Protective Effect Of Porcine Blautia Hominis LYH1 And Bacteroides Cellulosilyticus LYH2 On Colitis In Mice

Inflammatory bowel disease(IBD)is a common intestinal disease that harms humans and commercially farmed animals.Problems on animal gut health arising from the ban of antibiotics in the feed has been widely discussed,and the use of next-generation probiotics(NGP)with intestinal inflammation therapeutic effects may be one of the strategies of replacing antibiotics.Genera Bacteroides(Ba)and Blautia(Bl)contain a large number of NGP candidates.In this study,we first identified the biological character of Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 previously isolated from the gut of healthy weaned pigs.Following that,we evaluated the immunomodulatory effects of the two strains using an intracellular infection model in murine macrophage(RAW264.7).Finally,using a mice model with dextran sodium sulphate(DSS)-induced colitis,we further investigated effects of the two strains and their metabolites on colitis and preliminarily revealed underlying mechanisms.Our findings may provide new ideas for developing strategies of replacing antibiotics and preventing of IBD.Experiment 1 Identification and Biological Characteristics of Pig-derived Blautia hominis LYH1 and Bacteroides cellulosilyticus LYH2In this experiment,we first identified physiological,biochemical,genetic and biological characteristics of pig-derived Bl.hominis LYH1(deposited in China Center for Type Culture Collection,CCTCC NO:M 2021753)and Ba.cellulosilyticus LYH2(deposited as CCTCC NO:M 2021754).Then,the antibiotic resistance and antibacterial activities of the two strains were investigated in vitro.Main findings are shown as follows:1.Bl.hominis LYH1 is oval-shaped,Gram-positive and asporulate,while Ba.cellulosilyticus LYH2 is rod-shaped,Gram-negative and asporulate.Both strains are anhemolytic and gelatinase-,oxidase-and catalase-negative.2.Bl.hominis LYH1 is sensitive to eight antibiotics such as enrofloxacin,sulfamethoxazole and rifampicin.It has weak sensitivity to vancomycin and is not sensitive to six antibiotics such as amoxicillin,lincomycin and clindamycin.Ba.cellulosilyticus LYH2is sensitive to 13 antibiotics such as polymyxin,enrofloxacin and tetracycline,and has weak sensitivity to amoxicillin,rifampicin,and ampicillin.3.Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 exhibit different antibacterial activities against Salmonella Typhimurium,Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.Ba.cellulosilyticus LYH2 exhibits the strongest inhibition on S.Typhimurium.4.Both genomes of Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 are enriched Carbohydrate-Active enzymes(CAZymes).Ba.cellulosilyticus LYH2,in particular,has the ability to degrade cellulose and hemicellulose.Antibiotic analogues are present in the metabolites of both strains.Experiment 2.Immunomodulatory Effects of Pig-Derived Blautia hominis LYH1 and Bacteroides cellulosilyticus LYH2 on Murine MacrophagesTo investigate the effect of the two strains to the immunity of macrophages,an intracellular infection model of murine mononuclear macrophage RAW264.7 cell line was established using S.Typhimurium.Results showed that:1.Both live Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 as well as their metabolites reduced intracellular replication of S.Typhimurium(P<0.05).2.Metabolites of Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 increased the viability of S.Typhimurium-infected RAW264.7 cells(P<0.05).3.Metabolites of Bl.hominis LYH1 downregulated the relative expression of genes encoding inflammatory cytokines(IL-6,IL-17A,and TNF-α)and nitric oxide synthase(i NOS)(P<0.05),while metabolites of Ba.cellulosilyticus LYH2 downregulated the expression of TNF-αand i NOS(P<0.05).The results showed that pig-derived Bl.hominis LYH1 and Ba.cellulosilyticus LYH2could alleviate the cell inflammatory damage caused by S.Typhimurium by promoting the antibacterial activity of macrophages and decreasing their inflammatory response.Experiment 3.Mechanism of alleviating DSS-Induced Colitis in Mice by Pig-Derived Blautia hominis LYH1 and Bacteroides cellulosilyticus LYH2Sixty specific-pathogen-free six-week-old male C57BL/6J mice with similar bodyweight were randomly allocated into 6 groups,including control,DSS,DSS+each of the two strains(live bacteria),and DSS+metabolites from each of the two strains.The experiment was divided into two stages.Stage 1 lasted for 21 days,during which mice in the control and DSS groups were given sterile saline by gavage,while mice in the DSS+live bacteria group and DSS+metabolites group were given an equal volume of live bacteria(10~9 CFU/m L)or their concentrated metabolites every other day by gavage.In stage 2,mice except for the control group were given 3%DSS solution to induce acute colitis.After 7days,all mice were sacrificed and samples were collected.Results are shown as follows:1.Changes in bodyweight,colon length,and disease activity index(DAI)indicated a successful DSS-induced colitis mouse model.DSS challenge increased the expression of genes encoding inflammatory cytokines(TNF-α,IL-1β,and IL-6)in the colon of mice(P<0.05).Treatment with both strains increased colon length and reduced the expression of IL-1β,IL-6,and TNF-α(P<0.05)in colon compared to DSS group.Treatment of Bl.hominis LYH1 also tended to downregulate the expression of signal transducer and activator of transcription 1(STAT1)after infection(0.05<P<0.1).2.Compared with DSS group,metabolites of Ba.cellulosilyticus LYH2 increased the abundance of tight junction proteins,occludin,claudin-1,and ZO-1 in colon(P<0.05).3.Treatment of Ba.cellulosilyticus LYH2 increased the thickness of colonic mucus layer in colon compared to DSS group(P<0.05).4.Compared with DSS group,treatment of Ba.cellulosilyticus LYH2 reduced the proportion of CD3~+,CD4~+and CD8~+T lymphocyte subsets in the blood of mice with DSS challenge(P<0.05).5.Treatment of Ba.cellulosilyticus LYH2 increased the concentration of acetic acid,propionic acid,butyric acid,and total short-chain fatty acids(SCFAs)in the colon of DSS-induced colitis mice(P<0.05)compared to DSS group.In conclusion,live bacteria of pig-derived Bl.hominis LYH1 and Ba.cellulosilyticus LYH2 and their metabolites have a protective effect on DSS-induced colitis in mice in the current study,probably by relieving systemic immune activation and inflammatory responses through reducing macrophage polarization,secretion of proinflammatory cytokines and differentiation of T lymphocytes.They may also enhance intestinal barrier by improving the physical and chemical barriers,and improve the enteric environment by regulating the production of SCFAs.Our findings provide a reference for the development of NGPs and novel antibiotic substitutes.