Study On Biological Effects And Mechanisms Of Croton Crassifolius Geisel. And Its Active Components In Treating Ulcerative Colitis

BackgroundCroton crassifolius Geisel.(CCG),a folk drug in southern China,has the function of regulating qi and relieving pain,removing wind and dampness,reducing swelling.It could be used to treat gastric and duodenal ulcer.The research showed that the CCG had significant effect on animal models with various acute and chronic inflammations.The compound sanbaiji decoction could be used to treat Ulcerative colitis(UC)in clinic.UC,a chronic intestinal inflammatory disease,is recurrent,difficult to treat and complicated in etiology and pathogenesis.At present,first-line drugs are often accompanied by large dosage and adverse reactions,so it is urgent to develop a safe and efficient drug.Due to the good safety of Traditional Chinese medicine and its active ingredients,it has always been a research hotspot to discover new drugs.ObjectiveThe aim of this study is to evaluate the activity of CCG on UC mice and to elucidate the potential mechanism of its effect based on metabonomics.What follows is to explore the potential pharmacodynamic materials basis of CCG.Methods1.The chapter of the thesis was to evaluate the effect of CCG on UC mice.The mice were randomly divided into control group,model group,mesalazine group(100 mg/kg),CCG group(600 and 150 mg/kg),Cyperenoic acid group(50 mg/kg)and Teucvidin group(50 mg/kg).UC mice induced by Dextran Sulfate Sodium(DSS)were administrated by gavage for 10 days.On the 11th day of the experiment,serum was collected from the orbit,and thymus and colon were collected.Combined with the weight change,disease activity index(DAI),the colon length,the histopathological examination and the expression of inflammatory protein iNOS and COX-2 in colon,therapeutic effect of CCG on UC was evaluated.2.The chapter of the thesis was to analyse the metabolism of UC mice and the effect of CCG on metabolism in UC mice based on metabonomics.Metabonomics analysis was carried out on serum and colon samples of experimental mice based on UPLC-QTOF-MS.After data was preprocessed,the peaks were divided into big peaks,medium peaks and small peaks according to the response value.Removed the small peaks,and imported medium peaks into SIMCA-P software to perform PCA and OPLS-DA analysis.Mesium peaks with VIP>1.5 and |Pcorr|>0.5 combined large peaks were recognized as candidate metabolites,and then screened the metabolic biomarkers in serum and colon by One way ANOVA and FDR correction(Q<0.05).These potential biomarkers were identified by the metabolite fragmentation and HMDB database.The metabolic pathway enrichment analysis and biological interpretation of metabolites were carried out by the biological database network.3.The chapter of the thesis was to present preliminary research on the active components of CCG on UC mice.Comparing the pharmacodynamic index(body weight,DAI,colon length and pathological changes)between groups including model group,mesalazine group,CCGH group,Cyperenoic acid group and Teucvidin group,the effects of the main components of CCG on UC mice were analyzed.4.The chapter of the thesis was to evaluate the effect of Cyperenoic acid on UC mice and to elucidate the mechanism of Cyperenoic acid based on RT-PCR.The mice were randomly divided into control group,model group,mesalazine group(100 mg/kg),Cyperenoic acid group(100,50,25 mg/kg),which were administrated by gavage for 9 days.The serum,thymus and colon of mice were collected on the 10th day to evaluate the effect of Cyperenoic acid.Detecting the expression of genes of inflammatory protein(IL-1β,TNF-α,COX-2)and tight junction protein(Claudin-1,ZO-1,Occludin)with RT-PCR technology was used to analyze the mechanism of Cyperenoic acid on UC.Results1.The results showed that CCGH could relieve the weight loss,DAI,the degree of bloody stool,ulcer of colon,the proteins expression level of iNOS and COX-2 was decreased apparently on UC mice.2.Based on metabonomics,the pathological mechanism of UC mice and the pharmacodynamic mechanism of CCG on UC were elucidated.76 differential metabolites were screened and identified from UC mice,which were mainly involved in glycerolphospholipid metabolism,sphingolipid metabolism and unsaturated fatty acid metabolism.Based on One way ANOVA,the differential metabolites in UC mice were screened,among which 25 were significantly changed by CCG,indicating that CCG could significantly regulate glycerolphospholipid metabolism,sphingolipid metabolism and unsaturated fatty acid metabolism.This suggested that CCG could alleviate UC by regulating energy metabolism,inflammation and oxidative stress.Through pearman correlation analysis of different metabolites in UC mice,39 different metabolites were found to be closely related to the pharmacodynamic indexes of CCG.In addition to phosphatidylcholines,four metabolites including Indoleacetic acid,17-HDoHE,Uric acid and 6,7-dihydro-12-epi-LTB4 were related to the effect of CCG.Based on the integration of KEGG database and HMDB database,it was found that 17-hdohe and 6,7-dihydro-12epi-ltb4 could regulate PPAR α and PPAR γ.3.Pharmacodynamic evaluation of the main components on UC mice suggested Cyperenoic acid could be the active component in CCG.The results showed that high and medium dose of Cyperenoic acid had significant therapeutic effect on UC.The gene expression level of inflammatory proteins(IL-1β,TNF-α,COX-2)and tight junction proteins(claudin-1,occluding,ZO-1)was reversed after Cyperenoic acid administration.This suggested that Cyperenoic acid could alleviate UC by enhancing intestinal mucosal barrier and ameliorating intestinal inflammation.ConclusionCCG and its active component Cyperenoic acid could effectively alleviate UC.Based on metabonomics,it was found that CCG might act synergistically through energy metabolism,inflammatory response,and oxidative stress.CCG might regulate unsaturated fatty acids metabolism to activate PPARα/γ,thus reducing the inflammatory reaction and maintaining the steady state of glucose and fat metabolism,which needed further verification in later experiments.Cyperenoic acid might treat UC by eliminating inflammatory reaction and enhancing mucosal barrier.However,the molecular mechanism of Cyperenoic acid was still unclear,which had good research and development prospect.