| Objective: Intestinal barrier injury is a key pathogenic factor in the occurrence and development of inflammatory bowel disease(IBD).At present,IBD is difficult to cure and prone to relapse,so there is an urgent need to develop new therapeutic strategies.Lactobacillus plantarum(L.plantarum)can effectively improve the symptoms of IBD,but the specific mechanism is still unclear.In this study,we established a mouse model of colitis induced by dextran sulfate sodium salt(DSS),screened L.plantarum,and evaluated its effect on intestinal barrier and the regulation of intestinal flora and metabolites.Methods:(1)Lactobacillus plantarum from different sources were isolated and purified,and identified by bacterial morphology,biochemical indicators and 16 S r DNA.In vitro simulated gastric juice and bile salt medium were used to evaluate the acid and bile salt tolerance of the strains.(2)The body weight,disease activity index,colon length,intestinal mucosal permeability and colon tissue structure of DSS-induced colitis mice were observed to screen out the strains with better effect on alleviating colitis.(3)Immunohistochemistry(IHC)was used to detect the indexes related to cell junction and mucin in colon tissue of colitis mice to evaluate the regulatory effect of the strains on intestinal mechanical and chemical barriers.ELISA,IHC and q RT-PCR were used to detect the levels of inflammation-related cytokines and immune cell infiltration in serum and colon of mice to evaluate their regulatory effects on intestinal immune barrier.The microbiome and metabonomics techniques were used to analyze the effects on intestinal flora and metabolites in colitis mice.(4)The effects of sodium butyrate on the growth and migration of normal colonic epithelial cells were detected by CCK-8,plate clone formation and Transwell assay.q RT-PCR and immunofluorescence were used to detect the effect of sodium butyrate on tight junction related indicators of normal colonic epithelial cells.The body weight,disease activity index,colon length,intestinal mucosal permeability,intestinal barrier structure,and serum inflammatory factors were observed to evaluate the protective effect of sodium butyrate on the intestinal barrier of DSS-induced colitis mice.To explore the effects of sodium butyrate on intestinal flora and metabolites in colitis mice by using microbiome and metabolomics technology.Results:(1)L.plantarum C,L.plantarum S and L.plantarum F from three different sources were successfully isolated,purified and identified,and all showed good acid and bile salt tolerance.(2)The in vivo study confirmed that L.plantarum C had a good effect on alleviating colitis,which could significantly inhibit the weight loss,increase of disease activity index and colon shortening,reduce intestinal mucosal permeability and maintain the integrity of colon tissue structure in mice.(3)L.plantarum C significantly increased the expression of ZO-1,Claudin-1,Occludin,E-cadherin and MUC2 in the colon of colitis mice,indicating that L.plantarum C could improve the intestinal mechanical and chemical barrier.L.plantarum C significantly reduced the expression of pro-inflammatory cytokines IL-6,TNF-α and IL-1β at the protein level or m RNA level in the colon of colitis mice,as well as the level of pro-inflammatory cytokines in the serum.The m RNA expression of anti-inflammatory cytokines IL-10,TGF-β and IL-4 was significantly increased,while the protein expression of F4/80 were significantly decreased in colon tissue,indicating that the strain had a significant inhibitory effect on inflammation and played a role in balancing the intestinal immune barrier.(4)The results of microflora diversity analysis showed that the α diversity of intestinal flora was increased after L.plantarum C intervention.The relative abundance of butyric acid-producing bacteria such as Clostridiaceae＿Clostridium,Ruminococcaceae＿Ruminococcus,Butyricoccus,Alistipes and Coprococcus was increased,while the relative abundance of potential pathogenic bacteria such as Desulfovibrio and Turicibacter was decreased,so as to optimize the structure of the flora and regulate the imbalance of the flora caused by colitis.(5)Metabolomics results showed that the composition of metabolites in colitis mice was changed after L.plantarum C intervention,and the content of butyric acid and its derivatives was significantly increased.The pathways enriched in differential metabolites were mainly involved in lipid metabolism and amino acid metabolism.(6)The results of in vitro experiments showed that sodium butyrate could promote the growth and migration of normal colonic epithelial cells and the expression of tight junction proteins ZO-1 and Claudin-1.(7)The results of in vivo experiments showed that sodium butyrate intervention significantly inhibited the weight loss,increase of disease activity index and colon shortening,reduced intestinal mucosal permeability and serum inflammatory factor levels in colitis mice.The colon tissue structure was relatively intact,and the expression of tight junction proteins ZO-1 and Claudin-1 was increased.It is suggested that sodium butyrate can improve the symptoms of colitis and intestinal barrier.(8)The results of bacterial flora and metabolites analysis showed that sodium butyrate intervention in colitis mice can upregulate the relative abundance of butyric acid-producing bacteria such as Coprococcus,Butyricoccus,Ruminococcaceae＿Ruminococcus and Alistipes,and downregulate the relative abundance of potential pathogenic bacteria such as Desulfovibrio and Turiciactor,thereby improving the composition and structure of intestinal flora.The metabolite composition was changed,and the differential metabolite enrichment pathways were mainly involved in lipid metabolism and amino acid metabolism.Conclusions:(1)L.plantarum C was selected as the better strain to alleviate colitis;(2)L.plantarum C can improve the intestinal mechanical,chemical and immune barriers in colitis mice,balance intestinal flora and metabolites,and improve the intestinal barrier through butyric acid and its derivatives. |
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