| Objective: Inflammatory bowel disease(IBD)is an idiopathic intestinal inflammatory disease that often affects the ileum,colon,and rectum.The common pathological change in the intestine of patients with IBD is the damage of the intestinal mucosal barrier,which mainly due to the abnormal differentiation of intestinal epithelial cells.The existing research indicates that the differentiation of intestinal epithelial cells is mainly regulated by the Notch signaling pathway.Fucoidan is a sulfated carbohydrate mainly derived from brown algae,and its anti-inflammatory activity and so on the anti-oxidation,anti-tumor activities have been widely studied.Based on Notch signaling pathway and bioinformatics,this study investigated the protective effect of fucoidan on human normal colonic epithelial cells(NCM460)after inflammatory injury.Methods:1.Bioinformatics analysis.The GSE55651 dataset was downloaded from GEO database.We used the R language to preprocess the data and screened out the differential expression genes.The key target genes were screened out by DAVID database and PPI(Protein–protein interaction)network analysis.2.To establish cell inflammatory injury model,cell treatment and cell grouping.Culture NCM460 cell line in vitro and divide into 4 groups.(1)Blank group: this group contains no cells but only complete medium;(2)Control group: cells are cultured using complete medium;(3)LPS induction group: we use lipopolysaccharide(LPS,1mg / L)to intervene cells to construct inflammatory injury models:(4)LPS + low,medium,or high dose fucoidan group: we use LPS to construct inflammatory injury models,and then use low,medium or high(50,100,200 ?g / L)concentration of fucoidan culture medium to incubate the cells.3.Detection method.CCK8 assays were performed to detect the relative survival rate of each group of cells;ELISA methods were used to determine the levels of inflammatory factor IL-6 and TNF-? in the cell culture solution of each group;methods real-time fluorescent quantitative PCR(qRT-PCR)was used to analyze the expression of IL-6,TNF-?,Notch-1,Hes-1 and Math-1 mRNA in each group of cells.4.SPSS 20.0 software was used for statistical analysis.The experimental data were expressed as mean ± standard deviation((?) ± s).Comparisons among multiple groups were analyzed by single factor analysis of variance.Pairwise comparisons between groups were performed by LSD-t test.P < 0.05 was considered statistically significant.Results: 1.The results of bioinformatics analysis showed that we obtained 172 differential genes from 3 control samples and 3 LPS-induced inflammation damage samples,including 27 down-regulated differential genes and 145 up-regulated differential genes.GO analysis results suggested that these differences primarily participate in the processes of inflammation,immune response,and positive regulation of RNA polymerase II promoter transcription.The KEGG pathway enrichment analysis showed that these differential genes were mainly involved in TNF signaling pathway,cytokine-cytokine receptor interaction,etc.Through the PPI network analysis,4 candidates were confirmed: IL-6,STST1,IL-1b and IRF7.Combined with other relevant literature reports,we finally selected IL-6 for further research.2.The results of CCK8 assay suggest that LPS intervention can significantly inhibit the proliferation of NCM460 cells,and its cell survival rate is significantly lower than that of the control group(P < 0.05);while FUC can promote cell proliferation after inflammatory injury in a dose-dependent manner.The relative survival rate of cells after FUC treatment was significantly higher than that in the LPS induction group(P < 0.05).3.ELISA test results suggest that LPS intervention can significantly promote the secretion of inflammatory factors IL-6 and TNF-? in NCM460 cells.The level of inflammatory factors in the cell culture fluid of the LPS induction group is significantly higher than that of the control group(P < 0.05);After the cells were treated with low,medium,or high doses of FUC,the levels of IL-6 and TNF-?were significantly reduced relative to the LPS induction group(P < 0.05).4.The qRT-PCR results showed that compared with the control group,the expression levels of Notch-1,Hes-1,IL-6,TNF-?m RNA in LPS induction group were significantly increased(P < 0.01)and the mRNA expression level of Math-1 was =significantly reduced(P < 0.01).Compared with the LPS induction group,the expression levels of Notch-1,Hes-1,IL-6,and TNF-? m RNA in the low,medium,or high dose FUC groups were significantly reduced(P < 0.01),and the expression of Math-1 mRNA was increased significantly(P < 0.01).Conclusion: 1.FUC can obviously alleviate the inflammatory damage of cells induced by LPS,reduce the degree of inflammatory response,and increase the relative survival rate of cells.2.The therapeutic effect of FUC on LPS-induced cellular inflammatory damage may be related to the Notch-1 signaling pathway. |
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