| Objective:To investigate the function of Probiotics to control the balance of Th1 / Th2 cells of colitis in rats,Which was induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS), and further study the possible immune mechanisms and pathways of probiotics to prevention and treatment of Ulcerative colitis. Methods:34 specific pathogen-free(SPF) male rats, according to a completely random method divided into two groups, control group(A,n = 9) and model group(n = 25). The rats in model group were dealed with TNBS to build the model of Ulcerative colitis. The next day, two rats were pick up randomly from each group and then stained the colon tissue sections with HE method,by light microscope to verify colitis model was constructed successfully. model group were then randomly divided into three groups: B group : model group(n = 8); C group : probiotics intervention group(n = 8); D group: sulfasalazine intervention group(n = 8),and label to all rats.After model was completed, each rat of A group and B group using normal saline 1ml / day,intragastric infusion for one week; each rat of C group and D group daily dose dissolved in 1ml normal saline to gavage for one week. Observed the activity, mental status,coat color,eating, stool and weight changes of the rats every day. and recorded detail.Based on changes in body weight, stool and blood in the stool to compute DAI scores.On the 8th day, all rats were killed and cut out the colon, to rinse the colon immediately with ice-cold PBS until clean, and then use dried and clean filter paper to blot moisture.After macroscopic examination and score, according to the lable of each rat, part of the tissue of each rat was placed in 13% neutral formalin fixative fixed for 24 hours, and then to embed in paraffin, HE staining and immunohistochemistry to detect T-bet and GATA-3. The remaining part of the colon according to the different lable,weighed and wrapped by the aluminum foil, quickly put into liquid nitrogen(-196 ?) to frozen,and then transfer to ultra-low temperature freezer(-80 ?) for storage as soon as possible, in order to detect IFN-?, IL-12, IL-4, IL-10 by ELISA. Results: 1.A group rats throughout the experiment process activities better, eating normally, increase in weight, diarrhea and blood in the stool does not appear. The remaining three groups of rats TNBS row after modeling process are different degrees of activity and reduce the consumption, weight gain or reduced, diarrhea and blood in the stool. Both B group and D death a rat. After C group rats intervene by probiotics, compared with B group,the basic situation improved activity and increased consumption, body weight increased slowly relieve diarrhea and blood in the stool, the stool forming and visible, no obvious bloody stools. After feed on sulfasalazine, D group increased eating, weight gain, diarrhea and blood in the stool reduced stool forming, blood in the stool better. After the probiotic and sulfasalazine intervention, from the third day, the DAI scores in C group and D group compared with B group was statistically significant(P <0.05), there was no significant difference between C group and D group(P> 0.05).On the 6th day,Model group basic situation was worst, DAI score was highest.2. Colon tissue macroscopic and microscopic pathology Rating: A group of rats colon wall thickness and mucosal folds were normal,no edema and hemorrhage;Microscopic layers of the intestinal wall structural is integrity,the glands arranged in an orderly manner.B group a localized thickening of the colon,deformation of the lumen,tered distribution of mucosal folds.Visible edema, hemorrhage, erosion and ulceration of varying sizes.Microscope examine we found the epithelial necrosis, goblet cells decreased, glands rupture and row cases of disorder,In addition,crypt abscesses can be obvious.After treated by probiotics and sulfasalazine,C and D group the wall thickness was uniform, mucosal folds was clear, no blocking, without congestion and edema.observed under the microscope, the damage of epithelial cells and glands was reduced, less inflammatory cells infiltration,ulcer healing on varying degrees.Both of these improvement which compared to B group was significant difference(P <0.05).there was no obvious statistically difference between C group and D group(P> 0.05).3.The expression of IFN-?,IL-12, IL-4 and IL-10 in four groups colon tissues:the B group rats colon IFN-? and IL-12 measured by ELISA,the two Th1 cytokines were significantly increased,the changes compared with A group was obvious statistically difference(P <0.01); Th2 cytokines IL-4 and IL-10 content were significantly reducing in group B,there was a significant difference which compared with A group(P <0.01).The three group C, D and B compared : C and D group compared with B group, IFN-? and IL-12 content were significantly decreased, and the difference was statistically significant change(P<0.05); IL-4 in colon tissue content in both of the intervention group were significantly higher,the change difference compared with B group was statistically significant(P <0.05); the expression of IL-10 in the two treatment groups were increased,but only C group change difference was statistically significant(P <0.05).4.Compared with A group,B group expression of T-bet is significantly increased, the difference was statistically significant(P <0.05); after the intervention of probiotics and sulfasalazine, T-bet in the C Group and D Group expression intensity compared with B group, the difference was statistically significant(P <0.05); Compared C group with D group,there was no significant difference(P > 0.05), but To be viewed from mean, C group(0.39) is lower than D group(0.41). Compared with A group, the expression of GATA-3 in B group was slightly increased, but the difference was not statistically significant(P> 0.05);after the intervention of probiotics and sulfasalazine, the expression of GATA-3 in group C and group D intensity increased which Compare with B group, the difference was statistically significance(P<0.05),Compared C with D,the expression of GATA-3 in C group was higher than D group, the difference was statistically significant(P <0.05).Conclusions:1.TNBS combined with ethanol maybe the best way to lead to ideal model of Ulcerative colitis in rats. 2.The model of Ulcerative colitis in rats Construct by TNBS combined with ethanol,the cytokines IL-12 and IFN-?of Th1 expression increased, the cytokines IL-4 and IL-10 of Th2 expression decreased.3.Probiotics can promote the production of Th2 cytokines IL-10 and IL-4 in rats deal with TNBS and ethanol,inhibit Th1 cytokines IL-2 and IFN-? production, this indicates that probiotics can adjust the balance of Th1/Th2 cytokine,and thus play an anti-inflammatory and immunomodulatory effect.4.Probiotics can induce Th2 cell-specific transcription factor GATA-3 expression and inhibit the expression of Th1 cell-specific transcription factor T-bet, indicating that probiotics by influence the expression of cellular transcription factor to regulate the balance of Th1 / Th2 cells. |
PDF Full Text Request