Effect Of Polygala Root Aqueous Extract On The Experimental Colitis Of Mice And Its Mechanisms

Background and objective:Inflammatory Bowel Disease (IBD), including Crohn's disease (CD) and ulcerative colitis(UC), of which the etiology and pathogenesis is unknown , is a characteristically chronic disease resulting from the combined contribution of environmental factors, genetic factors, microbial factors, and the intestinal immune system. The role of immune factors is becoming more and more important. It's difficult to treat and there are no known cures at the present. Current treatment strategies endeavor to control the underlying inflammation, and conventional anti-inflammatory medicines including 5-aminosalicylic, glucocorticoid, immuno -suppressive agent and immunomodulator, are used widely. But they have many adverse effects. So it is important to find the effective therapy medicine in field of IBD research.According to its clinical symptoms, IBD belongs to "diarrhea" and "dysentery" in traditional Chinese medicine. The polygala root, which can benefit intelligence, calms the nerves and has the function of reducing engorgement and anti-inflammation, and so on. The polygala root is a common medicine, of which the mechanism is unclear. In order to know the effect of the polygala root aqueous extract, experimental trinitrobenzene sulfonic acid (TNBS) colitis models were used .Materials and methods: 1. The establishing of experimental colitis modelsTo establish the colitis models, male BALB/c mice were received 100μ1 enemas of lmg TNBS in 50% ethanol. The weight, survive, diarrhea, disease activity index (DAI) score, colon macrophage and microphage histological score were observed.2. Observed the effect of polygala root aqueous extract on the TNBS modelsExperimental colitis was induced by TNBS, and its severity was evaluated bygross colon injury, histological damage, body weight and disease active index. Administered the polygala root aqueous extract (2g crude drug /kg and 4g crude drug /kg) on the TNBS models once a day after 24 hours of making experimental model. We studied the effect of the different dose of polygala root aqueous extract on all these parameters.3. Investigated the mechanism of polygala root aqueous extract on the TNBS modelsAll colonic surgical specimens were collected immediately after being resected. Expression of interleukin-4(IL-4) mRNA and interferon(IFN)-γmRNA in colon mucosa was observed by RT-PCR. The rest of specimens were opened longitudinally, rinsed, examined for gross morphological changes, and a representative full-thickness sample was obtained. The colon was opened longitudinally, washed several times in Sodium Chloride including of penicillin and streptomycin to remove feces and debris, and then incubated in 0.5 mM EDTA in calcium-free and magnesium-free HBSS for 20 minutes at 37°C in swing bed to abstract intraepithelial lymphocytes and epithelial cells. It was repeated twice after thorough washing. And then the tissues were cut into small pieces. The tissues were digested then put in RPMI 1640 containing 1 mg/ml type I collagenase for 120 minutes at 37°C in 5% CO2-humidified atmosphere. Lamina propria mononuclearcells (LPMCs) were finally isolated from the interface of a 40%/70% Percoll gradient. The cells were stimulated for 48 hours with Abs to anti-mouse CD3 and anti-mouse CD28, followed by the collection of culture supernatant and analysis of the IFN-γand IL-4 content by enzyme linked immunosorbent assay (ELISA). Established standard ELISA curve of IFN-γand IL-4, IFN-γand IL-4 in cell culture supernatants was detected and quantified using a specific sandwich ELISA and 96-well plates.4.statistical analysis was performed with SPSS-13.0 software. All data was demonstrated with x±s. The data was analyzed by paired t-test for independent sample. P<0.05 was considered significant difference. Results:1. The experimental results suggested that mouse with the TNBS/ethanol enema developed diarrhea and blood stool. The weight decreased significantly. The scores of DAI, gross colon macrophage and microphage histological were increased. Further research indicated that 10 days after the enema, the inflammation still evidently existed in mice's colon. According to the histological characters, mice with TNBS colitis were remarkably similar to human CD.2. The results indicated that the polygala root extractive significantly suppressed the various manifestations of experimental colitis as was demonstrated by substantial reduction in the macroscopic colonic damage, preservation of the microscopic colonic structure, reduction of weight loss. It suggested that they could prevent and treat TNBS inducing colitis. It is not significant between the high and low dose of the polygala root extractive.3. Expression of IFN-γmRNA and IL-4 mRNA in colon mucosa was observed by RT-PCR. In the model group, the level of IFN-γmRNA was significantly increased than that of normal control group (.P<0.01), but expression of IL-4 mRNA was significantly lower (.P<0.01). Compared with the TNBS model group, the expression level of IFN-γmRNA in colon mucosa, the polygala root aqueous extract therapy groups, was significantly decreased; but IL-4 mRNA was significantly increased. It has no significant diversity between the two therapy groups. It is quantified to use a specific sandwich ELISA. LPMCs culture supernatants cytokine. The TNBS model group, the LPMCs culture supernatants basic level of IFN-γwas significantly increased than that of normal control group, but IL-4 production was significantly lower. Compared with the model group, the level of IFN-γ, in which the polygala root aqueous extract therapy groups' LPMCs producted, was significantly decreased; but IL-4 production was significantly increased. Conclusions:1. Applicated 1 .0mg TNBS in alcohol on a BALB/c mouse can induce the mice's acute colitis successfully.2. These studies provide that administration polygala root aqueous extract are effective on treating experimental colitis. There are not dose-effect relationship between Two different doseage polygala root aqueous extract groups.3. The mechanisms of which the polygala root aqueous extract treats the TNBS colitis may be through up-regulating the activation of IL-4, and inhibiting the expression level of IFN-γand releasing cytokines and mediators which are related to the colon mucosal inflammation.