydrochloride subcutaneous injection induced phlegam and blood stasis syndrome rats with administration of GX(2,4,8 mL·kg-1)for 14 days.Then combined phlegam and blood stasis syndrome was scored based on traditional Chinese medicine(TCM)syndrome scoring scale;H&E staining was used to observe the pathological of aorta tissue;automatic hemorheometer was used to measure the blood viscosity and erythrocyte aggregation index;serum levels of TG,TC,LDL-C and HDL-C were determined by enzyme method and the expressions of ICAM-1 and VCAM-1 in aorta tissue were detected by immunohistochemistry.Western blot was used to measure the expression of proteins involved in PI3K,Akt,p-PI3K and p-Akt in aorta tissue.UPLC-Q-TOF-MS non-targeted metabolomics method was used to study the mechanism of action of GX in the treatment of phlegm blood stasis-cardiovascular diseases,and the serum metabolic profile of the rat model of GX in the treatment of phlegm and blood stasis syndrome was characterized.Combined with QI platform,the serum metabolites in control group,model group and GX group were analyzed by multivariate statistical analysis to screen the differential metabolites.The metaboanalyst software package was used to analyze the pathways involved in differential metabolites,and the"Target-biological pathway-metabolic pathway-metabolite" network was constructed with the combination of network pharmacology techniques.Results:1:Based on the HPLC-Q-TOF-MS analysis and database,69 chemical constituents of GX and 163 targets of GX for the treatment of phlegm and blood stasis-related cardiovascular diseases were obtained.Then,key targets such as serine/threonine kinase 1(Aktl),tumor necrosis factor(TNF),interleukin 6(IL6),vascular endothelial growth factor A(VEGFA),cellular tumor antigen p53(Tp53)were screened.Pathway analysis showed that the targets of GX in the treatment of phlegm and blood stasis-relate cardiovascular diseases were mainly involved in PI3K/Akt signaling pathway,sphingolipid metabolism,platelet activation,HIF-1,Rap 1 and other signaling pathways.In addition,molecular docking analysis showed that apigenin,cucurbitacin D,linolenic acid and kaempferol and other key components had potential binding ability with Aktl,TNF,IL6,VEGFA and Tp53.2:In the animal experiments,compared to the phlegm and blood stasis syndrome group,GX could significantly improve the traditional Chinese medicine syndrome score,blood lipid,vascular endothelial structure disorders and reduce serum ET-1 level,increase serum NO and eNOS levels,which could restore aortic endothelial function.In addition,the expression of adhesion factors ICAM-1 and VCAM-1 in aorta could be significantly reduced,whicn could improve the vascular endothelial injury of aorta.Western blot revealed that GX could significantly decrease the phosphorylation levels of PI3K and Akt in aorta.3:Metabolomics detected 129 kinds of potential biomarkers in rats with phlegm-stasis interjunction syndrome,and GX could regulate 54 kinds of metabolites in linoleic acid metabolism,sphingoid metabolism,tricarboxylic acid cycle and other metabolic pathways.Network pharmacological screening of the above 54 metabolites revealed that 9 metabolites of phlegm-stasis internode associated with cardiovascular disease in GX callbacks were associated with 69 targets,involving biological pathways such as cholesterol metabolism,fatty acid metabolism,lipopolysaccharide response,inflammatory response,glucose homeostasis and metabolism.These results indicated that GX could improve cholesterol metabolism,fatty acid metabolism,lipopolysaccharide response,inflammatory response,glucose homeostasis and metabolism by interfering with the metabolism of linoleic acid,sphingosine,docosahexaenoic acid,rosemary acid,succinic acid,adenine,L-phenylalanine,L-valine and other biological pathways in rats with phlegm-stasis interjunction syndrome.Play the role of treating phlegm-stasis interjunction cardiovascular disease.Conclusion:This study also clarified that the reversal of pathological of phlegm and blood stasis syndrome rats may be related to GX inhibiting PI3K/AKT signaling pathway,which could improve vascular inflammation and vascular endothelial function injury.GX regulates the expression of associated targets such as Aktl,TNF,IL6 and VEGFA by interfering with the metabolism of linoleic acid,sphingosine,docosahexaenoic acid,rosemary acid,succinic acid,adenine,L-phenylalanine and L-valine in serum of phlegm-stasis interjunction rat model.Synergistic improvement of cholesterol metabolism,fatty acid metabolism,lipopolysaccharide response,inflammatory response,glucose homeostasis and metabolism and other biological pathways,play a role in the treatment of phlegm-stasis interjunction cardiovascular diseases.