The Study Of Protective Function That BBR Play In The DSS-induced Colitis In Mice

[Purpose]In order to establish mouse model of colitis,we observe the effects that the BBR have on tissues, cells, affect the molecular level in mice with colitis.This research will provide evidence for the development of BBR as a new use in the treatment of colitis.[Methods]1 Establishmet of experimental animal modelThe establishment of model will be divided into two stages, the first stage is the DSS-induced colitis stage, the second stage is treatment stage, different experimental groups have different treatment in two stages. The C57 male mice were random Ly divided into four groups by weight and sex, and every group had eight mice.Respectively, the control group without any treatment of,the group administered by high dose of Berberine,he group administered by medium dose of Berberine,the group non-administered by Berberine.Apart from the control group, other three groups were fed with 2% DSS, and DSS would be removed the fifth day. Medieum and high doses of the drug were administered on sixth day as 50 mg / kg, 100 mg / kg. Monitored and recorded the weight, loose stools, blood in the stool and other changes in mice.2 HE staining evaluationWe took colon tissue on the tenth day, fixed tissues with 10% neutral formalin,embedded in paraffin with embedded machine, cut slices with slicer, then dewatered and hydrated, stained according to the instructions of HE staining kit, obseved and photographed under a fluorescence microscope, and scored HE staining results as the scoring criteria.3 Effects of Berberine on intestinal floraWe took feces on the eighth day in mice, dissolved into three concentration gradient(10,1,0.1 mg / m L)with sterile PBS, each concentration coating 3 boards, each board coated plate 600μL fecal bacteria solution, cultured in 37℃ oven overnight, and then observed and counted the number of colonieson the second day. Finally,we calculated the number of colonies and analyzed the Berberine,s impact on fecal colonies in mice.4 Flow cytometric analysis of the proportion of inflammatory cells in each group in mice colitisColon tissues of three groups were taken(not including the high-dose group) on tenth day, and lamina propria mononuclear cells separated(LPMC)of the colon,marked with two group of flow antibody(Ly6c-PEcy7 and CD11b-APC, Gr-1-FITC and CD11b-APC),incubated on ice for 30 min in dark, washed additional antibody with 1640 mediumincluding 3% serum, and finally fixed with 2% paraformaldehyde, temporarily put4 ℃ refrigerator stored.When the cells were transferred to fixed good flow of sample tubes,we started the flow analysis instruments and software, debugging cell grouping, collected cells to 1 × 105, stopped detection, accessed to data.5 Q-PCR to detect the expression of inflammatory cytokinesWe collected colon tissues on the tenth day, a small part of the tissue was ok.We grinded the tissue to a powder in liquid nitrogen and then added chloroform and Trizol, took water layer, added an equal volume of isopropanol and centrifuged to get RNA precipitate,then washed precipitation with 75% alcohol ethanol, and finally dissolved precipitate with water uncluding RNA enzyme to obtain the RNA,saved at- 80 ℃. After digestion by DNA enzyme to remove DNA, we treated the RNA with 70 ℃ metal bath for 10 min, and prepared for 20μL reaction system as reverse transcription kit,and then got c DNA in PCRby reverse transcription,Finally,we perfomed by Q-PCR, and analyzed data by IQ5 software.[Results]1 Changes of phenotypes in the annimal model1.1 Weight trendsWe recorded daily body weight of mice, and drew the body weight curve with Graphpad Prism 6 software and found body weight of mice gradually recovered which given Berberine,and weight of mice which given high-dose BBR recovered better than lower ones,but body weight of body weight none-administered BBR continued to decline,maintained poor body state.1.2 Stool changeFeces of mice groups non-administered Berberine changed as aggravation as colitis,which normal form gradually became shapeless loose stools;groups of high and lower doses of BBR in the stage of colitis gradually formed loose stool, while in the treatment stage,the stool form slowly returned to near normal, and high doses groups, loose stools recovered better.The blank control group of mice,s feces shape had no changes.1.3 Anal bleeding changesAnal bleeding of mice groups non-administered Berberine changed as aggravation as colitis,which occult bleeding gradually become visible bleeding;groups of high and lower doses of BBR in the stage of colitis gradually form visible bleeding, and in the treatment stage, bleeding improved recovery, and mice given high doses of drug got better than the other experimental drug group. Control group without any treatment of mice had no abnormal situation.1.4 Colon morphological changesColon of mice without Berberine became thicker and shorter, while the groups with middle and high doses of Berberine, the colon recovered close to normal after the treatment of the drug, and the colon from high dose of BBR recovered better than lower ones.2 HE staining changesColon tissue morphology of negative control group was intact, structure stratification was significantly, no damage, no inflammation; colon tissue of model mice had serious damage, basic structure of the mucosa was destroyed, a large number of goblet cells was missed, infiltration of lymphocyte in lamina propria was visible, edema and hyperemia in muscularis mucosa was obvious; while the medium and high dose of groups of mice had less damage, structured was organizational, infiltration inflammatory cell in lamina propria was decreased, goblet cells was relatively increased, edema and hyperemia in muscularis mucosa was not found, tissue morphology has been significantly restored.3 Effects of Berberine on intestinal floraThe number of colonies rarely in negative control group, only 104 orders of magnitude;the number of colonies of mouse feces after the administration of Berberine significantly reduced(P<0.01), we decided that Berberine has the effect of plant antibiotics to inhibit propagation of intestinal flora after injury, and thus play a protective function.4 Flow cytometric analysis of the proportion of inflammatory cells in each group in mice colitisIn a ratio of mononuclear cells, there were no significant difference between the medium dose of group and model group; while in the proportion of neutrophils, the medium dose of group was significantly lower than the model group. We speculated that the main mechanism of inhibition of BBR in colitis may inhibit the migration and aggregation of neutrophil.5 Q-PCR to detect the expression of inflammatory cytokinesExpression of repair factor TGF-β increased,and pro-inflammatory cytokine TNF-α,IL-23 expression decreased in the group which given Berberine.[Conclusion]Berberine have a protective effect on DSS-induced colitis,Berberine can reduce weight decrease,loose stools and blood in the stoolcaused by colitis, so that normal colon morphology,tissue damage improved, the proportion of inflammatory cells neutropenia,would be lower with decreased expression of inflammatory cytokines, and increased repair factor expression, while inhibition of intestinal bacteria invasion, eventually colitis can be cured.