Exploration On The Immunological Mechanism Of Huangqin Decoction Regulating The Balance Of Th17/Treg And Th1/Th2 Cells In UC Model Mice Based On Network Pharmacology

1 Background and objectiveUlcerative colitis(UC)is a recurrent chronic inflammatory bowel disease,and its etiology has not been fully elucidated.Huangqin Tang(HQT)is widely used clinically in the treatment of UC.Compared with chemical drugs,its composition is complex and can play a role in the overall treatment of UC with multiple components,multiple targets and multiple pathways.Network pharmacology combined with molecular docking technology can achieve efficient and rapid screening of active ingredients in the research of traditional Chinese medicine compound prescriptions,build a biological system network,and study the intervention of drugs on diseases from a holistic perspective.This study first screened the potential active compounds and related targets of HQT through network pharmacology combined with molecular docking technology,and explored the mechanism of HQT in the treatment of UC.Furthermore,through the UC mice model,and the use of Flow Cytometry,Immunohistochemistry,Enzyme-Linked Immunosorbent Assay(ELISA)and other test methods to verify the Th17/Treg,Th1/Th2 cell balance immune mechanism closely related to the pathogenesis of UC obtained in the preliminary network pharmacology research.In order to combine the previous research,a more comprehensive clarification of the mechanism of HQT treatment of UC.2 Research methods and results2.1 Explore the potential mechanism of HQT in the treatment of UC based on network pharmacology and molecular docking technologyDownload the GSE53306,GSE36807,and GSE24287 data sets from the GEO database to calculate and analyze the differential genes and merge the genes from the UC-related disease database;The chemical components and targets of the four traditional Chinese medicines in the HQT are discovered.Cytoscape_v3.7.1 software was used to construct the molecular network of HQT for the treatment of UC,and the core components of HQT were screened according to the topological parameters;based on the STRING database,the PPI network of HQT for the treatment of UC was constructed,and the core targets of HQT for the treatment of UC were screened according to the topological parameters.The core target and the core components obtained above are molecularly docked,and the binding mode and affinity between the small molecule and the target protein are analyzed.Use R_v3.5.2 and Cytoscape_v3.7.1 software to perform GO and KEGG enrichment analysis on the intersection of disease and drug.After deduplication of the genes from the disease database and the differential genes of the GEO database,a total of 3569 UC disease targets were collected,and 138 HQT active ingredients were retrieved and 324 targets were intersected by HQT active ingredients and UC.The active ingredients were screened out including 18 nuclear ingredients including Quercetin,Liquiritigenin,Wogonin,Baicalein,Baicalin and 15 core targets including IL6,TNF,IL4,IL10,IL1B,18 active compounds and protein targets IL6,TNF,IL4,IL10,IL1B,IL17A,IL2,FOXP3 have strong white affinity.The enrichment of P<0.05 obtained by GO enrichment is 1654,104,102 in BP,CC,and MF,respectively,with a total of 1856 items.The biological process is mainly related to the response to oxidative stress and to the molecular origin of bacteria.The reaction is related to the reaction to lipopolysaccharide,and the molecular function is mainly related to the oxidoreductase activity and protein phosphatase binding.83 KEGG signaling pathways with P<0.05 were obtained through the Clugo plug-in enrichment,which mainly involved Th17,Thl and Th2 cell differentiation,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,JAK-STAT signaling pathway,etc.Related signals and other pathways.2.2 The effect of HQT on the balance of Th1/Th2 and Th17/Treg cells in UC mice induced by DSSDrinking 2.5%DSS freely for 5 days induces UC mice model;Flow Cytometry measures the ratio of Th17/Treg cells and Th1/Th2 cells in mice mesenteric lymph nodes(MLNs);CBA and ELISA methods measure IL-4,,IFN-γ,IL-2,IL-6,IL-17A,IL-10,TNF-α,TGF-β in colon tissue;Immunohistochemical method to detect IFN-γ,IL-4,IL-17A,Foxp3 in mice Expression in colon tissue.The results show that HQT can increase the Thl and Treg ratio in UC mice MLNs and the content of related cytokines in mice colon tissue,reduce the Th2 and Th17 ratio in UC mice MLNs and the content of related cytokines in mice colon tissue,and reduce Thl7/Treg,Thl/Th2 cell ratio.In addition,HQT can inhibit the expression of pro-inflammatory factors IL-2,IL-6,TNF-α,IFN-γ,and IL-17A,and increase the expression of anti-inflammatory factors TGF-β,IL-4,and IL-10.3 ConclusionBased on the methods of network pharmacology and molecular docking technology,the 18 active ingredients of HQT for the treatment of UC were screened out,regulating IL6,TNF,IL4,IL10,IL1B,IL17A,IL2,FOXP3 and other target proteins,and regulating IL-6,TNF-α,IL-4,IL-10,IL-1β,IL-17A,IL-2,IFN-y and other cytokines affect the differentiation of Th1,Th2,Th17,Treg cells,and then regulate Th17/Treg cells,Th1/Th2 cells Balance,inhibit intestinal immune abnormalities,stabilize the intestinal environment,regulate the release levels of pro-inflammatory cytokines and anti-inflammatory cytokines to play a therapeutic role in UC.At the same time,it can also inhibit the activation of Jak-STAT signaling pathway,Toll-like receptor signaling pathway,and NOD-Like receptor signaling pathway,etc.Regulate the differentiation of Th1,Th2,Th17,Treg cells,regulate the intestinal mucosal immunity,protect the intestinal microbiota,reduce the damage of the intestinal barrier,and play a role in the treatment of UC.Using in vivo animal experiments,low Cytometry,Immunohistochemistry,ELISA,it was found that HQT can regulate the imbalance of Th1/Th2 and Th17/Treg cells in UC mice,regulate the abnormal intestinal immunity of UC mice,and stabilize the intestinal environment.HQT can also inhibit the expression of pro-inflammatory factors IL-2,IL-6,TNF-α,IFN-γ,IL-17A,and increase the expression of anti-inflammatory factors IL-4,IL-10,TGF-β to regulate The release level of inflammatory cytokines and anti-inflammatory cytokines,thereby reducing the occurrence of intestinal inflammation,and then exerting a therapeutic effect on UC mice.The mechanism of HQT in the treatment of UC may be related to the restoration of Th1/Th2,Th17/Treg cell balance and related cytokine levels.