Study On The Pharmacodynamic Material Basis And Mechanism Of Tongxie Yaofang In The Treatment Of Ulcerative Colitis

Objective:The aim of this study was to establish a comprehensive and systematic analysis method for the identification of chemical components in the decoction of Tongxie Yaofang(TXYF)and its components into blood,and to reveal the pharmacodynamic material basis and molecular mechanism of TXYF in the treatment of UC through network pharmacology and molecular docking,combined with HT-29 cell inflammation model induced by LPS.Methods:1.Medicated serum preparation: the SPF 30 male SD rats were randomly divided into 3 groups,namely,YXDZ group,TXYF group,the blank group.The YXDZ group was given metharazine solution intragastric administration,the TXYF group was given TXYF decoction intragastric administration,and the blank group was given equal volume of distilled water intragastric administration,once a day for 7 consecutive days.Blood was collected from abdominal aorta to obtain medicated serum of rats for use.2.In vitro and in vivo chemical composition analysis: UPLC-LTQ-Orbitrap-MS technology was used to establish the analysis method for TXYF decoction samples and medicated serum samples of rats after oral administration of TXYF decoction,and to identify the chemical components in TXYF decoction and the prototype components in serum after its administration into blood.3.Network pharmacology and molecular docking: To obtain the TXYF prototype components migrating to blood targets using TCMSP and Swiss Target Prediction Database;Ulcerative colitis was searched through Genecard,Drugbank and OMIM databases to predict disease targets.The intersection of blood prototype component targets and disease targets was selected,and the obtained potential targets were analyzed using STRING database for PPI network analysis.GO functional enrichment analysis and KEGG pathway mapping were performed for potential targets.The network diagram of "constituents migrating to blood-target-pathway" was constructed using Cytoscape3.9.1 software.Auto Dock Tools 1.5.7 and Pymol software were used to verify the molecular docking between key components of TXYF and core targets.4.Cell experiment verification: The prepared rat medicated serum was used to interfere with the LPS-induced HT-29 cell inflammation model,and the cell supernatant and cells of each group were collected for detection after 24 h of intervention.The contents of TNF-α,IL6 and IL1β in the supernatent of each group were detected by ELISA,and the expressions of p38 MAPK,p-p38 MAPK and p65(cytoplasm and nucleus)in the cells of each group were detected by WB.Results:1.Composition identification: A total of 90 compounds were identified from TXYF decoction under positive and negative ion modes,mainly flavonoids,coumarins,monoterpenoid glycosides,chromones and lactones.In addition,the sources of Chinese medicines for all compounds identified in Tongxie Yaofang were assigned.Among them,9 compounds were from Atractylodis Macrocephalae Rhizoma,21 compounds were from Paeoniae Radix Alba,24 compounds were from Citri Reticulatae Pericarpium,29 compounds were from Saposhnikoviae Radix,and 7 compounds were from at least two Chinese medicines.On this basis,12 prototype compounds were identified from TXYF medicated serum,which were paeoniflorin,albiflorin,cimifugin,salicylic acid,hesperetin,5-O-methylvisammioside,isosinensetin,atractylenolide Ⅲ,3,3’,4’,5,6,7,8-heptamethoxyflavone,tangeretin,atractylenolide Ⅰ and nobiletin.2.Network pharmacology and molecular docking results: A total of 231 blood component targets and 840 UC-related targets were obtained through the database.PPI analysis screened out 10 core targets including TNF,IL6,IL1β and VEGFA,and the core targets were overwhelmingly related to inflammation.GO enrichment analysis and KEGG pathway mapping indicated that the therapeutic effect of TXYF on UC mainly involved the regulation of inflammatory response,response to oxidative stress,inflammatory response and other biological processes,as well as AGE-RAGE,TNF,IL-17,MAPK,EGFR and other signaling pathways.6 key components,such as atractylenolide Ⅰ,albiflorin and nobiletin,were screened out from the constructed "constituents migrating to blood-target-pathway" network diagram.Molecular docking results suggested that the key components of TXYF were closely bound to the core targets,among which atractylenolide Ⅰ and nobiletin may be the effective components of TXYF in the treatment of UC.3.Content changes of inflammatory factors in cell supernatant: Compared with the normal group,the contents of TNF-α,IL6 and IL1β were significantly increased in the LPS-induced model group(P < 0.0001 or P < 0.001),and after the intervention of10% medicated serum,the contents of IL6 were not significantly decreased(but also decreased)in the 10% medicated serum compared with the model group.The contents of above inflammatory factors were significantly decreased(P < 0.05 or P < 0.01).4.Changes of protein expression of p38MAPK/NF-κB signaling pathway in cells:Compared with the normal group,the expression of p38 MAPK and p-p38 MAPK in HT-29 cells was significantly increased(P < 0.001),the nuclear shift of p65 protein was also significantly increased(P < 0.0001),but the expression of p65 protein in the cytoplasm was significantly decreased(P < 0.0001).After the intervention of 10%medicated serum,compared with model group,the expression of p38 MAPK and pp38 MAPK protein was significantly decreased(P < 0.01 or P < 0.05),and the nuclear shift of p65 protein was also significantly decreased(P < 0.01).However,the expression of p65 protein in the cytoplasm was significantly increased(P < 0.0001).Conclusion:An analytical method for rapid,comprehensive and systematic identification of the chemical components in TXYF decoction and its components into blood was established.The mechanism of action and active components of TXYF in the treatment of UC were predicted by means of serum pharmacochemistry combination network pharmacology and molecular docking.The potential molecular mechanism of TXYF treatment of UC may be the inhibition of p38MAPK/NF-κB signaling pathway.This study provides certain theoretical basis for the pharmacodynamic material basis,mechanism of action and clinical application of TXYF in the treatment of UC,and provides reference for further development of potential drugs for the treatment of UC.