Based On ~1HNMR And 16SrDNA Technology, The Effects Of Moxibustion On Colon Metabolites And Intestinal Flora Of Crohn’s Disease Rats Were Investigated

Objective:This experiment used 2,4,6-Trinitrobenzene sulfonic acid(TNBS)enema on SD rats model,the preparation of Crohn’s disease(CD Crohn’s disease)in the rat model of moxibustion on the clinical curative effect of Crohn’s disease model in rats,using 16 S r DNA technology and nuclear magnetic resonance hydrogen spectrum detection and discusses moxibustion intestinal flora of Crohn’s disease model rats and the effect of colon tissue metabolites.Methods:40 SPF male rats,were randomly divided into normal group(10rats)and model group(30 rats)after one week of adaptive feeding.The rats model of CD intestinal fibrosis was prepared by referring to the method of Morris2,4,6-trnitrobenen Sulpician aid TB prescription.After verifying the model,the rats in the original model group were randomly divided into model group,moxibustion group and western medicine group,with 10 in each group.The normal group did not give any intervention;The model group was fixed without treatment;Moxibustion group conducted moxibustion intervention,and selected “tianshu point” and “shangjuxu point” by operating acuponits,and treated them once a day for 15 minutes each time for a total of 7 days;In the western medicine group,mesalazine was gavaged once a day for a total of 7 days.1.Observe the general behavior changes,body weight,fecal characteristics,and blood in the stool of the rats in each group,and calculate the disease activity index of the rats,and observe the colon histopathological changes of the rats in each group by gross and HE staining.2.16 S r DNA technique was used to detect the changes of intestinal flora abundance and diversity of rats in each group,and the changes of intestinal flora of rats in the CD model of moxibustion intervention were observed.Through LEf Se analysis,to find out the significant difference in bacterial flora after moxibustion intervention.3.The changes of metabolites in the colon tissues of rats in each group were detected by h NMR.The nuclear magnetic spectrum was pretreated and normalized by Mest Re Noya software.Metabolic patterns and differential metabolites of colon tissues were analyzed by multivariate analysis methods such as PCA and PLS-DA,and the characteristic metabolites in rat faces samples were screened by combining VIP values in the PLS-DA model.Through the biological function analysis,to find out the relevant metabolism.Results:1.After successful CD modeling through TNBS-induced rats,the model rats demonstrated dry hair,laziness,weight loss and anorexia.Loose stool,thin stool and bloody stool were observed.The DAI score was significantly increased.After the intervention of moxibustion and mesalazine,there was a relatively improvement of mental status of rats assigned to moxibustion and western medicine group.The weight gain was seen compared with model group.Fecal characteristics and bloody stool were also improved compared with the model group.There was a significant decline in the DAI score(P<0.01).Macrography was used to observe the colon morphology of rats in each group: The colon in the blank group had normal morphology with soft intestinal mucosa,reddish color,thin intestinal wall,and had no congestion and edema.Irregular morphology of intestinal wall,ulceration of the lateral mucosa,adhesion and thickening of the intestinal wall,erosion,bleeding,and high degree of edema of the intestinal mucosa,thickening like a polyp can be seen in model group rats.However,after treatment in moxibustion and western medicine group,recovery in colon morphology occurred and mucous membrane was smoother than model group.The intestinal wall became thinner with alleviated local congestion and edema.There was no obvious ulcerative erosion surface observed.The result of HE staining in each group showed that model group rats demonstrated disordered colon tissue structure,vague layer,distorted colon glands,necrosis and shedding of goblet cells,deformity of crypts and inflammation infiltrates of entire intestinal wall with transmural inflammatory reaction,congestive edema of intestinal mucosa and thickening of mucosal muscle layer,as compared with normal blank group rats.After moxibustion and western medicine therapy,comparatively complete intestinal mucosal structure,clearer intestinal tissue layer were discovered.Colon glands structure restored and thickening intestinal mucosal muscular layer improved.Compared to model group,improvement of inflammatory cells infiltrate and congestive edema were also observed.2.Intestinal flora 16 S r DNA high-throughput sequencing results: Alpha diversity analysis results showed that moxibustion had an positive regulatory effect on the diversity of intestinal flora.ANOSIM similarity analysis and Beta diversity analysis results indicated that there was a significant difference of intestinal flora between the normal blanc group,model group,moxibustion group and western medicine group.LEf Se analysis results showed that moxibustion was able to regulate the intestinal flora of bacteroidaceae,parabacteroides,clostridium,paraprevotella,lachnospiraceae|g,anaerostipes,oscillospira,ruminococcaceae and so on.3.Compared with normal group rats,metabolites of colon tissue in CD model group rats can change methanol,betaine,leucine,isoleucine,lysine,proline,hypoxanthine,alanine,acetate glycine,valine,choline,creatine and inositol.Additionally,the concentration of methanol,betaine,choline,creatine and inositol were significantly lower than those in normal group(P<0.05).there was a significant increase in the concentration of leucine,isoleucine and lysine(P<0.05)as compared with normal group.The main metabolite pathways affected were taurine,hypotaurine,alanine,aspartic acid,glutamic acid,arginine,proline,glycine,serine,and threonine metabolism,valine acid,leucine and isoleucine synthesis,etc.whereas,after moxibustion intervention,To certain extent,it can also significantly revert betaine,choline,creatine,inositol leucine,isoleucine and lysine of CD model rats and restore the disordered metabolic status to normal status.Conclusion:1.Moxibustion can improve the general condition,DAI score and colon pathology of TNBS-induced CD model rats.2.Moxibustion had certain regulating effect on the imbalance of intestinal flora in CD model rats,among which Clostridium,Paraprevotella,and vibrio butyrate in the family heliconiaceae(Lachnospiracealg.Anaerostipes),fibrillation spirillum(Oscillospira),microbacteria(Ruminococcaceae),and rothia(Roseburia)may be the key bacteria in acupuncture therapy for CD.3.Moxibustion partially restored the metabolic disorder state of the CD model rats by resetting some metabolites,such as leucine,isoleucine,lysine,taurine,inositol,creatinine,betaine,etc.Moxibustion may improve CD syndrome was by affecting the integrity of the intestinal barrier,affecting the intrinsic and adaptive immune response,reducing intestinal inflammation,relieving intestinal oxidative stress,reducing intestinal oxidative damage,and promoting intestinal epithelial cell proliferationsyear.