The Effects Of Fecal Microbiota Transplantation And Probiotic Treatment On The Occurrence And Development Of Dextran Sulfate Sodium-induced Colitis In Mouse Model

Objective To evaluate the effects of fecal microbiota transplantation(FMT)and probiotic treatment on the occurrence and development of dextran sulfate sodium(DSS)-induced colitis,we established the mouse model and treated the mice with FMT and probiotic to study the role of interventions in developing DSS-induced colitis and underlying mechanisms.Methods 1.Todetermine the ideal mouse model of acute colitis,C57BL/6J,C57BL/6N and BALB/c were fed with sterile distilled water which contained with 3%or 5%DSS for 6 days.Mice were examined once a day to record the weight loss,stool consistency and the presence of fecal blood then the disease activity index(DAI)was charactered.Fecal occult blood was measures by otoluidine.2.Along with drinking sterile distilled water containing 3%DSS,the C57BL/6J mice were given FMT which come from normal mice.Feces was cultured 2-3 hours under anaerobic condition and fecal residue was removed by simply filtering before intragastric administration.3.The C57BL/6J mice were fed with sterile distilled water containing 5x108 CFU/mL HT121-2 Lactobacillus one week prior to 3%DSS water treatment,the mice were continued to be inoculated orally with 5 × 1 08 CFU/mouse/day through the whole experiment period.4.The gene expressions in colonic tissue were examined using the real time PCR and the distribution and function of IL-17A-producing cells and IL-22-producing cells were analyzed using flow cytometry.Results 1.The establishment of the DSS-induced mouse colitis:(1)The body weight of three strains of mice were compared with their respective normal control mice,the weight lose of C57BL/6J started ealier and were greater than the C57BL/6N and BALB/c mice.The percentages of weight loss of C57BL/6J,C57BL/6N and BALB/c mice at day 6 were 21.79±2.58%,12.52±3.97%and 2.88±4.76%respectively.(2)The stool scores of colitis group in C57BL/6J were significantly increased from day 3 to the end of experiments compared with non-colitis control group,while no difference was observed between colitis and control group in both C57BL/6N and BALB/c mice model.(3)The scores of presence of fecal blood and DAI in C57BL/6J colitis group were significantly greater than control group during the whole period of experiment.The presence of fecal blood in C57BL/6N and BALB/c delayed and occurred at day 4.(4)The length of colons in C57BL/6J and BALB/c colitis group were significantly shorter than control group while there is no difference in C57BL/6N strain.All above,we concluded that C57BL/6J mouse is the ideal DSS-induced colitis model to conduct the following evaluation experiments.2.Evaluation of FMT intervention:Our data show that FMT intervention significantly prevented the weight loss of colitis mice at day 6 compared with the non-treated group(11.49±3.03 vs 21.79±2.58%,P<0.01).The scores of stool consistency in FMT group at day 5 were significantly lower than it in non-treated group.The scores of presence of fecal blood and DAI in FMT group were significantly lower than those in non-treated group from day 2.The mean of colon length in FMT group were also significantly longer than it in non-treated group(4.90±0.39 vs 4.41 ±0.41 cm,P<0.01).The FMT also attenuated the histopathology change compared with the non-treated group.The result of genes mRNA expressions in colon tissue showed that DSS induced many genes expressions to varying degree.It was noted that FMT intervention caused significantly upregulation of Claudin2 gene compared with non-treated group(0.75±0.32 vs 0.36±0.20,P<0.05).All these results suggested that FMT intervention obviously attenuated the symptom of colitis and pathology of colon.FMT might affect the development of colitis through mechanism of modulating host gene expression such as Cludin2 gene to maintain the intact of epithelial.3.Evaluation of HT121-2 Lactobacillus intervention:The results showed that the scores of stool consistency and DAI in probiotic-treated group at day 3 and 4 were significantly lower than it in non-treated group.Probiotic treatment had no effect on weight loss and scores of fecal blood in DSS-induced colitis mouse model.The mean of colon length at day 6 in probiotic-treated group was significantly longer than it in non-treated group(4.7±0.35 vs 4.38±0.40 cm,P<0.05).The probiotic also attenuated the histopathology change compared with the non-treated group.We observed that the mRNA expression of antimicrobial peptide Reg3y in colon was significantly increased in probiotic-treated group compared with non-treated group(156.24±49.46 vs 89,25±46.20,P<0.05).All these results suggest that probiotic treatment plays a role in preventing the development of DSS-induced colitis in mouse model and the upregulation of antimicrobial peptide Reg3y induced by probiotic treatment might be involved in the mechanism of Lactobacillus intervention.4.The pilot study of distribution and function of type 3 innate lymphocytes in DSS-induced mouse colitis:The present results show that DSS treatment caused the decrease of percentage of lymphocytes in mouse intestinal intraepithelial and recruitment of more lymphocytes in lamina propria.The percentages of IL-22 and IL-17A-producing cells in Lineage+ CD4+ were all less than 1%in lamina propria,suggesting that the cell source of IL-22 and IL-17A-producing cells were not Lineage+ CD4+ cell in lamina propria of DSS-induced colitis.We further found that the percentages of IL-22 and IL-17A-producing cells in Lineage-CD 127+ cells were 43.34%and 18.04%respectively,which means that the main cell sources of IL-22 and IL-17A-producing cells are Lineage-CD127+ innate lymphocytes in lamina propria of colitis mouse model.Conclusions 1.C57BL/6J was the ideal strain for DSS-induced colitis model.2.Both FMT and probiotic treatment attenuated the symptom of colitis and pathology of colon.DSS colitis induced several genes upregulated including IL-17A,IL-22,Claudin2,TNF-?.FMT and Lactobacillus intervention might affect the host colonic gene expression in a different way.3.The main cell sources of IL-22 and IL-17A-producing cells are Lineage-CD127+innate lymphocytes in lamina propria of DSS-induced colitis mouse model.