| Objective:This dissertation focus on the regulation effects of ulcerative colitis model rats’ gut micro-flora and urine metabolic through electro-acupuncture treatment, and the regulation mechanism is also discussed through host global metabolic profiles, to explain the role of acupuncture treatment on ulcerative colitis, provide new and more adequate experimental basis in order to reveal the mechanism of acupuncture treatment on ulcerative colitis, and reveal this multi-target, multi-level complex system in acupuncture treatment and a comprehensive understanding of the efficacy of acupuncture, provide in-depth theoretical reference and experimental evidence.Method:39healthy male SD rats were randomly divided into control group and UC model group. Ulcerative colitis model rats were induced by Dextran sulfate Sodium (DSS) with40g/L concentration, after succeed, the UC model rats were separated to UC model group and treatment group by randomly by second time. The treatment group were electro-acupunctured with Zu Sanli(ST36) and Guan Yuan(RN4), the treatment would be implemented for5days. No treatment was acted on the blank control group and model group.1. After electro-acupuncture treatment, general observation was implemented, fecal occult blood were tested, disease activity index (DAI) were scored, and tissue morphology was observed by light microscopy as well as ultrastructure was evaluated by transmission electron microscopy (TEM).2. The feces of rats were analyzed in each group from gut micro-flora’s view with the application of DGGE method, and the dominant bands were evaluated and identified by analysis. The experiment helps to constitute clear intestinal flora characteristics and changes and to look for the dominant flora and its related changes after treatment. 3. The researches were investigated on the UC rats’ urine metabolic phenotype and key metabolites changes after electro-acupuncture treatment with the application of’H NMR metabolomics. The regulation mechanism is discussed through host global metabolic profiles to improve gut micro-flora with electro-acupuncture treatment.Results:1. The efficacy study of electro-acupuncture treatment’s on ulcerative colitis:1) After DSS induced UC model was setup, the model group were with poor general condition, fecal appearance of pus and blood stool, DAI score increased significantly; Electro-acupunctured rats’mental status were improved; the stool, diarrhea and other conditions were improved obviously; the DAI score decreased, and the difference between groups was statistically significant (p<0.05).2) The results of the optical microscope showed that, comparing with the control group, ileocecal and distal colonic mucosal layer structure damaged, produced ulceration. submucosa congestion and edema, inflammatory cell infiltration in the mucosal layer, large amounts of free of neerotic tissue in the model group. Glands structure and arrangement of intestinal mucous in the electro-acupuncture therapy group were relatively complete and orderly. It was also found that submucosa was without apparent hyperaemia oedema, only a small amount of infiltration of inflammatory cells, lymphocytes and less fibroblasts.3) The results of electrical microscope observation showed that, comparing with the control group, ileocecal and distal colon tissue in the model group were sparse and short, in addition, epithelial cells in the model group were fulled with large vacuoles, the mitochondria were swelling, and inflammatory cells were infiltrated. After acupunctured, microvilli were relatively ordered, mitochondria were normal, nucleoplasm and cytoplasm were evenly distributed, rough endoplasmic reticulum were abundant in gut tissue of the control group.2. The regulation effects research on UC rats’ gut micro-flora after electro-acupuncture treatment:1) DGGE pattern analysis showed that the abundance and diversity index of the intestinal microecology of the ulcerative colitis rats were obviously decreased. The difference between groups were statistically significant (p<0.05); and at the same time, the abundance and diversity index of gut micro-flora in the treatment group obviously increased comparing with the model group, and the difference between groups was statistically significant (p<0.01).2) Clone sequencing results showed example lane dominant bacteria were identified and classified to Bacteroidetes. Firmicutes and Proteobacteria. These were the same as gut intrinsical micro-flora components. According to the edge strip identification results of identification. intestinal flora is mainly composed of the total bacterial content of Bacteroides and Proteobacteria decreased in the model group, while Firmicutes in model group were rising. After electro-acupuncture treatment, the content of the tree top type microflora clined to the normal direction. The contents of Lactobacillus sp. and Lachnospiraceae bacterium in model group were significantly reduced (p<0.05) acomparing with control group. Electro-acupuncture treatment can increase the content of Lactobacillus sp.(p<0.05)and Lachnospiraceae bacterium (p<0.05).3. The urine metabolic phenotype research:1) There was an obvious difference between control group and model group with multivariate statistical analysis. PCA analysis showed that, the samples in the electro-acupuncture group located between the control group and model group, indicating acupuncture can alter the metabolic of host orgnism and make the metabolic phenotype of the urine sample be close to control group, which suggested electro-acupuncture treatment perform effectively in treating ulcerative colitis.2) The plot effects with the control group were analyzed using the factor loading graph, it showed electro-acupuncture had the presence of benign regulation function on the deviated3-D-hydroxybutyrate, hippurate and acetate (p<0.05).3-D-hydroxybutyrate,hippurate and acetate were recognized as the key metabolites of electro-acupuncture treatment for ulcerative colitis.Conclusion:1. From the view of the animal behaviors and pathological morphology in UC model rats after electro-acupuncture treatment, symptom and pathological changes are improved.2. Electro-acupuncture has a positive adjustment on both gut micro-flora diversity and the expression of beneficial bacteria in UC model rats. Lactobacillus sp. and lachnospiraceae bacterium were probably considered as key bacteria in the treatment process.3. Effects of electro-acupuncture treatment on UC in rat urine key metabolites have the presence of benign regulation function,in which3-D-hydroxybutyrate. hippurate and acetate are recognized as key metabolites in electro-acupuncture treatment for ulcerative colitis. Electro-acupuncture has a protective effect on intestinal mucosal, and can promote cell to utilize the energy, which may be the influence of acupuncture on intestinal micro-flora and the main mechanism.4. Electro-acupuncture has a protective effect on intestinal mucosal. and can promote cell to utilize the energy, which may be the influence of acupuncture on intestinal micro-flora and the main mechanism. |
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