| ObjectiveOn the basis of previous research,the mechanism of action of Astragalus-patrinia drug pair on repairing intestinal mucosal barrier of ulcerative colitis(UC)was studied from three aspects of network pharmacology,in vivo and in vitro experiments,in order to develop effective targeted drugs for UC.Method1.Network pharmacology research:Firstly,the Chinese Medicine System Pharmacology Analysis Platform(TCMSP)was searched to obtain the active ingredients and related targets of the Astragalus-patrinia drug pair,and then the Gene Cards database and the OMIM database were used to obtain the targets of ulcerative colitis.The intersection target of the two was obtained by drawing Venn diagram,which could be used as the potential target of Astragalus-patrinia drug pair for the treatment of UC.The intersection targets were uploaded to the STRING v11.0database,and a protein-protein interaction(PPI)network map was constructed.The Metascape database was used to conduct gene biological process(GO)enrichment analysis and signaling pathway(KEGG)enrichment analysis on the intersection targets,and Autodock vina 1.1.2 was run for molecular docking.The above methods were used to screen the signal pathways and possible targets of Astragalus-patrinia drug pair in the treatment of UC.2.In vivo experimental research:60 C57BL/6 male mice were randomly divided into 6 groups and drank 2.5% DSS solution freely for 7 days to establish a UC mouse model.On the 3rd day of drinking DSS solution,the mice were given intragastrically.After 8 days of administration,the mice were sacrificed.The changes of mouse body weight,disease activity index,colon length and spleen index were observed and evaluated;the pathological changes of colon tissue were observed by HE and PAS staining;the contents of serum inflammatory factors were detected by ELISA and q RT-PCR;q RT-PCR and WB were used to detect the expression of genes and proteins related to Occludin and Claudin-1;q RT-PCR,WB and immunohistochemistry were used to detect JAK1/STAT6/SOCS1 pathway gene and protein expression;the correlation of Occludin and Claudin-1 gene expression with JAK1,STAT6,SOCS1 gene expression was analyzed.and the mechanism of Astragalus-patrinia drug pair on repairing the intestinal mucosal barrier in UC mice was explored.3.In vitro experimental research:Combined with previous studies,LPS 20ug/m L intervention of HT-29 cells for 24 h induced the establishment of inflammatory cell model.After 24 h intervention of different concentrations of Astragalus membranaceus herb,cell viability was detected by CCK-8method,and the optimal intervention concentration of Astragalus-patrinia drug pair was screened.The concentration was divided into low,medium and high dose groups.After 24 hours of intervention in LPS-induced HT-29 cells,ELISA and q RT-PCR were used to detect inflammatory factors IL-6,IL-1β,IL-18,TNF-α and IL-13 expression,WB method was used to detect Occludin and Claudin-1 protein expression,q RT-PCR and WB technology were used to detect JAK1/STAT6/SOCS1 m RNA and protein expression after intervention of Astragalus-patrinia drug pair,and the effect of Astragalus-patrinia drug pair on LPS was observed.To observe the protective effect of Astragalus membranaceus herb on LPS-induced HT-29 inflammatory cell model,and to further explain the mechanism of Astragalus-patrinia drug pair on repairing intestinal mucosal barrier of UC.Result1.Network pharmacology results:Quercetin,luteolin and kaempferol are the key components in the network,and AKT1,TP53,VEGFA,etc are the key targets in the treatment of ulcerative colitis.The enrichment analysis involves the response to inorganic substances,the response to lipopolysaccharide,the apoptosis signaling pathway,the response of cells to organic cyclic compounds,etc.;the KEGG pathway enrichment analysis results include the JAK-STAT signaling pathway involved in the previous study.Molecular docking results showed that AKT1,CASP3,JUN,IL1 B,PTGS2 had good binding ability with quercetin,luteolin and kaempferol,and TP53,VEGFA,MYC could be combined with quercetin,luteolin and kaempferol.The signaling pathway proteins JAK1 and SOCS1 have good binding ability with quercetin,luteolin and kaempferol,and STAT6 can be combined with quercetin,luteolin and kaempferol.2.In vivo test results:The UC mouse model was successfully replicated in vivo,and the treatment of Astragalus-patrinia drug pair could effectively reduce the DAI score,weight loss,colon shortening,spleen index,etc.In addition,it can significantly down-regulate the overexpression of DSS-induced inflammatory factors IL-6,IL-1β,IL-18,and TNF-α(P<0.05-P<0.0001),and up-regulate the anti-inflammatory factors IL-13 and Occludin,Claudin-1 expression(P<0.05-P<0.0001),significantly decreased the gene expression of JAK1 and STAT6(P<0.05,P<0.01,P<0.0001),down-regulated p-JAK1 and P-STAT6 protein expressions(P<0.05-P<0.001),and up-regulated SOCS1 gene and protein expressions(P<0.05-P<0.0001)in a dose-dependent manner.The results also showed that Occludin and Claudin-1 were negatively correlated with JAK1 and STAT6,and positively correlated with SOCS1.Occludin and Claudin-1 were correlated with the gene expression of JAK1,STAT6 and SOCS1(P>0.05).3.In vitro test results:Follow-up experiments were conducted with 2.25mg/ml,4.5mg/ml and 9mg/ml of Astragalus-patrinia drug pair as low,medium and high intervention concentrations.The results showed that: The expressions of IL-6,IL-1β,IL-18 and TNF-α were decreased in medium-dose,high-dose and mesalamine groups,while the secretion of IL-13 was increased(P< 0.05-P< 0.0001).The expression of Occludin and Claudin-1 was up-regulated in high dose group and mesalamine group(P<0.01-P<0.0001).JAK1 and STAT6 gene expression were down-regulated in the high dose group and mesalazine group,SOCS1 gene expression was up-regulated(P<0.05-P<0.0001),P-JAK1 and P-STAT6 protein expression was down-regulated,SOCS1 protein expression was up-regulated(P<0.05-P<0.0001).There was no statistical difference between them(P>0.05).Conclusion1.The network pharmacology KEGG pathway enrichment analysis found that the Astragalus-patrinia drug pair is mainly involved in the JAK-STAT pathway and other signaling pathways.The molecular docking results showed that the main components quercetin,luteolin,kaempferol and pathway proteins JAK1,SOCS1,STAT6 has a good binding ability,suggesting that Astragalus-patrinia drug pair may target the treatment of UC by regulating the JAK1/STAT6/SOCS1 pathway.2.The gene expression levels of Occludin and Claudin-1are correlated with JAK1,STAT6 and SOCS1,suggesting that the expression of Occludin and Claudin-1 can be regulated through the JAK1/STAT6/SOCS1 pathway,which may be a new target for repairing the intestinal mucosal barrier in UC point.3.Astragalus-patrinia drug pair has a significant protective effect on DSS-induced UC mouse model and LPS-induced HT-29 cells,which may up-regulate the expressions of Occludin,Claudin-1 and IL-13,down-regulate the expression of pro-inflammatory factors IL-6,IL-1β,IL-18,and TNF-αthrough the JAK1/STAT6/SOCS1 pathway.so as to repair the intestinal mucosal barrier of UC and play a protective role. |
PDF Full Text Request