Study On The Mechanism Of Huangqin Decoction In Treating Ulcerative Colitis Based On Network Pharmacology And Bioinformatics

Objective:To predict the active ingredients,potential targets and metabolic pathways of Huangqin decoction in the treatment of ulcerative colitis in mice by network pharmacology and bioinformatics GEO chip,and to detect the mechanism of Huangqin decoction in the treatment of ulcerative colitis in mice and its effect on intestinal bacteria by molecular biology techniques and 16srDNA sequencing techniques.Methods:TCMSP database was used to screen the active ingredients and targets of four kinds of Huangqin Decoction.GeneCards,PharmGkb,TTD,DrugBank database and GEO database were used to screen the disease gene information.Wayne diagram was drawn for the targets of diseases and traditional Chinese medicine components and PPI network was constructed.CytoNCA and BisoGenet in R4.0.4 software and Cytoscape 3.8.0 software were used for screening and topological analysis and network construction.GO,KEGG enrichment analysis and KEGG relationship network construction were performed with R language.Finally,the main active ingredients,core targets and metabolic pathways of Huangqin decoction in the treatment of ulcerative colitis were obtained.Dextran sulfate sodium was used to construct a mouse model of ulcerative colitis and the model was evaluated in terms of behavior,body weight change,fecal occult blood test,disease activity index,colonic histomorphological observation and colonic histopathological examination.After the model was established,the mice were randomly divided into blank group,model group,positive group,high dose group of Huangqin decoction,and low dose group of Huangqin decoction.After 21 days of treatment,the colon tissues of mice in each group were collected to observe the morphological lesions of colon tissues,HE staining was used for histopathological examination of colon tissues,immunohistochemistry was used to detect the changes of IL1β,IL6,IL10 and TGFβ cytokine contents in colon tissues,and 16srDNA sequencing technique was used to detect the changes of intestinal bacteria in mice in each group,in order to study the therapeutic effect of Huangqin decoction on ulcerative colitis model mice,the changes of inflammatory factors and the effects on intestinal bacteria.Results:165 active ingredients in Huangqin decoction were obtained by joint search of network pharmacology and bioinformatics,mainly including flavonoids,alkaloids,and terpenoids,with 2836 related targets and 160 intersection targets.The GEO database screening resulted in 910 differential genes,including 575 up-regulated genes and 335 down-regulated genes in the experimental group.The components with the highest number of connected disease nodes obtained by the construction of the "active component-UC target" network were quercetin,wogonin,acacetin,kaempferol,and naringenin,and the PPI network was constructed to obtain key factors such as IL6,IL1β,FOS,and STAT3.GO and KEGG enrichment analysis and KEGG network construction revealed that Huangqin decoction was mainly enriched on signaling pathways such as TNF and IL17 in the treatment of ulcerative colitis,and the core genes were IL6,IL1β,FOS,and JUN.Animal experiments showed that Huangqin decoction could improve the behavioral characteristics such as mental status of model mice,reduce the positive expression of fecal occult blood test,and increase the body weight of mice.The length of colon in the positive drug group and Huangqin decoction treatment group was significantly increased compared with the model group,and the color of colon tissue in the Huangqin decoction medium dose group tended to be normal,and the length was closest to the blank group.HE staining showed that inflammatory cells were significantly reduced and glands were arranged more regularly in the medium dose group of Huangqin decoction compared with the positive drug group and the high dose group of Huangqin decoction.Immunohistochemistry showed that there were significant differences in IL6 and IL10(P<0.01)and IL1β(P<0.01)in the blank model group.Compared with the blank group,the contents of IL1β and IL6 were significantly increased and the content of IL10 was significantly decreased in the model group(P<0.01).Compared with the model group,IL1β was significantly decreased in the high and low dose groups of Huangqin decoction(P<0.05),the IL1β content was the least in the medium dose group of Huangqin decoction,IL6 was significantly down-regulated in the medium dose groups of positive and Huangqin decoction(P<0.01),IL10 was significantly up-regulated in the high and medium dose groups of positive and Huangqin decoction(P<0.01),and the content was the most in the high dose group,followed by the low dose,high dose,and positive drug groups.There was no significant difference in TGFβ content among the groups(P>0.05).2705010 gut bacteria sequences were measured by 16srDNA technique.Species taxonomy showed that the abundance of Bacteroidetes and Proteobacteria in the model group was slightly more than that in the other groups,the abundance of gram negative bacillus was the lowest at the class level,and the abundance of γ-proteobacteria was higher than that in the other groups.After treatment,Alpha diversity Shannon and Simpson index analysis showed that the flora diversity in the model group was significantly lower than that in the blank group,and the flora diversity in the high dose group of Huangqin decoction was closest to that in the blank group and more stable.The rarefaction curve showed that the sequencing depth was enough to study the microbial diversity of each sample,and the distance matrix was significantly different from the community composition of the model group by PCoA analysis,and the high dose group of Huangqin decoction tended to blank group flora relatively.The analysis of differential bacteria showed that there were many Bacteroides species in the model group,the content of Lactobacillus species was more in each treatment group,and the abundance of Phylum Firmicutes was the most in the positive drug group,and there were differences between the model group and other groups in Vibrio,Erysiphaceae,Clostridium,Coprococcus,Bacteroides,Chlorobacteriaceae.The genus and family levels of Prevotella were expressed in the model.Among the positive drugs,the abundance expression of Pseudobutyrivibrio,Lactococcus,Bacillus,Clostridiaceae and Peptostreptococcaceae was higher.The highest abundance in the low-dose group of Huangqin decoction was Rikenella,and the abundance expression of Moraxellaceae and Acinetobacter was higher in the medium-dose group of Huangqin decoction.Conclusion:The use of network pharmacology and bioinformatics,molecular biology and 16srDNA sequencing technology can confirm that Huangqin decoction can reduce the inflammatory factors IL6,IL1β,increase IL10 and then affect the diversity of intestinal bacteria,so that the abundance of beneficial bacteria increased,the abundance of harmful bacteria decreased,which is involved in inhibiting the expression levels of IL6,IL1β protein factors in IL17,TNF and other signaling pathways,to play a role in the treatment of ulcerative colitis.