Study On Pharmacodynamic Material Basis And Mechanism Of Qizhiweitong Granules In Treating Chronic Non-Atrophic Gastritis

The formulation of Qizhiweitong granules(QZWT)is derived from the Sinisan in Shang Han Za Bing Lun by Zhang Zhongjing.It is a commonly used traditional Chinese medicine(TCM)compound preparation for the treatment of chronic non-atrophic gastritis(CNG).Due to the complex composition of QZWT,the mechanism of treating CNG has not been fully elucidated.However,it is difficult for traditional pharmacological research methods to reflect the integral action of TCM compounds and their interaction with the body.The research methods of metabolomics and network pharmacology are consistent with the whole concept of TCM and conform to the characteristics of multi-component,multi-target and multi-pathway action of TCM compounds,so they are particularly suitable for the study of the mechanism of action of complex TCM systems.Objective: Taking QZWT in the treatment of chronic non-atrophic gastritis as the research object,high resolution mass spectrometry combined with molecular network visualization analysis was used to mine the chemical composition information in depth,and the data of metabolomics and network pharmacology among different species were analyzed to explore the main active components and metabolic pathways,and then molecular docking technology and Enzyme-linked immunosorbent assay(ELISA)methods were used to verify the material basis and mechanism of action.Methods:1.Using UHPLC-Q-TOF-MS/MS technology and non-targeted metabonomics method,based on the mass spectrum cracking laws of seven representative Bupleurum pentacyclic triterpenoid saponins,the chemical components of Bupleurum chinense in QZWT were qualitatively analyzed by matching with Peak View software and self-built database.Progenesis QI and multivariate statistical analysis were used to process the data and screen the chemical markers of Bupleurum chinense DC.(BC)and Bupleurum scorzonerifolium Willd(BS).2.QZWT were qualitatively analyzed by UHPLC-Q-TOF-MS/MS method.The obtained mass spectrum data were uploaded to the Global Social Molecular Network of Natural Products(GNPS)for spectral library search and molecular network analysis.Various chemical components in QZWT were identified and the compound database were constructed.3.Combined with the compound database of QZWT,the components with OB > 30% and high IG as active components were screened in Swiss ADME,and other chemical components were searched and supplemented by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)database,literature mining and other methods to screen active compound targets.Disease-related targets were collected from Gene Cards,OMIM,Disgenet and Drugbank databases and corrected by Uni Prot database to obtain disease-related targets.Cytoscape and STRING software were used to construct component-target network and protein interaction(PPI)network to find the core target.The intersection targets of "active ingredient-disease" were imported into Metascape database for pathway annotation and enrichment analysis,and visual mapping was made using bioinformatics online platform to predict the mechanism of QZWT in the treatment of chronic gastritis.4.The rat model of CNG was established by hunger and satiety disorder combined with 0.1% free drinking of ammonia.The model was verified and the efficacy of QZWT was evaluated by histopathological section staining and biochemical index detection.The plasma and gastric tissue samples of rats were collected,Progenesis QI and SIMCA-P 14.1 software were used for data processing and analysis.The differential metabolites were screened under the condition of VIP>1,P<0.05,FC≥1.2 or ≤0.8.Bioaccumulation and metabolic pathway analysis were carried out by using online software Metabo Analyst 5.0 and online database Kyoto Encyclopedia of Genes and Genomes(KEGG),and the correlation between metabonomics and network pharmacology was analyzed by using the "Joint-Pathway Analysis" function module of Metabo Analyst 5.0.5.According to the set exclusion criteria,qualified patients with CNG and healthy volunteers were recruited,and the plasma,urine and saliva samples were collected according to the experimental groups.The data were collected by UHPLC-Q-TOF-MS/MS technology,and the data were processed and analyzed by Progenesis QI and SIMCA-P 14.1 software.The differential metabolites of each group were screened and identified,the metabolites enrichment analysis and metabolic pathway analysis of potential biomarkers were carried out.Metabo Analyst 5.0 was used to analyze the relationship between metabonomics and network pharmacology,and the bioinformatics network and protein interaction network map of metabolite-gene-protein were constructed.6.The levels of arachidonic acid(AA),phosphatidic acid(PA)and lysophosphatidic acid(LPA)in plasma and gastric tissue of rats were determined by competitive ELISA,and the levels of prostaglandinendoperoxide synthase 1(PTGS1)and prostaglandin-endoperoxide synthase 2(PTGS2)in rats were determined by double antibody sandwich method.The core active components kaempferol,quercetin,glycyrrhizin A and tangerine were selected as molecular docking ligands and docked with the selected core target proteins(PTGS1,PTGS2 and LPA2)to verify the pharmacodynamic material basis and mechanism of QZWT in the treatment of CNG.Results: 1.A total of 131 chemical components were identified in BC and BS,among which 35 were reported for the first time and 11 could be used as chemical markers to distinguish the two BR herbs.2.104 compounds were identified successfully in QZWT,including 36 flavonoids,20 alkaloids,12 triterpenoids,10 monoterpenoid glycosides,10 phenols,8 coumarins and 8glycosides.3.194 active chemical constituents were obtained,of which the main active components were kaempferol,quercetin,glycyrrhizin A and tangerine.There were 267 chronic gastritis related targets,and 112 drug and disease targets.PPI network showed that potential core targets were NPM1,HNRNPK,HSPA5,YWHAZ and HNRNPA1,respectively.4.In the analysis of plasma samples,14 different metabolites were found,the main metabolic pathways involved arachidonic acid metabolism,glycerol phospholipid metabolism,steroid hormone biosynthesis,etc.In the analysis of gastric tissue samples,29 different metabolites were found,and their main metabolic pathways involved glycerophospholipid metabolism,sphingolipid metabolism and arachidonic acid metabolism.Compared with plasma samples,metabolites in gastric tissue samples were more significantly expressed.Through the association analysis of the results of network pharmacology and metabolomics,it was found that the different metabolites in the two samples were mainly involved in arachidonic acid metabolism and glycerophospholipid metabolism.5.In the analysis of plasma samples of patients,75 different metabolites were found,and their main metabolic pathways involved sphingolipid metabolism,glycerolphospholipid metabolism,Linoleic acid metabolism and arachidonic acid metabolism.In the analysis of urine samples,71 different metabolites were found,and their main metabolic pathways involved sphingolipid metabolism,Arginine metabolism and tyrosine metabolism.In the analysis of saliva samples,32 different metabolites were found,and their main metabolic pathways involved sphingolipid metabolism and glycerolphospholipid metabolism.Through the association analysis between network pharmacology and metabolomics results of CNG patients,it was found that the main metabolic pathways of different metabolites in the three samples involved arachidonic acid metabolism,linoleic acid metabolism and arginine metabolism.A comprehensive comparison of human and rat metabonomic studies showed that the main metabolic pathways of QZWT in the treatment of CNG were mainly related to arachidonic acid metabolism and glycerolphospholipid metabolism.6.In rat plasma and gastric tissue samples,the levels of AA,PTGS1,PTGS2,PA and LPA in the model(CNG)group were significantly higher than those in normal control(NC)group(P<0.01),while the levels of each index in the QZWT group were significantly lower than those in CNG group(P<0.05).The main active substances of QZWT,such as quercetin,kaempferol,lycyrrhizin A and tangerine,have strong binding activity with core targets,such as PTGS1,PTGS2 and LPA2.Conclusion: In this study,by integrating molecular network technology,network pharmacology,non-targeted metabolomics,ELISA detection and molecular docking technology,and using a variety of statistical analysis methods,the pharmacodynamic material basis and mechanism of QZWT in the treatment of CNG were studied,and provided a scientific basis for scientific basis for clinical use of QZWT.At the same time,it provides data support for the study of differences among different species.This study provides a valuable example reference and methodological reference for the systematic study of the action mechanism of multi-components,multi-targets and multi-pathways of traditional Chinese medicine compound preparation.