ObjectionTo establish dextran sulfate sodium salt(DSS)-induced acute colitis murine model,study the protective effect of EBN on colitis and further explore the potential mechanism behind this experiment from Th17/Treg balance.Methods1.Establish DSS-induced acute colitis modelAcute colitis in mice was induced by different concentration DSS which were supplemented in drinking water for 7 days then drinking normal water for 3 days.During DSS treatment,body weight,stool consistency and stool occult were daily monitored.At the end of experiment,the entire colon was removed from sacrificed mice and its length was measured.2.Effects of EBN on DSS-induced acute colitisMale C57BL/J mice were randomly divided into six groups:normal control(control),model group(DSS),sulfasalazine group(SASP),high-dose EBN group(EBNH),mediumdose EBN group(EBNM)and low-dose EBN group(EBNL).The mice were pretreated with EBN or water for 42 days and drinking DSS on day 43 to induced acute colitis.The SASP group was intragastrical with sulfasalazine on day 43.The EBN groups were continue oral EBN until the end of the experiment.The normal and model groups were intragastric administration with distilled water.In pretreatment stage,the body weight of each mice was weekly recorded.During the modeling process,body weight,stool consistency and stool occult were daily monitored.After the experiment,the entire colon was removed from sacrificed mice and measured its length.Subsequently,distal colonic tissues were fixed in 4%paraformaldehyde and stained with H&E to evaluated histological change.In addition,homogenized colon tissue to detecting the activities of myeloperoxidase(MPO),and expression of IL-1β and TNF-α.3.Effect of EBN on Th17/Treg cell balance in IBD miceAt the end of the treatment,animals were scarified and collected colon tissue to evaluated the cytokine content(IL-17A,IL-10,IL-6 and TGF-β).Flow cytometry analysis proportion of Thl7 and Treg cell in spleen.The level of protein expression(IL-17A and Foxp3)was determined by immunohistochemical analyses and western blot.Results1.Establish DSS-induced acute colitis modelCompared with the normal group,the mice with a concentration of 2%showed a trend of weight loss from the sixth day(P<0.05),while the 3%group lose sharply from the seventh day(P<0.001).The DAI score of the 2%concentration group increased significantly from the third day(P<0.01),whereas the 3%DSS group from day 1 to day 10 was significantly higher than that of the normal group(P<0.05).The colon length of mice at each concentration was significantly shorter than the normal control group(P<0.001).2.The effect of EBN on DSS-induced acute colitisCompared with the normal group,the DSS group developed severe colitis manifestations,including rapid weight loss,diarrhea,bloody stool,increased DAI and shortened colon.DSS induced significant colon tissue injury,as epithelial destruction,intense inflammatory infiltration and crypt damage.MPO activity as well as content of IL-1β and TNF-α in colon was significantly increased compared with normal control group(P<0.05,P<0.05,P<0.05).Compared with the model group,groups treated with either SASP or EBN alleviated the extent of body weight loss,diarrhea,bloody stools,DAI and colon length shortening.Meanwhile,SASP and EBN group largely prevented colon tissue injury,including mild infiltration of inflammatory cells and an obvious relief in the structure destruction.The SASP and EBN group dramatically reduced the activity of MPO and production of IL-1β and TNFα(P<0.05,P<0.01,P<0.001).3.Effects of EBN on the balance of Th17/Treg cells in IBD miceCompared with the normal group,DSS-treated mice showed significant increased production of IL-6,IL-10,IL-17 whereas decrease TGF-β content(P<0.001,P<0.001,P<0.001,P<0.001).The proportion of Th17 cells and Treg cells in the spleen were increased on the DSS group compared with the normal control group(P<0.001).Western blot and IHC results both demonstrated increase in IL-17A level and Foxp3 level in DSS group as compared to the normal control group(P<0.001,P<0.001).However,SASP and EBN group significantly reduced the content of IL-6,IL-10,IL-17(P<0.001,P<0.01,P<0.05)increased TGF-β(P<0.001)production in colon tissue.The percentage of Th17 cells and Treg cells were reduced compared with the DSS group(P<0.001,P<0.01).IL-17A and Foxp3 levels were significantly downregulated following SASP and EBN treatment compared with DSS group(P<0.001,P<0.001).Conclusion1.The optimal induction concentration under the experimental conditions is 2%,which is selected as the simulated concentration for subsequent studies.2.The protective effect of EBN on colitis mice through alleviate the symptoms,reduce the activity of MPO,IL-1β,and TNF-α in the colon tissue of mice.3.The protective mechanism of the bird’s nest on DSS-induced acute colitis in mice may be related to the regulation of Th17/Treg-related inflammatory factors and protein expression to restore the balance of Th17/Treg cells... |