| ObjectiveWe constructed ulcerative colitis model by Dextran Sulfate Sodium Salfate（DSS）in this study,and to investigate whether CXCL2/CXCR2 participates in the protective effect of C3G on ulcerative colitis.It provides experimental basis for the research and development of anthocyanin health care products and IBD treatment drugs with C3G as the main component.Methods1.Protective effect of C3G on ulcerative colitis in mouse.Forty BALB/c female mouse of 6-8 weeks ages were randomly divided into five groups of each 8 after adaptixely fed for a week,include control group（Control）,model group（DSS）,low dose group（DSS+5μgC3G）,medium dose group（DSS+25μgC3G）,and high dose group（DSS+50μgC3G）.Except the control group,the rest four groups all drank 3.5%DSS solution continously for 7 days.Different intervention of C3G groups were injected with C3G solution intraperitoneally the day before induction of DSS.Body weight,fecal traits,and fecal occult blood were monitored daily during the experiment.The disease activity index was determined by scores of body weight loss,diarrhea and rectal bleeding.Histopathological changes of colon were analyzed by HE staining.The mRNA expression of inflammatory cytokines such as TNF-α,IL-1β and IL-10 in colon tissue were determined by qPCR.The protein expression of inflammatory cytokines and occludin in colon tissue were determined by Western blot.2.Mechanism of C3G on ulcerative colitis in mouse.Thirty-six BALB/c female mouse of 6-8 weeks ages were randomly divided into three groups of 12 each,including control group（Control）,model group（DSS）and C3G group（DSS+50μgC3G）,they were treated as above.The number of F4/80+CD11b+macrophages in the colon tissue was determined by flow cytometry.RNA-seq was used to examine the differential gene expression in the colon of each group.The mRNA and protein expression of the CXCL2 and CXCR2 was determined by qPCR and Western blot.RAW264.7 macrophages were divided into control group（control）,inflammation model group（LPS）,low dose group（LPS+50μmol/L C3G）,medium dose group（LPS+100μmol/L C3G）and high dose group（LPS+200μmol/L C3G）.Cells in the C3G intervention group were treated different concentrations of C3G for 1h,then cells in the C3G intervention group and inflammation model group were stimulated 10μg/ml LPS for 24h.The mRNA and protein expression of inflammatory cytokines（TNF-α,IL-1β）and CXCL2,CXCR2 in cells were determined by qPCR and Western blot.Results1.Protective effect of C3G on ulcerative colitis in mouse.（1）Effect of C3G on the clinical symptoms of ulcerative colitis in mouse.Compared with the control group,the body weight of model group was lower（P<0.05）,model group showed significant blood in stool,loose stool,DAI scores was higher（P<0.05）and the length of colon was shorter（P<0.05）.Compared with the model group,the body weight had signifificant recovery（P<0.05）,the shortening and edema of colon of high and medium dose C3G had improved significantly（P<0.05）.（2）Effect of C3G on colon tissue structure and Occludin in mouse.The structure of colon tissue in the control group was intact.The colons of model group showed failure of crypt structure,mucosal congestion,loss of goblet cells and high level of leukocyte.The degree of colon damage was reduced after intervention of C3G.Histological score of mouse colon:the model group was significantly higher than the control group（P<0.001）;the high dose group was significantly lower than the model group（P<0.001）and the low dose group（P<0.05）.The protein expression level of Occludin in mouse colon tissue:Model and low dose groups were lower than the control group（P<0.05）;medium and high dose groups were higher than model（P<0.05）and low dose group（P<0.05）.（3）Effect of C3G on the mRNA and protein expression of inflammatory cytokines in the colonic tissue.The mRNA expression of inflammatory factors in the colonic tissues of each group,compared with the control group,TNF-α of model group（P<0.01）,low dose（P<0.01）and medium dose（P<0.05）were significantly increased,IL-1β of model group was increased but not significant（P>0.05）,IL-10 of model,low,and medium dose groups were decreased（P<0.05）;Compared with the model group,TNF-α and IL-1β in the low and medium dose groups have no significant change（P>0.05）,TNF-α of high dose group was decreased（P<0.05）,IL-1β of high dose group was decreased but not significant（P>0.05）,IL-10 of high dose group was increased（P<0.05）.Protein expression of inflammatory factors in the colonic tissues of each group,Compared with the control group,both TNF-α and IL-1β of model group were significantly higher（P<0.01）,IL-10 of model group was decreased（P<0.05）;Compared with the model group,TNF-α of medium and high dose groups were decreased（P<0.05）,IL-1β of low dose（P<0.05）,medium dose（P<0.05）and high dose（P<0.01）were significantly lower,IL-10 of high dose group was increased（P<0.05）.IL-1β of medium and high dose groups were higher than low dose group（P<0.05）.2.Mechanism of C3G on ulcerative colitis of mouse.（1）F4/80+CD11b+macrophages in the colonic tissue was inhibited by C3G.The number of F4/80+CD11b+ macrophages in the model group was significantly higher than that in the control group（P<0.01）,C3G caused a significant decrease in F4/80+CD11b+ macrophages in the colon（P<0.05）.（2）Results of the transcriptome sequencing analysis of the colon in mouse.The mRNA expression of CXCL2 and CXCR2 in transcriptome sequencing results:CXCL2 and CXCR2 of model group were significantly higher than control group（P<0.001）,CXCL2（P<0.01）and CXCR2（P<0.05）of model group were significantly higher than C3G group,CXCR2 in C3G group was higher than control group（P<0.05）.（3）C3G inhibits the mRNA and protein expression of CXCL2-CXCR2 in the colonic tissue.The mRNA expression of CXCL2 and CXCR2 in the colonic tissue of each group,CXCL2（P<0.05）and CXCR2 of model group were significantly higher than control group（P<0.01）,and CXCL2 and CXCR2 of C3G group were lower than model group（P<0.05）.The protein expression of CXCL2 and CXCR2 of control and C3G groups were lower than that in the model group（P<0.05）.（4）C3G inhibits the expression of mRNA and protein of inflammatory cytokines in LPS-induced RAW264.7 macrophages.The mRNA expression of inflammatory factors between the groups,compared with the control group,TNF-α of inflammation model group was significantly increased（P<0.01）,IL-1β of inflammatory model group（P<0.01）,low dose group（P<0.05）,medium dose group（P<0.01）and high dose group（P<0.01）were significantly increased;Compared with the inflammation model group,TNF-α of medium dose group（P<0.05）and high dose group（P<0.01）were significantly decreased.IL-1β was reduced in both the medium and high dose groups（P<0.05）.There were no significant changes in the protein expression of TNF-α between the groups（P>0.05）.The protein expression of IL-1β between the groups were significantly different,the inflammation model group,low and medium dose group were higher than control group（P<0.01）,the low,medium and high dose groups were lower than inflammation model group（P<0.05）,the high dose group was lower than low dose group（P<0.05）.（5）C3G inhibits the LPS-induced mRNA and protein expression of CXCL2,CXCR2 in RAW264.7 macrophages.The mRNA expression of CXCL2 and CXCR2 between the groups were significantly different,the inflammation model group and the low dose group were higher than control group（P<0.01）,medium and high dose groups were lower than inflammation model group（P<0.05）,the high dose group was lower than low dose group（P<0.05）.The protein expression of CXCL2 and CXCR2 between the groups were significantly different,CXCL2（P<0.05）and CXCR2（P<0.01）of inflammation model group were higher than control group;The high dose group was lower than inflammation model group（P<0.05）.Conclusions1.Treatment with C3G alleviated the clinical symptoms of DSS-induced ulcerative colitis,regulated the levels of inflammatory cytokines in the colonic tissues,repaired colonic mucosal tissues.2.C3G may exert its protective effect on IBD by inhibiting the CXCL2/CXCR2 axis. |
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