Development Of Effect About Polysaccharides From Dicliptera Chinensis In The Treatment Of Ulcerative Colitis

Objective: Inflammatory bowel disease(IBD),which includes ulcerative colitis(UC)and Crohn’s disease(CD),is a chronic,spontaneous disease with multiple intestinal lesions.As known,NF-κB,MAPKs pathways and dysregulation of intestinal flora are associated with the pathogenesis of ulcerative colitis.The purpose of this study was to investigate the effect of DCP on ulcerative colitis in mice by regulating NF-κB and MAPKS-related pathways,and to observe the effect of DCP on the intestinal flora of mice with ulcerative colitis.Providing a theoretical basis for the development of polysaccharide from Dicliptera chinensis(DCP)in ulcerative colitis is our purpose.Methods: First,50 male C57BL/6 mice were divided into Control group,DSS group,DCP100 group,DCP200 group and 5-ASA group with 10 mice in each group.The mice model of chronic ulcerative colitis was established by drinking 2% DSS periodically.The mice were sacrificed after repeating 3cycles and being observed for 9 days to confirm the model was established.DAI score,body weight change rate,colon length,relative colon weight and H&E staining were used to observe the lesions and tissue injury of mice colon.The levels of inflammatory cytokines IL-1β,IL-6 and TNF-α in serum of mice were detected and analyzed by ELISA.MPO analysis was used to detect the content of myeloperoxidase in the colon tissues of mice.The protein expressions of iNOS,COX-2,NF-κB,p-NF-κB,JNK,p-JNK,p38,p-p38,IκBα and p-IκBα in mouse colon were detected and analyzed by Western blot.The expression of p-NF-κB in colon tissues of mice was detected and analyzed by immunohistochemistry.The mRNA relative expressions of inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-α in the colon of mice were detected and analyzed by RT-qPCR.Second,LPS was used to induce inflammation in mouse mononuclear macrophage leukemia cells(RAW264.7).MTT assay was used to detect the effects of different concentrations of DCP and experimental concentration of LPS on cell activity,and the appropriate concentration of DCP was selected.The protein expressions of iNOS,COX-2,NF-κB,p-NF-κB,JNK,p-JNK,p38,p-p38,IκBα and p-IκBα in RAW264.7 cells were detected and analyzed by Western blot.The mRNA relative expressions of inflammatory cytokines IL-1β,IL-6,IL-8,TNF-α,MCP-1,IFN-γ,COX-2 and iNOS in RAW264.7 cells were detected and analyzed by RT-qPCR.Third,the fecal samples of mice with ulcerative colitis in each group were collected.The contents of various bacteria in the samples and the differences of the bacterial flora among the mice were analyzed and detected by 16 S rRNA sequencing.Results: First,in vivo efficacy results showed that,at the macroscopic level,DCP could alleviate the body weight loss caused by DSS in mice,reduce DAI score,relieve colon length shortening and relative colon weight increase.At the microscopic level,DCP alleviated the colonic mucosal injury caused by DSS in mice,including crypt branching,crypt displacement and goblet cell reduction.Second,in vivo molecular biological experiments showed that DCP decreased the contents of inflammatory cytokines IL-1β,IL-6 and TNF-αin serum of mice with ulcerative colitis.DCP decreased the contents of inflammatory mediators such as iNOS,COX-2 and MPO and the mRNA relative expressions of inflammatory cytokines such as IL-1β,IL-6,IL-8and TNF-α in colonic tissues of mice with ulcerative colitis.DCP decreased the expression of p-NF-κB,p-JNK,p-p38 and p-IκBα in the colon of mice with ulcerative colitis.Third,in vitro experiments and molecular biology results showed that different concentrations of DCP(62.5,125,250,500,1000 μg/mL)and experimental concentration of LPS(1 μg/mL)did not affect RAW264.7 cell activity.DCP dose-dependently decreased the expression of inflammatory mediators(iNOS,COX-2)and the mRNA relative expression of various inflammatory cytokines(IL-1β,IL-6,IL-8,TNF-α,MCP-1,IFN-γ,COX-2 and iNOS)in LPS-induced RAW264.7 cells.Meanwhile,DCP decreased the protein expression of p-NF-κB,p-JNK,p-p38 and p-IκBαin LPS-induced RAW264.7 cells.Fourth,16 S r RNA sequencing results showed that DCP could alleviate the decrease in intestinal bacterial diversity caused by DSS,promote Akkermansia and inhibit Gram-negative bacteria such as Bacteroides,Enterobacteriaceae and Parasutterella.Conclusion: DCP can alleviate chronic ulcerative colitis induced by DSS in mice,and its mechanism is related to the regulation of NF-κB and MAPKs-related signaling pathways to regulate intestinal flora.