Study On The Pharmacodynamic Mechanism Of Huanghou Zhixie Dropping Pill On Damp-heat Ulcerative Colitis Model Mice

Objective:Objective:To verify whether Huanghou Zhixie Dropping Pill can treat damp-he at ulcerative colitis model mice and its possible pharmacodynamic mechanism.Methods:Firstly,the network pharmacology was used for prediction,and the TCMSP data base and analysis platform(http://tcmspw.com/tcmsp.php)were used to predict and analyze the active components and targets of each single herb of Huanghou Zhixie Dropping Pills.Ulcerativ e colitis-related target genes were obtained from the GeneCards database(https://www.genecard s.org/),and the "Network Analyzer" function of Cytoscape 3.6.0 software was used to construct a drug-compound-target-disease network.For analysis,use the STRING database(https://stringdb.org/)to construct a protein-protein interaction(PPI)network and select key targets.After inst alling the GO and KKEGG enrichment analysis R packages in the R software,GO functions are performed Enrichment and KEGG pathway enrichment analysis screened out the main signaling pathways of Huanghou Zhixie Dropping Pills in the treatment of ulcerative colitis.Then,anima l experiments were conducted.Balb/c mice were grouped into groups,and the relevant literature on the "Methodology of Animal Experiments in Traditional Chinese Medicine" and Dextran Sul fate Sodium(DSS)-induced ulcerative colitis in mice was firstly applied.High-sugar and high-fa t diet The Balb/c mice were made into a model of large intestine damp-heat type,and then the m ice were made into a large intestine damp-heat type ulcerative colitis model with DSS free drink.Mouse disease activity index(DAI),colonic mucosal injury index(CDMI),pathological tissue score,etc.were used to evaluate the therapeutic effect of Huanghou Zhixie Dropping Pills.The s erum and colon tissue of mice were detected by enzyme-linked immunosorbent assay(Elisa met hod).Absorbance at 450 nm wavelength in the group.After removing the spleen of the mice,the residual blood was blotted with filter paper,and then weighed.The spleen index was calculated using the formula:spleen index=spleen weight/body weight×100%,and the spleen index of each group was compared.Western Blot was used to detect the expression of COX2,P65,iNOS,PMAPK,MAPK,P-P65 proteins related to MAPK/NF-κB signaling pathway in colon tissue and t he tight junction proteins claudin-1,ZO-1,expression of occludin.16sRNA was used to detect a nd compare the feces of the mice in the blank group,the model group,and the Huanghou middle-dose group(treatment group)to evaluate the effect of Huanghou Zhixie Dropping Pills on the i ntestinal flora of large intestine damp-heat ulcerative colitis..Results:Huanghou Zhixie Dropping Pills were searched by network pharmacology,and it was found that there were 5 active ingredients in dried ginger;2 active ingredients in Magnolia officinalis;14 active ingredients in Coptis chinensis;These active ingredients have a total of 147 target genes,4622 related genes in ulcerative colitis,and 62 common genes with Huanghou Zhi xie Dropping Pills.The results of the active ingredient-target-disease interaction network showe d that there were 20 active ingredients in Huanghou Zhixie Dropping Pills that could interact wit h the above target genes.The STRING database can obtain the top 30 key target genes of the int eraction relationship,affecting a total of 79 biological processes,mainly by affecting biological processes such as binding(blinding),receptor activity(receptor activity)to treat ulcerative coliti s.It can affect 70 signaling pathways,among which PI3K-AKT signaling pathway,AGE-RAGE signaling pathway and MAPK signaling pathway are the key pathways.The mouse experiments’showed that the DAI score and CDMI score of the model group were higher than those of the b1 ank group.In terms of pathology,the normal structure of intestinal mucosa disappeared in the m odel group,the number of goblet cells in the tissue was greatly reduced,and a large number of i nflammatory cells proliferated,which proved that the ulcerative colitis model was successfully modeled.After the mice were divided into groups,the model mice were treated with Huanghou Zhixie Dropping Pills and mesalamine respectively.The comparative analysis of the results sho wed that Huanghou Zhixie Dropping Pills could significantly improve the weight loss,diarrhea,blood in the stool and colon damage in ulcerative colitis model mice.And the Huanghou middle-dose group showed a similar therapeutic effect to mesalazine.Determination of inflammatory f actors in serum and colon tissue showed that IL-1β,TNF-α,IL-6,and TGF-β inflammatory-relat ed factors in the model group were significantly higher than those in the control group(P<0.05).After Huanghou Zhixie Dropping Pill treatment,the contents of the above factors were significa ntly lower than those in the model group(P<0.05).Further comparison of Huanghou Zhixie Dro pping Pill groups found that the Huanghou middle dose group had the best effect(P<0.05),whic h was similar to the positive control group mesalazine(P>0.05).The contents of IL-4 and IL-10 in the model group were significantly decreased(P<0.05),and the contents of these factors were significantly higher than those in the model group after Huanghou Zhixie Dropping Pill treatme nt(P<0.05).Similarly,the Huanghou Zhixie Dropping Pills group had the best effect(all P<0.05).The spleen index score of the blank group was significantly lower than that of the model gro up,with a statistically significant difference(P<0.05).After Huanghou Zhixie Dropping Pill tre atment,the spleen index decreased,but the difference between the Huanghou medium dose grou p and the model group was statistically significant.Academic significance(P<0.05).Compared with the blank group,the expression levels of COX2,iNOS and COX2 in the model group were significantly increased(P<0.05);compared with the model group,Huanghou Zhixie Dropping Pills significantly decreased Expression of COX2 and iNOS(P<0.05).Compared with P65,M APK,P-MAPK and P-P65,Huanghou Zhixie Dropping Pills could inhibit the phosphorylation 1 evels of P65 and MAPK(P<0.05).For the measurement of claudin,it can be seen that the expr ession levels of claudin-1,ZO-1,and occludin in the model group were significantly lower than those in the blank group,and the difference was statistically significant(P<0.01).Huanghou Zhi xie Dropping Pills Compared with the model group,Huanghou Zhixie Dropping Pills could sign ificantly increase the expressions of claudin-1,ZO-1 and occludin(P<0.05).The analysis of inte stinal flora showed that the α diversity analysis showed that the richness and diversity of the inte stinal flora in the model group were significantly lower than those in the blank control group,an d the species difference analysis showed that the composition of the three groups of flora was qu ite different.The phylum Bacteroidetes decreased in the murine gut,while the phylum Proteoba cteria increased.β-diversity analysis found that the blank group,the model group and the treatm ent group did not have overlapping samples,indicating that there was a difference between the t hree groups of samples and the content of OUT was high.From the species composition heat ma p,LefSe analysis and random forest analysis,it can also be seen that the species in the three gro ups are quite different.From the ZIPI map,it can be seen that there are the top 5 key species wit h the highest abundance in the three groups,which are Firmicutes(Firmicutes),Bacteroidetes(Bacteroidetes),Proteobacteria(Proteobacteria),Tenericutes(Tenericutes),Verrucomicrobia(Ve rrucomicrobia),the changes of these key species are enough to affect the changes of the entire fl ora.The functional potential prediction shows that the core flora mainly affects the synthesis of amino acids,nucleotides and biosynthesis of factors,repair groups,electron carriers and vitamin s,carbohydrate degradation,carboxylate degradation,nucleoside and nucleotide degradation,Ba cterial resistance,fermentation,glycolysis,glycan biosynthesis,glycan degradation,nucleic acid formation,L-glutamic acid and L-glutamine biosynthesis,O-antigen building block biosynthesi s(E.coli)-Biological processes such as phospholipase,tRNA loading,etc.Conclusion:The results of network pharmacology indicated that Huanghou Zhixie Droppi ng Pills,like other traditional Chinese medicines,can treat ulcerative colitis through multiple tar gets,multiple pathways and multiple pathways.In the experimental study,the molecular mechan ism of Huanghouzhixie Dropping Pills in the treatment of ulcerative colitis was explored.First,DSS was used to establish a mouse ulcerative colitis model.By comparing the DAI score,CDM I score,and HS score,it can be seen that Huanghouzhi Xie Di Wan has a good effect on the treat ment of ulcerative colitis.Further research found that its mechanism of action is related to regul ating the expression of cytokines IL-1β,TGF-β,IL-4.TNF-α,IL-6 and IL-10,reducing the splee n index of mice,protecting the tight junction protein claudin-1,ZO-1,occludin is involved in re pairing the intestinal mucosal barrier,and mainly acts through the MAPK/NF-κB signaling path way.Through the comparative analysis of fecal flora,it can be seen that Huanghou Zhixie Drop ping Pills can promote the growth of probiotics and symbiotic flora,inhibit the colonization of o pportunistic pathogenic bacteria or harmful bacteria,and help maintain the steady state of intesti nal flora.Ulcerative colitis is treated by affecting biological processes such as synthesis and deg radation by affecting the number of core flora.