| Background:Ulcerative colitis is one of the common inflammatory bowel diseases characterized by inflammation confined to the mucosa.UC was previously more common in Western high-income countries,however,the epidemiological pattern has evolved over the past 20 years,with its incidence leveling off in Western countries and rising substantially in newly industrialized countries.UC is an intestinal barrier disease,and intestinal microbiota is a key factor in the pathogenesis of UC.In recent years,natural polysaccharides have attracted more and more attention due to their safety,anti-inflammatory and immunobiological activities.Nostoc commune Vaucher(N.commune)is a kind of blue-green algae with high food and drug value.Its polysaccharide accounts for 60% of the dry weight of N.commune,which is heteropoly sugar of 210 k Da with ultra-high molecular weight,suggesting that it has quite strong biological activity,and polysaccharide depends on intestinal flora metabolism.The protective effect of polysaccharide N.commune on UC and the important role of intestinal flora in it remain to be studied.Objective:1.To observe the effect of N.commune polysaccharide on the symptoms of acute colitis in mice.2.To explore the effects of N.commune polysaccharide on intestinal microbiota and its metabolic pattern,intestinal mucosa,epithelial barrier,and inflammation in acute colitis mice.3.ABX mice model was established to assess the role of intestinal microbiota in relieving acute colitis of mice by N.commune polysaccharide.4.To explore the mechanism of N.commune polysaccharide in alleviating acute colitis through transcriptome sequencing technology.Methods:1.Male C57BL/6J mice aged 8 weeks were selected to induce acute colitis model with2.5%DSS.N.commune polysaccharide was given 2.5%DSS 14 days after early intragastatic administration,and free drinking for 6 days;5-ASA was given intragastatic administration on the first day of DSS,and polysaccharide and 5-ASA were given intragastatic administration until the end of the experiment.Body weight,fecal character and color of mice were recorded during DSS induction.2.After the experiment,the mice were sacrificed,orbital blood was collected,colon tissue and colon contents were collected,spleen weight was weighed,colon length was measured,DAI score was performed,H&E staining was performed,and colon histopathology score was performed.3.The composition and structure of gut microbiota of N.commune polysaccharide-pretreated colitis mice were analyzed by 16 S r RNA sequencing.The contents of gut microbiota metabolites and short-chain fatty acids of N.commune polysaccharide-pretreated colitis mice were analyzed by non-targeted metabolomics,and Spearman correlation method was used to analyze the correlation between differential gut microbiota and differential metabolites.4.The number of goblet cells and the secretion of MUC2 in the intestinal mucosa of N.commune polysaccharide-pretreated colitis mice were detected by AB staining,PAS staining and immunohistochemistry.Western blot and immunofluorescence were used to detect the expression of tight junction proteins ZO-1 and Occludin in colon tissue.5.ELISA was used to detect the contents of TNF-α,IL-1β,IL-6 and TGF-β1 in serum.The expression levels of F4/80 and Ly-6G were detected by immunohistochemistry.The kit was used to detect the activity of MPO in colon tissue.6.The gut microbiota was eliminated after four weeks of drinking with four antibiotics,to and the ABX mice model was established.Further,N.commune polysaccharide was still given 14 days in advance,and then 2.5%DSS was given free drinking for 7 days.Polysaccharide was given until the end of the experiment.Colon tissue was taken,colon length was measured,H&E staining was performed,and colon histopathological score was obtained.The kit was used to detect the activity of MPO in colon tissue.7.Transcriptomic sequencing analysis of colon differential genes and related signaling pathways of N.commune polysaccharide-pretreated colitis.Results:1.The results of body weight showed that the mice with DSS induced acute colitis began to lose weight and activity decreased on the third day,and obvious blood in the stool began to appear on the fifth day,and the weight showed a trend of varying degrees of recovery nine days later.It was found that the colon of mice in DSS group was significantly shortened,and H&E showed serious tissue damage.N.commune polysaccharide significantly improved the weight loss,DAI score down,colon shortening and tissue damage of DSS induced acute colitis mice.2.Observed OTU,α-diversity(Shannon index and Inverse-Simpson index),β-diversity(PCo A analysis)and relative abundance at the phylum and genus levels showed that N.commune polysaccharide remodeled the composition and structure of gut microbiota of mice with acute colitis.Akkermansia＿muciniphila was the most significant species of intestinal microflora remodeled by N.commune polysaccharide.3.The results of non-targeted metabolomics analysis showed that N.commune polysaccharide could inhibit the biosynthesis of PCs and TXB 2 in mice with acute colitis,and reshape the metabolic pattern of gut microbiota of mice with acute colitis.4.AB staining,PAS staining and MUC2 immunohistochemical results showed that the administration of N.commune polysaccharide could increase goblet cell number and MUC2 secretion in acute colitis mice.Western blot and immunofluorescence results showed that N.commune polysaccharide could improve the expression of ZO-1 and Occludin in acute colitis mice.ELISA results showed that the serum levels of inflammatory cytokines TNF-α,IL-1β and IL-6 in mice with acute colitis were significantly decreased by N.commune polysaccharide,while TGF-β1 was significantly increased.Ly-6G and F4/80 immunohistochemical display,the administration of N.commune polysaccharide significantly reduced the invasion of neutrophils in the lamina propria without affecting macrophages in the mouse model of acute colitis.Our results also showed that N.commune polysaccharide could significantly reduce the activity of MPO in mice with acute colitis,indicating that the number of neutrophils decreased.These results showed that N.commune polysaccharide could alleviated colonic mucosal and epithelial barrier damage and reduced inflammation in acute colitis mice.5.The protective effect of N.commune polysaccharide in the Antibiotics treatment group was weakened.These results indicated that gut microbiota mediated the palliative effect of N.commune polysaccharide on acute colitis to some extent.6.The results of RNA-seq showed that the signaling pathways related to inflammation and immune function may be involved in the alleviating effect of N.commune polysaccharide in acute colitis mice.Conclusion:N.commune polysaccharide ameliorates acute colitis mice by enhancing the intestinal mucosal barrier function,including remodeling the composition and structure of gut microbiota and its metabolic pattern,alleviating the damage of colonic mucosal and epithelial barrier function and reducing inflammation in acute colitis,and may affect the signaling pathways related to inflammation and immune function. |
PDF Full Text Request