| Objective:Ulcerative colitis(UC) is a chronic, idiopathic inflamatory disease that can affect the colon. It’s characterised by relapsing and remitting mucosal inflammation. UC usually started in the rectum and extended to proximal segments of the colon. Over the last decade,the incidence of ulcerative colitis has been increasing in Asia. The incidence of ulcerative colitis in China now is 11.6 per 100 000), it has become a common disease of digestive system. However, the pathogenesis of UC is still not clear. It is now recognized that UC is a chronic immunologically mediated disease at the intersection of complex interactions between genetics, environment and gut microbiota.The main symptoms of UC are abdominal discomfort, diarrhea, bloody stools, malaise ,anorexia and weight loss. Among thoses, mucous and bloody stool is the characteristic presentation. Some Extra-intestinal manifestations (EIMs) such as osteoarthritis, cholangitis,skin lesions and conjunctivitis may be occurred in about 1/3 UC patients. Colonoscopy usually showed mucosal erythema, erosion, engorge, swelling, brittle, pellet feeling enhance, bleeding and ulceration in UC patients. Up to now, there is no effective method to prevent and cure this disease, leading to the delay recovery and poor long-term prognosis.The current treatment options are the combinations of 5-aminosalicylic acids (5-ASAs),hormones, immunosuppressive drugs and biologics. The primary aim of medical management is to induce and maintain remission with the long-term goals of preventing disability, colectomy, and colorectal cancer. Although, these therapeutic options have solved many clinical problems, However, the problems such as high cost and drug side effects could not be ignored, novel therapeutic interventions need to be developed urgently.Recent studies suggested that intestinal microecology is closely related to the occurrence and development of many diseases such as UC. The abundance of intestinal flora of the patients was significantly lower than that of the health people, and the structure of the bacterial community changed significantly. To treat diseases by changing the intestinal micro ecological efforts have achieved remarkable results, fecal bacteria transplantation (Fecal Microbiota Transplantation, FMT) as a technique of changing the intestinal flora is both ancient and innovative, it’s effective rate of the treatment of CDI was as high as 90%. Large number of studies have found that FMT has some therapeutic value for UC, but the mechanism is unknown. Researchers observed that in the early stage of UC,the colonic mucosal barrier function is damaged and the intestinal flora is disordered.It is suggested that intestinal mucosal barrier function is very important in the pathogenesis of UC. Recently, it has been found that the intestinal mucosal barrier function can be improved by adjusting the intestinal flora.Methods:1. Mice were randomized into four groups. There were 23 mice in each group. The control group did not have any treatment. The "Model" group was treated with only DSS.The "FMT prevention" group was treated with DSS plus FMT followed by extra time for recovery. The "FMT treatment" group was treated with DSS followed by FMT.2. FMT bacteria liquid was preparaed for both FMT prevention group and FMT treatment group;3. 16S rRNA analysis was used to analyze the structure of the bacterial flora of FMT,and to control the quality of FMT liquid;4. The disease activity index of each group was assessed.5. The fecal occult blood was tested in each group;6. HE staining was used to observe the histopathological changes of the colon ;7. The ultrastructure of colonic epithelium was observed by transmission electron microscope;8. Immunohistochemical staining was used to observe the expression of ZO-1 and Occludin in the colonic epithelium of mice ;9. Serum D- lactate levels were detected in each group;10. The content of TNF- alpha in serum of each group was detected;11. The intestinal flora was analysised in mice of each group;12. Iamage J and SPSS 19.0 software were used to analyze the data.Results:1.The general conditions of mice were observed in each groupModel group, FMT prevention group and FMT treatment group of mice induced by DSS began to appear tired, dull coat, soft stools and other symptoms, and gradual emergence of diarrhea, gross blood stool and rectal bleeding. After Discontinued the DSS,mice in the FMT treatment group were recovered significantly and it’s faster than those in the model group and the FMT prevention group.2. Changes of body mass were observed in each group.After DSS administration, except the control group, mice in other three groups showed a significant decline in body weight, body mass of FMT prevention group and model began to decline continuously at day 3, and reach the lowest point at day 10, FMT treatment group mice body weight dropped to the lowest point at day 7, and began to recovery gradually. At day 14, the body weight of FMT group was significantly higher than that of model group and FMT prevention group (P < 0.05).3. Changes of disease activity index were detected in each group.After DSS induction, disease activity index of mice in model group, FMT prevention group and FMT group were increased, and reached the peak at day 8, then decreased gradually. On the fourteenth day, disease activity index of mice in FMT treatment group was lower than that of FMT prevention group and FMT group (P < 0.01).4.Colon of mice in each group were detected in each group.After DSS induction, the colon of mice in model group, FMT prevention group and FMT treatment group became lower transparency, scattered hemorrhage, partial intestinal constriction, shortening and so on. After 7 days of FMT the length of mice in FMT treatment group was significantly longer than the model group and FMT prevention group(P<0.05), and without significant bleeding on the surface of the colon.5. Histological changes of mice colon were observed in each group.After 7 days of DSS induction, colon tissue of mice in model group, FMT prevention group and FMT showed infiltration of inflammatory cells, ulcer formation, epithelial cell shedding, after 7 days of treatment of FMT, mice of FMT group recovered better than FMT prevention group and model group .6. Ultrastructural changes of colon tissue of mice were detected in each group.After 7 days of DSS, the morphology of epithelial cells and the tight junctions between cells in FMT group were significantly better than those of model group and FMT group,after 7 days of intragastric administration of FMT, the morphology and intercellular tight junction of colonic epithelial cells in FMT treatment group were better than those of model group and FMT prevention group.7. Expression of ZO-1 and Occludin in colonic epithelial cell of mice were observed.ZO-1 and Occludin expressed in mice colonic epithelial of control group at day7 and day 14. The expression of ZO-1 and Occludin in colonic epithelial tissues of mice in model group was significantly lower than that of control group at day 7, then recovered at day 14;Compared with the model group, the expression of ZO-1 and Occludin in colonic epithelial tissue of mice in FMT group was significantly increased at day seventh (P < 0.01),and at day 14 the expression of ZO-1 and Occludin had no significant difference with model group;The expression of ZO-1 and Occludin in colonic epithelium of mice in FMT treatment group was significantly higher than that in model group (P < 0.05) at day 14.8. The level of serum D- lactic acid of mice was detected in each group,The serum D- lactate level of mice in model group was significantly higher than that of the control group (P <0.01) at day 7 and day 14 .At day 14, the serum D- lactate level of FMT treatment group was higher than that of the control group, but was significantly lower than the model group (P < 0.01), suggesting that the colonic mucosal barrier function restored.9. The level of serum inflammatory factor TNF- alpha was detected in each group.The serum TNF- a level of mice in model group was higher than that of control group at day 7 and day 14 (P < 0.01). After 7 days of FMT the serum TNF-α levels of mice in FMT treatment group was higher than those in control group, but compared with the model group were significantly decreased (P < 0.01), with the reduction degree of inflammation.10. The quality of FMT bacterial solution for transplantation was controlled .The results showed that the number of bacteria contained in each FMT bacterial fluid was the same order of magnitude, and the structure of the flora was basically coincide at the genus level, so the structure of FMT bacterial flora was stable.11. The intestinal flora of mice was analysised in each groupThe fecal microflora structure was changed compared with before DSS administration in mice induced by DSS for 7 days. The Lactobacillus and Bacteroides decreased significantly and Helicobacter species increased. The proportion of Lactobacillus was not restored, but the increased level of Bacteroides decreased, and Helicobacter species continued to increase after discontinuation of DSS administration and natural recovery for 7 days. The intestinal bacteria in mice of FMT prevention group changes are similar with the model group after 7 days of transplantation, but Lactobacillus spp. and Allobaculum increased significantly when compared with the model group after discontinuation of DSS administration and natural recovery for 7 days. Whereas Bacteroides and Helicobacter species reduced. After fecal bacteria transplantation the fecal flora structure of mice in FMT treatment group changed, compared with the model group appeared more obvious increase of Lactobacillus Bacteroides and Helicobacter species decreased, showing direction of control group.Conlusion:1. After 7 days DSS administration, The intestinal flora of mice in the model group were disordered and intestinal mucosal barrier were damaged;2. In the 14 day period, symptoms, colon tissue inflammation and intestinal mucosal barrier of mice in FMT prevention group recovery is not obvious, after FMT transplantation the intestinal flora disorder has no obvious improvement, but out of DSS drinking water for 7 days recovery, FMT intestinal flora is obviously better than that of model group,suggesting that in the presence of DSS, FMT may lead some bacteria colonized in the intestine of mice as "seed bacteria" form, and multiply rapidly after stopping DSS.3. After FMT treatment, the intestinal flora, colon gross appearance, histological changes, inflammation index and intestinal mucosal barrier of tight junction protein ZO-1 and Occludin expression of mice in DSS model group were significantly restored. And the symptoms of mice were significantly improved ,these results indicated that the effect of FMT on DSS induced mice colitis was remarkale.4. FMT may play a key role in the treatment of colitis in DSS mice by regulating the structure of intestinal flora and promoting the recovery of colonic mucosal barrier function in mice. |
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