| Objective:This experiment used TNBS to make enema model of SD rats,and successfully prepared Crohn's disease(CD)model,then observed the regulation of acupuncture and moxibustion on the changes of intestinal flora in CD rats.The diversity of intestinal flora Alpha,the structure and composition of phylum-level flora,and the specific differentiated species at family level were compared and analyzed.To explore the relationship between the treatment of CD by Acupuncture and moxibustion and the occurrence of intestinal flora,and to provide favorable experimental basis for clinical prevention and treatment of CD by Acupuncture and moxibustion.Method:Thirty-two SD rats were randomly divided into four groups: HE(blank group),HA(model group),HB(acupuncture group)and HC(moxibustion group).CD intestinal fibrosis rat model was prepared by Morris 2,4,6-trinitrobenzene sulfonic acid(TNBS)method.After successful modeling,the rats in the acupuncture group and moxibustion group were intervened.The operation points were selected as "Tianshu" and "Shangjuxu" points,once a day for 15 minutes each time,for a total of 7 days.The blank group and the model group were fixed for 15 minutes by the same method,but no other treatment such as acupuncture and moxibustion was given.The body weight,fecal characteristics,stool blood and disease activity index(DAI)of rats were recorded during the experiment.After treatment,the feces of rats were collected and the intestinal microorganism samples of 32 rats were analyzed by 16 S rDNA high throughput sequencing method.Colon specimens of rats were collected and stained with HE.Colon pathological changes of rats in four groups were observed under biomicroscopy.Result: 1.After TNBS induction,CD model rats showed dry hair,laziness,weight loss,anorexia,loose stool,loose stool and blood stool.DAI score increased significantly.After acupuncture and moxibustion treatment,the weight of rats increased,stool characteristics,stool blood and other conditions improved,and DAI score decreased significantly(P < 0.01).After TNBS induced CD in rats,the general condition of CD model rats was poor,feces appeared purulent and blood stool,DAI score increased significantly.2.The colon morphology of rats was observed by naked eye: the colon morphology of rats in blank group was normal,and the intestinal mucosa was soft,light red,and the intestinal wall was thin,which was normal tissue.In model group,the intestinal wall of rats was irregular in shape.The intestinal mucosa adhered to the opposite mucosa to form ulcers,congestion and swelling,intestinal wall adhesion,intestinal wall thickening,erosion,bleeding and ulcer,intestinal mucosa was highly edematous and became massive,such as pebbles.After treatment with acupuncture and moxibustion,the colon morphology was restored,the mucosa was smoother than the model,the local congestion and edema were obviously alleviated,the intestinal wall became thinner,and no obvious ulcer erosion was observed.3.The pathological changes of colon in four groups were observed under biomicroscopy: compared with the blank group,the model group had blurred colon tissue,disordered structure,congestion and edema of intestinal mucosa,bending and deformity of colon glands,necrosis and exfoliation of goblet cells,crypt deformity and infiltration of inflammatory cells,thickening of mucosal muscular layer.After the treatment of acupuncture and moxibustion,the colon tissue morphology was restored and the structure was more complete than that of the model group.4.High-throughput sequencing of 16 S rDNA of intestinal flora: Diversity analysis: Compared with the blank group,the diversity and abundance of intestinal flora in CD model group decreased,while the diversity and abundance of intestinal flora recovered after acupuncture and moxibustion treatment(P < 0.05 or P < 0.01).Analysis of dilution curve: The results of dilution curve constructed by Chao1,Observed species,Shannon and Simpson index show that Chao1,Observed species,Shannon and Simpson index increase at OTU level with the increase of sequencing data.When the amount of sequencing data reaches a certain value,abundance and diversity index decrease,and the curve tends to be flat.Community composition and significant difference analysis: At the gate level,community structure analysis showed that the number of sclerenchyma and Bacteroides decreased while the number of Proteus increased in the model group.After treatment with acupuncture and moxibustion,the number of Bacteroides increased and the number of Proteus decreased significantly.Significant differences in species were found among Enterobacter,Lactobacillus and Bacteroides at the scientific level.The main manifestations were that the number of Enterobacter increased significantly in the model group(P<0.01),while the number of Bacteroides and Lactobacillus decreased significantly(P<0.01).After the treatment of acupuncture and moxibustion,the number of Enterobacteria decreased significantly(P<0.01),the number of Bacteroides and Lactobacillus increased significantly,and moxibustion increased significantly.(P < 0.05).Conclusion: 1 Acupuncture and moxibustion can improve the general condition,activity index and pathological changes of CD model rats induced by TNBS.2 Acupuncture and moxibustion have a benign regulatory effect on intestinal flora disorders,among which bacteroides,Enterobacter and Lactobacillus may be the key bacteria in the treatment of CD by Acupuncture and moxibustion. |
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