Effect Of Human Milk Oligosaccharides On Gut Microbiota And Short-Chain Fatty Acids In Mice With DSS-Induced Colitis

Inflammatory bowel disease(IBD)is a type of chronic relapse and remitting disease related to imbalance of the mucosal immune system and intestinal ecosystem,its incidence in China is increasing quickly year by year and showing a low age trend.Although the etiology and pathogenesis of IBD is unclear now,recent years studies suggest that intestinal flora imbalance and metabolic disorders play an important role in the development of IBD.Human milk oligosaccharides(HMO)can promote intestinal health by increasing the colonization of beneficial bacteria,resisting pathogen infection,and promoting immune maturation.Particularly,2'-fucosyllactose(2'-FL),as the most abundant oligosaccharide in human milk,has been allowed to be added to infant formula powders and foods for special medical purposes.However,it is unclear whether HMO and 2'-FL diet could ameliorate IBD in larger children and adults.Therefore,we established DSS-induced mouse model to evaluate the effects of supplementation of HMO or 2'-FL on inflammatory response,intestinal flora composition and SCFAs production.The research results will help to clarify the biological mechanism of HMO involved in intestinal anti-inflammatory and homeostasis regulation,and provide strong theoretical support for the "dietary intervention" of HMO.1.Effect of HMO on the symptoms of DSS-induced colitis miceFirst,the dextran sodium sulfate(DSS)-induced colitis mice model was established to investigate the effect of HMO or 2'-FL on intestinal inflammation.After one week acclimation,all C57BL/6 mice(6 weeks,female)were randomly assigned to 5 experimental groups(n = 10 per group): control group,DSS colitis group,HMO group(400mg/kg BW),low doses of 2'-FL groups(200mg/kg BW)and high doses of 2'-FL groups(400mg/kg BW).The intestinal inflammation was assessed by body weight,DAI score,immune organ index,colon length,and colon tissue pathological section.Results indicated that compared with the control group,DSS induced mice exhibited severe intestinal inflammatory response,which manifesting in weight loss,DAI score arise,higher spleen index and lower thymus index,colon shorten,colonic crypt damage and inflammatory cell infiltration,up-regulated MPO activity,IL-6 and TNF-? levels,down-regulated IL-10.Either HMO or 2'-FL consumption attenuated the inflammatory response,and the 2'-FL group was dose-dependent.2.Effect of HMO on the gut microbiota of DSS-induced colitis miceThe 16 S r DNA genes V3–V4 region of bacterial was sequenced using Illumina Mi Seq to analyze the effects of HMO or 2'-FL on the intestinal flora of mice with colitis.Results showed that compared with the control group,DSS induction reduced both of diversity and richness.However,oral treatment of HMO significantly improved the above indicators,but 2'-FL treatment had no significant effect.PCo A analysis revealed that the bacterial community structures of HMO group were very similar to those of the control,suggesting that the HMO had a good regulatory effect on intestinal community disorders induced by DSS.At the phylum level,DSS group had a lower Bacteroidetes,whereas higher Firmicutes and Proteobacteria proportion than the control group.Compared to the DSS group,HMO treatment effectively increased the proportion of Bacteroidetes,and significantly reduced the proportion of Firmicutes and Proteobacteria.And 2'-FL treatment improved the relative abundance of Bacteroidetes and effectively decreased the Proteobacteria proportion,but significantly increased the relative abundance of Firmicutes.At the genus level,it was found that the abundance of Streptococcus,Escherichia-Shigella and Lactococcus were drastically increased in the DSS group compared to the control,and the proportion of Lactobacillus,unclassified Muribaculaceae,Lachnospiraceae family and Akkermansia decreased.Both HMO and 2'-FL treatment remarkably reversed the upward trend of Streptococcus,Escherichia-Shigella,Lactococcus.Administered with HMO effectively increased the genera of unclassified Muribaculaceae,Bacteroides and Ruminococcus torques group,but consumption of 2'-FL dose-dependently up-regulated the abundance of Lactobacillus.In addition,the correlation analysis between dominant bacteria and inflammatory biomarkers showed that the relative abundance of Lactobacillus and unclassified Muribaculaceae were negatively correlated with pro-inflammatory biomarkers,but positively correlated with the antiinflammatory cytokine IL-10(P<0.05).In contrast,the proportion of Streptococcus,EscherichiaShigella and Lactococcus was positively correlated with the pro-inflammatory biomarkers overall and significantly negatively correlated with the level of IL-10.Moreover,PICRUSt analysis result showed that the bacteria genes related to infectious diseases(such as Shigellosis,Pathogenic Escherichia coli infection)and the microbial gene contents related to the cancers pathways were decreased in HMO and 2'-FL group,respectively,while the bacteria genes related to the polysaccharide metabolism(glycosaminoglycan degradation and Glycolysis / Gluconeogenesis)was increased compared to DSS group.3.Effect of HMO on the short-chain fatty acids(SCFAs)of DSS-induced colitis miceMeanwhile,The SCFAs levels in all the treated-group were quantitatively determined by gas chromatography-mass spectrometer(GC-MS).The results showed that compared with normal mice,the content of SCFAs in the DSS group was significantly reduced(P<0.05),while HMO intervention significantly increased the the contents of acetate,propionate,butyrate,valerate,isobutyrate and isovalerate(P<0.05).2'-FL increased the level of SCFAs overall in a dose dependent manner,in which the contents of acetate,propionate,valerate,isobutyrate and isovalerate were significantly increased in the 2'-FL-H group(P<0.05).And correlation analysis results showed that Lactobacillus was positively correlated with propionate and isovalerate,the undefined genera of Muribaculaceae was significantly and positively correlated with butyrate(P<0.05).Rresults showed that the efficacy of HMO and 2'-FL in the prevention of DSS-induced colitis may be attributed to a decrease in the abundance of opportunistic bacterial pathogens and an increase in the proportion of beneficial bacteria,and the regulation of microbial metabolites SCFAs,which are beneficial for promoting intestinal microecological stability and metabolic balance.Notably,when given at high doses,the individual 2'-FL has similar effects as HMO.This study provides strong support for HMO or 2'-FL as a special food ingredient to prevent or against IBD in children or adults.