The Effect Of Berberine On The Secretion Of Cytokines In Intestinal Epithelial Cells

Objective:We use IBD model induced by DSS through berberine treatment,isolate IEC and detect several cytokines of IEC.We select cytokines that can play an important role in regulating Th2 and Th17 function,such as IL-6,IL-18,IL-23,IL-25 and IL-33,and detect several representative cytokines of LPL,such as IL-4,IL-10,IL-17A and IFN-?for understanding the possible regulating role of the cytokines released by intestinal epithelial cells,and also exploring the new mechanism that the secretion of cytokines effected by berberine in intestinal epithelial cells can indirectly regulate the immune function of intestinal immune cells.Methods:1.Model establishment and experimental group:C57BL/6 mice were used to establish the animal model of DSS induced acute enteritis:within five days,the experimental group(berberine+DSS)and the control group(DSS)were free to drink 3%DSS(with PBS solution),and the blank control group(PBS)mice were free to drink PBS solution.Experiment group was given berberine at 8 am every day,and the control group and the blank control group were given PBS every day(with the same dose).Three groups of mice were killed on the sixth day.2.Referring to the Cooper HS method:mice were measured the weight,and observed the fecal traits and defecate.3.Identification of histological results:Models of acute enteritis induced on the sixth day,after the death of the three groups of mice,near the heart 1/2 colon tissue 0.5 cm are soaked in 4%paraformaldehyde solution,the dehydration,paraffin embedding,sectioning and using microscope observed model organization morphological structure and inflammation after HE staining.We score the section according to the classification standard of inflammation pathological and histological score evaluation.4.Identification of intestinal epithelial cells and detection of cytokines:Acute enteritis model on the sixth day,we kill the mice,separation and extraction of intestinal epithelial cells(IEC);using flow cytometry to identify the IEC and counting cells;using Real-time PCR and Western blotting to detect cytokine IL-6,IL-18,IL-23,IL-25 and IL-33 expression levels.5.Identification of intestinal lamina propria lymphocytes and detection of cytokines:Acute enteritis model on the sixth day,we kill the mice,separation and extraction of intestinal lamina propria lymphocytes(LPL);using flow cytometry to identify the LPL and counting cells;After LPL stimulated 48h in vitro,the expression levels of IL-4,IL-10,IL-17A and IFN-?were detected by ELISA.Results:1.DSS induced acute enteritis in mice was successful.Compared with the PBS group,the mice in the disease model had IBD symptoms,such as reduced activity,body weight loss,mental malaise,excretion of stool,the index of disease activity increased,intestinal tube was shortened,narrow and deformed,and dilute stool.However,compared with the DSS group,the berberine+DSS group had less symptoms.The results showed that berberine had reduced the symptoms of acute enteritis in mice.2.HE staining showed that goblet cells disappearance and a large number of inflammatory cell infiltration in disease model mice compared with PBS mice.The DSS group has more serious damage than the berberine+DSS group.The more area of goblet cells disappeared,the more recess disappeared,inflammatory cells infiltrated into the muscularis layer,and the mucous was thickened with edema(P<0.01).The results showed that berberine reduced the degree of intestinal inflammation in mice.3.In the flow cytometry surface staining experiment,the proportion of CD3~-B220~-cells of the purified intestinal epithelial cells were over 95%,and the CD45.2 was single peak,while in the unpurified intestinal epithelial cell,the proportion of CD3~-B220~-cells accounted for only 43.5%,and CD45.2 shows two peaks.The results showed that centrifugation was effective in the purification of intestinal epithelial cells.4.In the flow cytometry,the isolated intestinal epithelial cells were stained with FITC-CK18,and the peak was found to shift to the right,and the positive rate was over 85%.The results showed that the cells were intestinal epithelial cells.5.The Real-time PCR test results showed that compared with the DSS group,the mRNA expression level of IL-6 and IL-23 in the intestinal epithelial cells of berberine+DSS group decreased significantly(P<0.01),IL-25,IL-33 mRNA expression level decreased significantly(P<0.05),IL-18mRNA expression level increased significantly(P<0.05).These results suggest that in DSS induced acute enteritis,berberine can reduce the cytokines mRNA expression level of intestinal epithelial cell,such as IL-6,IL-23,IL-25 and IL-33,and enhance the expression level of IL-18.6.The results of Western blotting showed that compared with the DSS group,the protein expression level of IL-6,IL-23 and IL-25 in the intestinal epithelial cells of the Berberine+DSS group decreased significantly(P<0.05),the protein expression level of IL-33decreased significantly(P<0.01),IL-18 protein expression level was obviously increased(P<0.05).This result is the similar with the Real-time PCR result.It is further confirmed that berberine can down-regulate the expression level of IL-6,IL-23,IL-25 and IL-33 in intestinal epithelial cells,and enhance the expression level of IL-18.7.In the flow cytometry surface staining experiment,a large number of lymphocytes(64.1%)were detected in the sample of the intestinal lamina propria lymphocyte,of which 46.1%were CD3~+T cells.We gated the CD3~+T cells,51.4%were CD3~+CD4~+T cells and 29.0%were CD3~+CD8~+T cells,and the groups were reasonable.This result can prove that the sample is the lymphocytes of the intestinal lamina propria.8.ELISA results showed that compared with the DSS group,the expression level of IL-4 in the intestinal lamina propria lymphocytes of the berberine+DSS group decreased significantly(P<0.01),the expression level of IL-17A decreased significantly(P<0.05),while the expression level of IFN-?increased significantly(P<0.001)and the expression level of IL-10 obviously increased(P<0.05).The results showed that in the acute enteritis induced by DSS,the use of berberine can reduced the cytokines secreted by Th2 and Th17 cells,enhanced the cytokines secreted by Th1 and Treg cells.And this change is the similar with the production level of such cytokines in the intestinal epithelial cells.Conclusion:1.Berberine inhibited the expression of IL-6,IL-23,IL-25 and IL-33 in intestinal epithelial cells and increased the expression of IL-18 in the inflammatory state.2.Berberine plays the role of immune-regulation through regulating the immune response of the intestinal lamina lymphocyte by influencing the secretion of cytokines in intestinal epithelial cells and alleviates DSS induced intestinal mucosal inflammation in mice.