| Ulcerative colitis(UC)is a chronic intestinal disease with unclear pathogenesis and recurrent episodes,which seriously affects the normal life of affected animals.The incidence of UC increased due to the changes in eating habits.Medications commonly used for treating UC are usually immunosuppressive and recurrence rates are also high,thus new methods for UC treatment with little side-effect are badly in needed.Chinese herbal medicines possess the properties of low toxicity,low residue,and easy accessibility so they are widely used in clinical practice.Previous studies showed that purslane and Pulsatilla Decoction soup showed anti-inflammatory,antioxidant,and immunomodulatory effect on UC.Therefore,in the present study purslane and Pulsatilla Decoction soup were used in combination to analyze its effect on UC.Network pharmacology was applied to comprehensively evaluate their potential pharmacological effects.Histopathological analysis,molecular biology,and intestinal flora changes were assessed to evaluate the modulatory effect of SPD on UC which will provide a reference for the treatment of UC with Chinese herbal medicine.Methods: 1.The main active ingredients and action targets of SPD(Supplenmented Pulsatilla Decoction)were obtained by systematic pharmacological analysis platform.The main targets of UC were screened based on the pharmacology analysis platform of traditional Chinese medicine system.The main targets of UC were screened according to the disease storage database,and the intersection targets were the potential targets of SPD treatment for UC.Cytoscape software was used to construct the ingredient-target-disease interaction network and the frequency of protein interaction was constructed using STRING,and then DAVID software was used to conduct gene ontology annotation(GO)and signaling pathway(KEGG)enrichment analysis,so as to preliminately screen out the potential targets of supplemented pulsatilla decoction in regulating UC,and prepare for future studies.2.According to the results of preliminary experiment,3.5% DSS was selected to establish UC model,and salicylazosulfapyridine(150mg/kg)was selected as positive drug treatment group(T),and three SPD treatment groups with low(L,4.37 g/kg),medium(M,8.75 g/kg)and high(H,17.29 g/kg)doses were set up.The efficacy of the treatment was assessed by weight change,DAI score,histopathology and other phenotypic changes.Colon tissues were collected,detected the gene expression of cytokines(TNF-α,IL-1β and IL-10)and barrier-related molecules(occludin and MUC2)by real-time quantitative PCR assay(RT-q PCR).The expression of IL-6,IL-1β and TNF-α proteins was measured by ELISA,and detected the level of MDA,SOD,CAT and GSH antioxidant indexes in serum.3.16 S rDNA sequencing analysis and differential analysis was performed at the gate level and genus level to explore the effects of DSS modeling and treatment in each treatment group on the changes of intestinal flora in mice and to evaluate the therapeutic effects of SPD on UC from the aspect of the changes of microflora.Results: 1.60 ingredients and 411 drug targets in SPD were identified by screening the active ingredients of Chinese medicine.There were 43 intersecting targets with 975 targets of UC.The "component-target-disease" network analysis showed that SPD may mainly regulate the UC by modulating Alb,IL-6,TNF,Casp3,Egf,IL-10 and other key targets through quercetin and lignocaine.GO function annotation and KEGG enrichment results found that SPD may modulate UC mainly through TNF-α,MAPK and HIF-1 signaling pathways.2.The UC model was successfully established using 3.5% DSS,and the mice showed obvious weight loss,hematochezia and other symptoms.The results are as follows:(1)Compared with model group,body weight and disease activity index(DAI)were significantly increased in medium and high dose SPD treatment group,and colon shortening caused by DSS was significantly relieved(P < 0.05).(2)HE stained sections showed that,compared with the model group,medium and high dose SPD treatment significantly improved the colon tissue damage caused by DSS.(3)Compared with model group,the m RNA levels of pro-inflammatory cytokines TNF-α and IL-1β were significantly decreased by medium and high-dose SPD treatment(P < 0.05),while the m RNA levels of anti-inflammatory cytokines IL-10 were increased by medium and high-dose SPD treatment(P < 0.05).Moreover,m RNA levels of barrier proteins Occludin and MUC2 in colon tissue were significantly increased in high-dose SPD treatment group(P < 0.05).(4)ELISA results showed that compared with model group,the expressions of pro-inflammatory cytokines TNF-α and IL-6 protein in colon tissue of high-dose SPD treatment group were significantly down-regulated(P < 0.05),and the protein expression level of IL-1β was down-regulated to a certain extent.(5)Compared with model group,the serum MDA content in different doses SPD treatment groups was significantly decreased(P < 0.05),and the contents of SOD,CAT and GSH in serum in high dose SPD treatment group were significantly increased(P < 0.05),so as to improve the antioxidant level of the body.3.16 S rDNA sequencing results showed that the species of flora in UC group significantly decreased and the structure significantly changed(P < 0.05),and the drug treatment could significantly improve their levels and make them tend to the level of normal control group.In addition,the analysis of phylum level and genus level showed that the abundance of Proteobacteria in the control group significantly higher than other groups(P <0.05).A total of eight genera were enriched by screening and analysis of dominant groups at the genus level,namely Bacteroides、Parabacteroides、Akkermansia、Lactobacillus、Parasutterella 、 Helicobacter 、 Clostridium＿sensu＿stricto＿1 and Odoribacter.Among them,The abundance of Bacteroides and Parabacteroides in the model group was significantly higher than in the other groups,suggesting that it may be a landmark microorganism for assessing drug efficacy.This also indicated that the supplemented pulsatilla decoction might regulate UC by regulating intestinal flora.Conclusion: In this study,the feasibility of SPD treatment for UC was analyzed based on the network pharmacology,and the UC model was successfully established.It was found that SPD treatment of compound Chinese medicine can inhibit the expression of pro-inflammatory cytokines,increase the levels of barrier related proteins and antioxidant enzymes,and regulate the composition of intestinal microflora.Clinical symptoms,disease activity index and tissue damage of colon caused by ulcerative colitis were improved in UC mice.This study provides a theoretical basis for the clinical application of supplemented pulsatilla decoction,and also provides a potential new strategy for the treatment of UC. |
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