Studies On The Effect Of Low Dose Of Cyanidin-3-O-glucoside On Dextr An Sulfate Sodium-induced Colitis And Its Mechanisms

Research background:Inflammatory bowel disease(IBD)is a chronic disease of gastrointestinal tract in which overactivation of inflammatory response is implicated.IBD is extremely easy to relapse,and its incidence was increasing year by year worldwide.Patients with long-term IBD will be easily induced to colorectal cancer if they are not treated timely and effectively.Exploring the pathogenesis and intervention methods of IBD is important for the treatment of IBD and the prevention of CRC.Cyanidin-3-O-glucoside(C3G)is a powerful anti-inflammatory agent,widely existing in fruits and vegetables.A lot of the biological benefits of dietary anthocyanins have been reported,including anticancer activity,anti inflammatory activity,neuroprotective activity,prevention of cardiovascular disease,anti-obesity,and anti-diabetic activity.However,few studies reported the mechanism of the anti-inflammatory activity in autoimmune colitis.Therefore,this study took mice as the colitis model,C3G as the treatment drug,and CD169+ macrophages as the treatment target,to explore the regulatory role of C3G in IBD and related mechanisms.Research purposes:This study is mainly focused on the efifect of C3G on IBD mouse model and the mechanisms involved in the inflammation process.These are necessary to seek appropriate therapeuticmethod and elucidate the critical mechanism of IBD:in order to prevent further inflammation progression.Research methods:C57BL/6 mice were given 3.5%DSS solution for 7 days,lug C3G or PBS were intraperitoneally injected every 2 days on day 0,3,6 of DSS administration.First,mice weight changes were recorded during model induction,the incidence of mice was observed after sacrifice.H&E staining was used for assessingthe severity of colitis in mice,as well as,the change of inflammatory cytokines in colons and mesenteric lymph nodes(mLNs)were measured by Q-PCR to clear the clinical effect of C3G injection in colitis.Immunofluorescence staining and flow cytometry were used to analyze the percentage changes of CD169+ macrophages in colon tissues,mesenteric lymph nodes and abdominal cavity,so as to clarify the important role of CD169+macrophagesin mice colitis with the treatment of C3G.After that,the changes of CCL22 and regulatory T cells(Treg)in colon and mesenteric lymph nodes were detected by Q-PCR and flow cytometryto explore the regulatory effect of C3G.Moreover,in vitro experiments further clarified the direct effect of C3G on macrophages.Finally,Q-PCR,immunofluorescence staining and flow cytometry were used to investigate the molecular regulatory mechanism of C3G in colitis through CD169+ macrophage pathway.Results:First,C3G significantly alleviated the clinical symptoms of colitis in mice.Experimental data show that C3G injected mice did not show the significant weight losscomparing with the colitis mice and PBS injected mice,the colon length and pathological conditionof C3G injected mice is much better than PBS injection group.Expression of inflammatory factors in colon and mesenteric lymph nodes such as IL ?6,IL-1?,IL-18 and TNF-a was significantly reduced.These results indicated that C3G significantly alleviates DSS-induced colitis in mice,and plays an important regulatory role in the occurrence of colitis.Secondly,C3G regulated mice colitis through CD 169+ macrophage pathway.Immunofluorescence and flow cytometry results showed that after C3G injection,the percentages of CD 169+ macrophages in colon and mesenteric lymph nodes weresignificantly reduced,while the percentages of immune-tolerance related factors and Tregs were significantly increased,and the activation of macrophages was also inhibited.Further study found that C3G injection caused the significant reduce of CD169+ macrophages percentage in abdominal cavityand inhibition of the activation of abdominal macrophages.Finally,invitro experiments showed that C3Ginhibited the activation of macrophagesdirectly.Moreover.C3G inhibited the induction of type I interferon,suggesting that C3G plays an important regulatory role in the induction of CD 169+ cells.Conclusions:The study demonstrated firstly strong effects of C3G to attenuate DSS-induced colonic damage in mice.The effector C3G on DSS-induced colitis clearly showed the decrease of CD 16d+ macrophages in both colons and mLNs.Moreover,immune-tolerance related factors and Treg were significantly increased.The increase of CD 169+ cell induced by type ? IFN could be inhibited by C3G.All these data suggest that the regulatory mechanism of C3G on colitis is mainly mediated by CD 169+ macrophages and type ? IFN pathway,on the other hand,it is regulated by Treg and related immune tolerance factors.