Anti-inflammatory And Mechanism Of Sulforaphane On DSS-induced Acute Colitis In Mice

ObjectiveInflammatory bowel disease(IBD)is a common disease associated with intestinal innate immune and adaptive immune disorders,including ulcerative colitis(UC)and Crohn's disease(CD),has now become a global disease in the 21 st century.Sulforaphane(SFN)extracted from broccoli has been shown to prevent inflammation,but the exact molecular mechanism is still unclear.We aim to investigate the effect and potential mechanisms of SFN in inhibiting the colitis.Part ? Anti-inflammatory effects of SFN on DSS-induced colitis in mice Methods 1.In this study,the acute colitis model was induced by dextran sulfate sodium salt DSS)in C57BL/6 mice.The change of body weight,stool frequency,blood in he stool were recorded every day from 0 day.Then the mice were sacrificed at 0 days and the colonic macroscopic changes and histological changes of the ice were recorded.2.The iNOS/Arg-1 of M1/M2 macrophage polarization markers in mouse colon tissues were detected by RT-PCR with the mRNA expression levels of miR-155,QKI,AHR.3.The protein of Nrf2,QKI,STAT1,I?B-?,NF-?B p65,NLRP3 in colon tissue were detected by Western blot.Results 1.The mice showed significant weight loss,diarrhea and blood in the stool with the administration of 2% DSS for 7 days.The pathology was mainly characterized by colonic shortening,erosion or ulceration and severe neutrophil infiltration.After treatment with SFN,the weight loss in DSS-induced acute colitis could be significantly improved and the score of disease activity index(DAI)was significantly reduced.2.RT-PCR showed SFN significantly inhibited the M1 polarization of macrophages in acute colitis with the iNOS decreased and promoted the M2 polarization macrophages with the Arg-1 increased.3.Histopathology demonstrated the range of damage in colon tissue was significantly reduced and the destruction of colonic crypt structure,the loss of goblet cells and the infiltration of neutrophils were significantly improved with the treatment of SFN.4.RT-PCR showed miR-155 increased and QKI and AHR decreased in DSS induced mice,which were improved with the treatment of SFN.5.Western blot analysis showed that Nrf2,QKI and I?B-? were decreased in acute colitis tissues,while NF-?B p65 and NLRP3 were increased,which were improved with the treatment of SFN.Part ? SFN promotes anti-inflammatory effects by inhibiting M1 macrophage polarization Methods 1.In order to verify the potential mechanism and pathways of SFN inhibiting the DSS-induced inflammation,we chosed RAW 264.7 as the cell line.2.CCK8 assay was used to detect the cytotoxic effect of different concentrations of SFN on RAW 264.7,and the optimal concentration of SFN to stimulate macrophages was selected.3.RAW 264.7 macrophages were treated by SFN with the stimulation of LPS combined with interferon gamma(INF-?)or IL-4 in vitro.4.The mRNA expressions of iNOS,Arg-1,miR-155,QKI and AHR in macrophages were detected by RT-PCR with different stimulation.5.The protein expression of Nrf2,QKI,STAT1,NF-?B p65 and NLRP3 were analyzed by Western blot with different stimulation.Results 1.CCK8 assay was used to analyze the toxicity of different concentrations of SFN on RAW 264.7 cells for 24 h.It was found that there was no proliferation inhibition when the maximum concentration of SFN was 10 ?M.2.RT-PCR showed SFN could inhibit the mRNA expression of iNOS or promote the mRNA expression Arg-1 significantly with stimulated by LPS+IFN-? or IL-4 in macrophages.3.RT-PCR demonstrated SFN inhibited the expression of miR-155 significantly in macrophages stimulated by LPS+IFN-?.4.RT-PCR showed SFN could increase the expression of QKI and AHR whether stimulated by LPS+IFN-? or IL-4 in macrophages.5.Western blot showed SFN could improve the protein expression of Nrf2 and QKI stimulated by LPS+IFN-? in macrophages.6.Western blot demonstrated SFN could increase the activation of STAT1 in macrophages stimulated by LPS+IFN-?.7.Western blot showed that SFN could inhibit the activation of NF-?B p65 and the protein expression of NLRP3 in macrophages stimulated by LPS+IFN-?.Conclusion 1.SFN can improve the weight loss,DAI score,macroscopic score and colonic inflammation-related tissue score significantly in colitis mice,as well as the colonic shortening,erosion,ulceration and epithelial injury.Histopathology showed the damaged area of colonic mucosa was reduced and the degree of ulceration,destruction of crypt structure and depletion of goblet cell depletion and neutrophil infiltration were significantly alleviated.2.SFN alleviated colitis in mice via restraining miR-155 in colon,regulating QKI/Ahr/STAT1 signal axis and subsequent NF-?B p65/ NLRP3 signaling pathway,promoting polarization of anti-inflammation M2 phenotype.