| Crohn's disease(CD)and ulcerative colitis(UC)are collectively referred to as inflammatory bowel disease(IBD),which manifests as chronic inflammation of the gastrointestinal tract caused by a series of genetic and environmental factors.It is generally believed that inflammatory bowel disease(IBD)is caused by a mucosal immune response disorder of the intestinal microflora of genetically susceptible individuals.However,traditional anti-inflammatory drugs,such as 5-aminosalicylic acid and glucocorticoids,are not effective for all patients,and some have certain side effects.Therefore,a large number of studies have begun to focus on the development of safe and effective new strategies for the treatment of IBD.Probiotics are one of the methods for the treatment of IBD,but the exact mechanism of action is still unclear.In this study,L.plantarum 17-1 was used to intervene in healthy mice for 21 days,and then dextran sodium sulfate(DSS)was used to induce an experimental colitis model.The results showed that colitis mice fed with L.plantarum 17-1,it can alleviate the symptoms of weight loss and colon shortening in colitis mice.Observing the colon tissue slices of colitis mice,it was found that the colon mucosal layer of the mice in the DSS treatment group had local necrosis,fibrous tissue proliferation accompanied by inflammatory cell infiltration,and crypt structure was damaged,the integrity of the intestine was destroyed,and the local submucosal edema was accompanied by inflammatory cell infiltration.However,the colon of the mice in the Lactiplantibacillus plantarum group was relatively intact without obvious lesions.It shows that adding Lactiplantibacillus plantarum can alleviate the phenotypic changes of colitis caused by DSS.ELISA kit method was used to detect immune factors in colon tissue,and it was found that the levels of pro-inflammatory factors TNF-?,TNF-?,IL-1?,IL-6 and IL-17 in the colon of the DSS group were higher than those in the control group(P<0.005),The level of anti-inflammatory factor IL-10 was lower than that of the control group(P<0.001),while mice fed a diet containing 0.05%L.plantarum 17-1,the pro-inflammatory cytokine TNF-?(P<0.001),TNF-?(P<0.001),the content of IL-6(P<0.001)and IL-17(P<0.001)were significantly reduced,and the anti-inflammatory cytokine IL-10 was significantly increased(P<0.001).In addition,the consumption of L.plantarum 17-1 can also significantly alleviate the decrease of antioxidant enzymes GSH-PX,GSH,CAT and SOD caused by DSS,as well as the increase of oxidation products MDA,ROS,etc.,and enhance the body's antioxidant capacity.In summary,it shows that L.plantarum 17-1 can alleviate the inflammatory response in mice with colitis through immunomodulation and antioxidant activity.To further study the relationship between bacteria and the intestinal tract,fresh feces were collected from the 1st to 8th day during the modeling period,and use 16S rDNA gene sequencing technology to study the changes in the intestinal flora of DSS-induced colitis mice.The results showed that after 21 days of intervention with L.plantarum 17-1 significantly improved the abundance of Ruminococcaceae_UCG-014,norank f Muribaculaceae,Lachnospiraceae_NK4A136_group,norank f Lachnos piraceae,Lachnospiraceae_UCG-006,Roseburia,Akkermansia.The abundance of Escherichia coli and Staphylococcus in the DSS treatment group increased,while the SCFA production of mice in the Lactiplantibacillus plantarum treatment group increased.L.plantarum 17-1 can increase the diversity of intestinal flora in mice with colitis,promote the growth of beneficial flora,effectively inhibit the growth of pathogenic bacteria in the intestine,promote the recovery of intestinal flora,so as to alleviate the intestinal flora disorder caused by DSS.The above experimental results found that L.plantarum 17-1 can alleviate inflammation by regulating the level of intestinal immunity,so in this paper,RAW264.7 mouse macrophages were used as the common immune cells in vitro,and LPS was used to induce macrophages with the final concentration of 50 ?g/ml to establish the cellular inflammation model,and use the CCK-8 method to detect the effect of different concentrations of L.plantarum17-1 bacterial suspension on the survival rate of mouse macrophages RAW264.7.We found that the original concentration of 1 × 1010 CFU/ml of viable suspension inhibited the growth of cells at 1 × 109 CFU/ml,which may be due to the non-drug pathogenic death of the cells due to the competition between the viable bacteria and the cells.Then the same method was used to determine the effect of different concentrations of heat-killing bacteria on cell survival.The concentration of heat-killing bacteria of 1×108 and 1×109 CFU/mL had a proliferation effect on cells,but the effects of different concentrations were unstable.The results of previous experiments showed that L.plantarum 17-1 live bacteria and heat killed bacteria had the tendency of inhibiting LPS immune damage at appropriate concentration,which could effectively alleviate the immune stress caused by E.coli lipopolysaccharide.However,the effect was not obvious when the viable bacteria was 1 ×109 CFU/ml and the heat killed bacteria was 1×1010 CFU/ml.we speculated that it might be due to the effect of its metabolite exopolysaccharide.L.plantarum17-1 preserved and identified in the laboratory was used as the research object.After the activation and fermentation,TCA was used to remove protein,alcohol precipitation,dialysis and freeze-drying to obtain crude extracellular polysaccharide.Then the protein was removed by Sevage method,decolorized by D4020 macroporous resin,DEAE Sepharose fast flow After separation,the content of EPS was determined by phenol sulfuric acid method,and the elution curve was drawn.Finally,the separated EPS was used to act on macrophages,and the proliferation activity of macrophages was detected.The results showed that the proliferation effect of EPS on mouse macrophages was much better than that of living bacteria and heat killed bacteria.At a concentration of 170 ?g/mL,the proliferation ability of polysaccharides on macrophages reaches the highest level of 283.6%.In summary,it can be preliminarily speculated that the mechanism of Lactiplantibacillus plantarum 17-1's mitigation effect on mouse colitis may be achieved through EPS regulation of immune response. |
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