| Background and ObjectiveUlcerative colitis(UC)is a chronic recurrent inflammatory bowel disease(IBD)involving colonic and rectal mucosa.Its incidence is increasing year by year around the world and showing a younger trend.At present,most drugs commonly used in clinic can cause a variety of adverse reactions,and some drugs are inconvenient for administration.Therefore,there is an urgent need for a new effective,safe,convenient and economical treatment to replace them.Lactobacillus Reuteri is one of the natural probiotics in the gastrointestinal tract of humans and animals,which can regulate intestinal flora and intestinal immunity.With the continuous progress of research,Lactobacillus Reuteri with different functions has also been found.MethodsPart one:HI120 bacteria were cultured and co-incubated with fluorescent dye AF 647 DIBO,and then administered intragastrically in C57BL/6J mice.24 hours,48 hours and 72 hours after intragastric administration,one mouse in each group was killed,the gastrointestinal tract was taken out and photographed by in vivo imaging.Additionally,another mouse in each group was anesthetized and photographed in vivo.Part two:40 BALB/c mice were randomly divided into NC group,UC group,HI120 group and HI132 group.NC group was not allowed to make the model.In other groups,high-pressure water was drunk for the first 7 days,and 3%DSS was drunk for the next 7 days.In addition to daily fixed intragastric administration of 0.025mL linoleic acid,the four groups of mice were respectively administrated physiological saline or corresponding bacterial powder solution for 14 consecutive days.The body weight and fecal occult blood of mice were measured every day after modeling to calculate the DAI score.On day 14,the mice were killed,tissues of the colon,mesenteric lymph nodes and Peyer’s patch were collected for HE staining,immunohistochemistry,Western Blot,immunofluorescence and other experiments.The contents of cecum were quickly collected and cryopreserved for subsequent 16s rDNA sequencing analysis.Results1.The in vivo imaging and organ imaging of mice in HI120 group showed high intensity fluorescence in the gastrointestinal tract after 24 and 48 hours of intragastric administration,and some fluorescence could still be observed in organ imaging after 72 hours.2.The body weight loss of mice in UC group was significantly higher than that in NC group(P<0.001),and the weight loss in HI120 group was lower than that in UC group(P<0.05).The DAI score in UC group was the highest,and the DAI score in HI120 group was significantly lower than that of UC group and HI132 group(P<0.001,P<0.01).3.Colonic HE staining in UC group showed that there were a lot of inflammatory cell infiltration,destruction of crypt structure,disorder and destruction of glands,while no obvious inflammatory cell infiltration were observed in HI 120 group and NC group.The epithelial cells were intact,and the lamina propria glands and crypts were normal and arranged neatly.4.Immunohistochemistry showed that the expression of COX-2 in UC group was the highest,and the expression in HI120 group was significantly lower than that in UC group and HI132 group(P<0.001,P<0.01).The expression of Muc-2 and Occludin in UC group were significantly damaged,and the expression of Muc-2 and Occludin in HI120 group were significantly increased compared with those in UC group and HI132 group(P<0.001,P<0.001;P<0.001,P<0.001).5.The results of Western Blot showed that the expression of COX-2 and NF-κB p65 was the highest and the expression of PPARy was the lowest in UC group,and HI120 could significantly up-regulate the expression of PPARy and down-regulate the expression of COX-2 and NF-κB p65.6.Immunofluorescence showed that IL-17 and FoxP3 could be co-located in CD4+T cells.The number of CD4+IL-17+Th17 cells in UC group was the most and the fluorescence intensity was the strongest.CD4+FoxP3+ Treg cells in UC group were less,and the fluorescence intensity were weaker;the number of Th17 cells decreased and the number of Treg cells increased in HI120 group compared with UC group and HI132 group.7.16s rDNA flora analysis showed that in UC group,the abundances of Verrucomicrobia and Tenericutes decreased while Proteobacteria increased,the abundances of Lactobacillus,Roseburia,Alistipes and Anaerofustis decreased and Desulfovibrio increased.The abundance of Bacteroidetes in HI120 group increased,the abundances of Desulfovibrio decreased while Lachnospiraceae_NK4A136_group,Alistipe,Quinella and Ruminococcus were significantly increased and the abundance and diversity of intestinal flora increased significantly in HI120 group.Conclusion1.HI120 could survive and colonize in the intestinal tract of mice for at least 72 hours.It can reduce the DAI score and significantly reduce tissue injury and inflammation,effectively treat DSS-induced ulcerative colitis,which is speculated to be related to the significant down-regulation of NF-κB p65 and COX-2 expression in the PPARγ pathway activated by CLA.2.HI120 can significantly up-regulate the expression of Muc-2 and Occludin proteins and enhance the intestinal mucosal barrier.3.HI120 can improve the inflammatory response by regulating the immune balance of Th17/Treg cells.4.HI120 can improve the microflora imbalance of UC mice by regulating intestinal flora. |
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