Study On The Mechanism And Change To Intestinal Microfloraand Metabonomics Of The Ulcerative Colitis Rat Treated With Anchang Decoction

Part 1 Research on the Intervention Mechanism of Anchang Decoction on Colonic Mucosa metabolic changes in Rats with Ulcerative ColitisBackground and Objective: Ulcerative colitis(UC)is a chronic inflammatory bowel disease.Recent advances in its pathogenesis and the search for effective prevention and treatment drugs are still limited.Anchang decoction is compound Chinese herbs that we have applicated and researched in clinic more than 20 years.It has definite clinical curative effect.Although there is a certain research foundation for the study of the relation between UC and UC,the mechanism of it is still unclear.Metabonomics and intestinal microecology have been the focus of attention in recent years,but few studies have been reported in UC.In this study,metabonomics was used to investigate the mechanism of the effect of Anchang decoction on UC.1.Methods: using 2,4,6-trinitrobenzene sulfonic acid(TNBS)model,TNBS dose of 90mg/kg,UC rat model was established.2.selecting the best formula to make model,randomly divided into model group,Mesalazinegroup,Bifidobiogen group(the positive drug control group),Anchang decoction low-,middle-and high-dose group(treatment group).3 each groups was sacrificed 1/3 survival rats at 5th,10 th,15th days after treatment,observe the general condition of the rats and the feces during the experiment,the disease activity index(disease activity,index,DAI)were taken;mucosa of rats for pathological analysis,the colon injury index(colon macroscopic damage index,CMDI)evaluation of mucosal injury rats,Geboes index of colonic mucosa pathology;the lesions of colonic mucosa by GC-MS detection(GC-MS),and analysis method,orthogonal partial least squares discriminant analysis using principal component(OPLS-DA),the first principal component of the OPLS-DA model VIP value(threshold >1)combined with the t test,P value(P<0.05)to find the differences of metabolites.1.Results: The UC rat model was consistent with the disease of UC in terms of disease symptoms,colon damage and pathological findings,and the model was best at 10 days after modeling.2.Chromatogram and mass spectrum library were matched and identified,and 47 accurate and accurate metabolic compounds were obtained.The treatment group and positive drug control group both had therapeutic effects on UC rats.3.By differential foreign body screening,we identified 10 potential metabolic markers of colonic mucosa in UC rats,namely adenine,cytosine,uridine,glycine,glucose,xylose,β-alanine,nectar Sugar,lactic acid,sarcosine.4.An Chang decoction can decrease adenine,cytosine,uridine,lactic acid,glycine and glucose,mannose,xylose,enhance sarcosine and β-alanine content,the callback rate was significantly higher than the positive drug group(P < 0.05),in which the middle dose Anchang Decoction group pullback best.Conclusion: 1.The UC rat model was established by using 2,4,6-trinitrobenzene sulfonic acid,and the Anchang decoction groups,Mesalazine group and Bifidobiogen group Both have therapeutic or relieving effects.2.There are 10 kinds of endogenous metabolites differences in colonic mucosa of UC rats: adenine,cytosine,glycine,uridine,mannose,lactic acid,alanine,glucose,creatinine and xylose,they may serve as potential molecular markers for diagnosis of UC metabolism.3.Anchang decoction may reduce the contents of adenine,cytosine,uridine,lactic acid,glycine and glucose,and increase the content of mannose,xylose,creatine,and alanine to regulate intestinal microecology.4.The regulating effect of Anchang decoction groups to 10 different endogenous metabolites in UC rats was better than that in the positive group.Part 2 The effect of the decoction on intestinal flora in rats with ulcerative colitisBackground and Objective: In recent years,the pathogenesis of UC may be closely related to genetic,immunological,intestinal microecology and other factors.The intestinal flora,acetic acid,endotoxin is an important component of the intestinal microflora,intestinal micro ecological stability is important for maintaining the normal intestinal function,and intestinal microflora imbalance may be one of the pathogenesis of UC.In this study,we compared the intestinal microflora and the changes of acetic acid and endotoxin in UC rats,and explored the micro ecological characteristics of intestinal tract in UC rats and the intervention mechanism of Anchang decoction.Methods: 1.According to the experimental one used the 2,4,6-trinitrobenzene sulfonic acid(TNBS)method to make the model and divided into groups.After tenth days,the rats were fed with intragastric administration.After 7 days,the rats were killed and the samples were taken.2.The lesion of colon contents extracted from the fecal bacterial genome DNA of the extracts of each bacterium was detected by real-time fluorescent quantitative PCR using SYBRGreen fluorescent dye method,each bacterium content by Delta-delta Ct relative quantitative method and quantitative analysis method to detect directly produce histograms;the content of acetic acid in liquid phase chromatography;detection of rat intestinal endotoxin limulus reagent method.Results: 1.Changes of intestinal flora in UC model group: compared with the normal control group,the relative expression of UC model group showed decreased in the Lactobacillus,Bifidobacterium,Clostridium,Bacteroides fragilis,Bacteroides(p<0.05),and in the Bacteroides,Bacteroides,Escherichia coli,Bacillus thuringiensis,Enterobacteriaceae,Enterococcus faecalis,Enterococcus faecalis showed increased(p<0.05).2.Changes of intestinal microflora in the Mesalazine group and Bifidobiogen group: compared with UC model group,the relative expression of Mesalazine group and Bifidobiogen group showed increased in Lactobacillus,Bifidobacterium,Clostridium,Bacteroides fragilis,Bacteroides multiforme,Bacteroides ovum,Bacteroides(p<0.05),while the expression level was lower than that of the normal control group(p<0.05);There was no obvious differences between Mesalazine group and the UC model group in the expression level of the Bacteroides simplex(p>0.05),The expression level of Bifidobiogen group was higher than that of UC model group(p<0.05);in the genus Fusarium,Botox,Bacteroides,Bacteroides,Enterococcus faecalis,Enterococcus faecalis and Escherichia coli,the relativeexpression levels of Mesalazine group and Bifidobiogen group were lower than that of UC model group(p<0.05);in the genus Bacteroides,the Mesalazine group was no significant difference with the UC model group(p>0.05),the Bifidobiogen group was lower than the UC model group(p<0.05).3.The changes of intestinal flora in Anchang decoction treatment group(Anchang decoction low-,middle-and high-dose group): compared with UC model group,the relative expression levels of the treatment group was rise in Lactobacillus,Bifidobacterium,Clostridium,Bacteroides fragilis,Bacteroides mult(p<0.05),and showed reduce compared with normal control group(p<0.05);In the treatment group,the expression level of Escherichia coli,Enterobacteriaceae,Enterococcus faecalis,Escherichia coli and Escherichia coli was lower than that in the UC model group(p<0.05);The low dose of Anchang decoction in Bacteroides simplex was no significant difference higher to UC model group(p>0.05);The middle dose and the high dose was higher than of the UC model group(p<0.05).4.Comparison of intestinal microflora changes in Anchang decoction treatment group: The middle dose of Anchang decoction Lactobacillus,Bifidobacterium,Fusobacterium,Bacteroides fragilis,Bacteroides bacillus,Bacteroides multiforme,Botox,Bacteroides bacillus,Enterobacteriaceae,Enterococcus faecalis,E.coli was closer to the blank control group than the other two groups(p<0.05);The high dose group was more close to the other two groups in the genus Bacteroides,Clostridium,O.coli and Gibbidium(p<0.05).5.Comparison of intestinal microflora changes between Anchang middle decoction group and Mesalazine group、Bifidobiogen group:The relative expression level of the middle dose of Anchang Decoction group was more close to the normal control group in Lactobacillus,Bacteroides fragilis and Bacteroides than Bifidobiogen group(p<0.05),more close to the normalcontrol group in Bifidobacterium,Clostridium,Bacteroides bacillus,Botox than Mesalazine group(p<0.05),and in Escherichia coli,Enterococcus faecalis,Enterococcus faecalis,Dendrobacter,Bacteroides bacillus,Osteobacterium,Bacteroides multiforme and Bacteroides was closer to the normal control group compared to Mesalazine group and Bifidobiogen group(p<0.05).6.Acetic acid and endotoxin model group was significantly higher than the control group and the treatment group(p<0.05),but there was no significant difference between the treatment groups(P > 0.05)Conclusion: 1.The changes of intestinal flora in UC rats were significantly different and characteristic,Bifidobacterium,Lactobacillus,Bacteroides spp.,Clostridium,Bacteroides ovatus,Bacteroides thetaiotaomicron,simplex Bacteroides,Bacteroides vulgatus relative expression levels decreased;hard bacillus,Bacteroides,Clostridium botulinum,Enterococcus faecalis and Enterococcus faecium Gibbs to Escherichia coli,in the relative expression content increased,This change may play a role in the pathogenesis of UC.2.Anchang decoction had obvious regulation effect on the whole intestinal microflora of UC rats and was consistent with the combination of Mesalazine and Bifidobiogen,and the effect of Anchang decoction on UC intestinal flora may be the mechanism of its treatment of UC;3.Compared with the high dose and low dose of Anchang decoction,the middle dose had a more significant effect on the symptoms and intestinal microflora of UC rats,it is possible to improve the current clinical application dose.5.The changes of acetic acid and endotoxin in UC rats may be the cause or manifestation of UC.At the same time,both Mesalazine and Bifidobiogen and Anchang decoction could have some regulation significance,but there was no obvious regulatory difference between the treatment groups.