| Objective: 1.To investigate the anti-colitis mechanism of resveratrol through the regulation of Wnt/?-catenin signaling pathway.2.To investigate the role of miRNA-31 in inhibiting the development of colitis byresveratrol.Methods:1.DSS induced colon colitis experiment: 28 C57BL/6 mice were randomly divided into four groups including control group,DSS group,DSS+Res group and Res group.The experiment lasted for 3 weeks.Mice in DSS group and DSS+Res group were given 1% DSS at libitum during week 1 and 3 while with normal sterile water during week 2.The mice in control group and Res group only drank normal sterile water for 3 weeks.The mice in DSS+Res group and Res group were given 0.2ml 8mg/ml resveratrol once a day at the third week,and the other two groups were given the same amount of 0.5% CMC-Na once a day during the third week.The mice were weighed daily and their activities and state of feces were recorded.After that,the mice were euthanized,the spleens were weighed,and the colonic length was measured.H&E staining was used to observe the pathological changes of the colon,and the expression of miR-31 in colonic tissue was detected by qPCR.The expression of ?-catenin and Cyclin D1 was measured by Western Blot.The expression of ?-catenin,TCF1/TCF7,Cyclin D1 was detected by immunohistochemistry.2.HCT116 cells culture experiments: HCT 116 cells were divided into two groups: control group and resveratrol treated for 24 h group(Res group).Then HCT 116 cells were treated with resveratrol at the concentration of 10mg/ml and cultured in incubator for24 h.24 hours later,collect the cells.The expression of ?-catenin,LRP-6,FZD3,c-Myc,Cyclin D1 in HCT 116 cells was detected by Westren Blot.3.miRNA-31 knock-down and over-expression experiments: HCT116 cells were divided into control group,miRNA-31 mimic group(miR-31-mimic group),miRNA-31 inhibitor group(miR-31-inhibitor group),Res group and miR-31-inhibitor+Res group.After transfection,the cells were incubated in the incubator for 48 hours.(Res group was not transfected and resveratrol was added after 24 hours of cell incubation.Resveratrol was added to miR-31-inhibitor+Res group 24 hours after transfection and then incubated for 24 hours).After 48 hours,the cells were collected.The expression of LRP-6,FZD3,?-catenin,c-Myc,Cyclin D1 in HCT 116 cells was detected by Westren Blot.Results:1.General reaction and weight changes of mice: Compared with the control group,the mice in the DSS group decreased significantly in weight and activity during the modeling period.They began to have defecation in the second week and blood stool in the third week.After administration of resveratrol,the weight loss of mice was better than that of DSS group,and it was good in spirit,normal in activity,lightened in defecation,and formed in defecation.2.Mouse colon length / body weight ratio: After the colon was removed,it was cut and flattened,and the length of colon was measured with a ruler.The colon length / mouse weight ratio was analyzed statistically.The results showed that the colon length / body weight ratio in DSS group was significantly lower than that in control group(p<0.05).There was no significant difference in colon length / body weight ratio between DSS+Res group and control group.There was no significant difference in colon length / body weight ratio between Res group and control group.The colon length / body weight ratio in DSS+Res group was significantly higher than that in DSS group(p< 0.05).3.Spleen weight / body weight ratio of mice: After euthanasia,the spleen of the mice was resected and weighed,and the changes of immune function of mice were observed.The ratio of spleen weight / mouse weight was analyzed statistically.The results showed that the spleen weight / body weight ratio of DSS group was significantly higher than that of control group(p<0.01).There was no significant difference in spleen weight / body weight ratio between DSS+Res group and control group.There was no significant difference in spleen weight / body weight ratio between Res group and control group.The ratio of spleen weight / body weight of DSS+Res group was lower than that of DSS group(p<0.05).4.The results of mouse colon H&E staining: The results of H&E staining showed that in DSS group,the inflammation of colon was the most serious,the infiltration of neutrophils and other inflammatory cells in mucosa and submucosa were observed under microscope,the intestinal wall was thickened,the crypt structure was seriously destroyed,and the goblet cells were reduced.After resveratrol administration,the inflammation of colon in mice was significantly less than that in DSS group,the epithelial structure was basically intact,and the infiltration of inflammatory cells was reduced.The score of pathological injury of colon tissue in each group showed that the injury of DSS group was more serious than that of control group(p<0.0001).After administration of resveratrol,the tissue damage of mice was obviously alleviated(p<0.01).5.The miR-31 expression of mouse colon: The results of qPCR showed that the expression of miRNA-31 in colon tissue of DSS group was higher than that of control group(p<0.05).There was no significant difference in the expression of miRNA-31 between DSS+Res group and control group.The expression of miRNA-31 in colon tissue of Res group was lower than that of control group(p<0.05).The expression of miRNA-31 in colon tissue of DSS+Res group was lower than that of DSS group(p<0.05).The results showed that the expression of miRNA-31 was up-regulated in colitis mice,and resveratrol could down-regulate the expression of miRNA-31 in colon tissue of colitis mice.6.Expression of Wnt/?-catenin signal Pathway protein in colon of mice detected by western blot: The results of western blot for mouse colon tissue showed that the expression of ?-catenin(p<0.05),Cyclin D1(p<0.05)in colon tissue of DSS group was significantly higher than that of control group(p<0.05).There was no significant difference in the expression of ?-catenin,Cyclin D1 between DSS+Res group and control group.There was no significant difference in the expression of ?-catenin,Cyclin D1 between Res group and control group.Compared with DSS group,the expression of ?-catenin,Cyclin D1 in DSS+Res group was significantly decreased(p<0.05).The results showed that Wnt/ ?-catenin signaling pathway was up-regulated in colon tissue of colitis mice.Resveratrol could down-regulate the Wnt/?-catenin signaling pathway in colon tissue of colitis mice.7.Expression of ?-catenin,TCF1/TCF7 and Cyclin D1 in Colon tissue of mice detected by immunohistochemistry: The results of immunohistochemistry showed that the expression of ?-catenin in the colonic epithelium of DSS group was significantly higher than that of control group(p<0.05).There was no significant difference in the expression of ?-catenin between DSS+Res group and control group.There was no significant difference in the expression of ?-catenin between Res group and control group.Compared with DSS group,the expression of ?-catenin in the colonic epithelium of DSS+Res group was significantly decreased(p<0.05).The expression of TCF1/TCF7 in the colonic epithelium of DSS group was higher than that of control group(p<0.0001).There was no significant difference in the expression of TCF1/TCF7 between DSS+Res group and control group.The expression of TCF1/TCF7 in the colonic epithelium of Res group was lower than that of control group(p<0.01).The expression of TCF1/TCF7 of DSS+Res group was lower than that of DSS group(p<0.0001).The expression of Cyclin D1 in the colon epithelium of DSS group was higher than that of control group(p<0.01).There was no significant difference in the expression of Cyclin D1 between DSS+Res group and control group.There was no significant difference in the expression of Cyclin D1 between Res group and control group.The expression of Cyclin D1 in the colon epithelium of DSS+Res group was lower than that of DSS group(p<0.05).8.Effect of resveratrol on the protein expression of Wnt/?-catenin pathway in intestinal epithelial cells: Proteins were extracted from HCT 116 cells.The results of western blot showed that the expression of ?-catenin(p<0.001),LRP-6(p<0.05),FZD3(p<0.05),c-Myc(p<0.001),Cyclin D1(p<0.05)were decreased after 24 h of resveratrol administration,which indicated that resveratrol down-regulated Wnt/?-catenin pathway in HCT 116 cells.9.Effect of miRNA-31 on Wnt signaling proteins: The results of western blot for HCT 116 cells showed that the expression of LRP-6(p<0.05),?-catenin(p<0.05)and c-Myc(p<0.05)were decreased after knock down of miRNA-31,while the expression of LRP-6,?-catenin,c-Myc protein was increased after overexpression of miRNA-31.These results suggest that the expression of miRNA-31 is associated with Wnt signaling pathway.There was no significant difference in the expression of Cyclin D1 and FZD3 protein among the three groups.10.Effect of knock down of miRNA-31 and give resveratrol to Wnt signaling protein: The expression of LRP-6(p < 0.001),c-Myc(p < 0.0001),FZD3(p<0.01),Cyclin D1(p<0.01)was significantly decreased in HCT-116 cells after knock down of miRNA-31 and administration of resveratrol compared with the control groups,and the expression reduction of four proteins compared to control group was significantly more than that in Res group: LRP-6(p < 0.001),c-Myc(p < 0.01)and FZD3(p < 0.05),Cyclin D1,(p < 0.05)compared to control group.The results showed that the inhibitory effect of miRNA-31 and resveratrol on the expression of Wnt signaling pathway related proteins was stronger.Conclusion:Resveratrol relived DSS-induced colitis,this action is related to down-regulation of Wnt/?-catenin signaling pathway,and the down-regulation of Wnt/?-catenin signaling pathway is related to miRNA-31 expression. |
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