| The intestinal barrier and bone metabolism of infants are critical,and a compromised gut barrier and disturbed bone metabolism directly increase the risk of immune-related and bonerelated diseases occurrences.The colonization of the gut microorganisms in infants occurs in parallel with their growth and development,and the relative abundance of Bifidobacterium in the intestine of infants has been negatively correlates with the development of diseases.It has been shown that Bifidobacterium can regulate the intestinal barrier damage and bone metabolism disorders in a variety of diseases or in non-healthy situations.Therefore,Bifidobacterium may play a positive role in the growth and health of infants by regulating intestinal barrier and bone metabolism.Bifidobacterium longum subsp.infantis is the dominant Bifidobacterium in the infant gut.However,whether and how supplementation of Bifidobacterium longum subsp.infantis in infants can regulate the intestinal barrier and bone metabolism remains unclear.Therefore,the purpose of this thesis was to investigate the effects and potential mechanisms of Bifidobacterium longum subsp.infantis on intestinal barrier and bone metabolism in growing mice,aiming to provide theoretical basis for the promotion of infant growth and health through dietary supplementation.The main contents of this thesis are as follows.The regulatory effects of B.longum subsp.infantis on the intestinal immune barrier of 3-week-old mice were compared.The results showed that the regulatory effect of B.longum subsp.infants on the intestinal immune barrier of 3-week-old mice exhibited strain specificity.CCFM1270 and CCFM1271 increased the ratio of colonic Ig G2a/Ig E in male mice(p<0.05).CCFM1269,CCFM1270,and CCFM1272 significantly increased the colonic Ig A content in both male and female mice(p<0.05).CCFM1271 significantly increased the colonic Ig A content in male mice(p<0.001).CCFM1269 significantly increased the levels of colonic IFN-γ and Ig G2 a,as well as the ratio of Ig G2a/Ig E in female mice(p<0.05).It also significantly increased the ratio of colonic Ig G2a/Ig E(p<0.05)and significantly reduced the level of colonic IL-4 in male mice(p<0.05).Furthermore,CCFM1269 regulated colonic JAK/STAT pathwayrelated genes and proteins in both male and female mice,and significantly increased the ratio of serum Ig G2a/Ig E(p<0.05)and the levels of IFN-γ(p<0.05),thus regulating the Th1/Th2 immune balance.CCFM1269 also significantly increased the content of colonic s Ig A in female mice(p<0.01),regulated the synthesis of Ig A genes in both colonic and Peyer’s patch,and affected the structure of the intestinal microbiota,then promoting the establishment of the intestinal immune barrier.However,CCFM1269 had no significant effect on the JAK/STAT pathway and Ig A synthesis pathway genes in neither jejunum nor ileum.CCFM1269 has a more pronounced role in regulating the immune barrier,and it may also affect intestinal interferon secretion and regulate the genes related to osteoclast differentiation and calcium signaling pathways.The mechanism for CCFM1269 regulating colonic IFN-β levels and intestinal mechanical barrier in growing mice was investigated.The results showed that CCFM1269 increased the level of colonic IFN-β in 3-week-old mice(p<0.05)but had no effect on colonic IFN-β in neither 4-week-old nor 5-week-old mice.Analysis of the pathways related to IFN-β production revealed that CCFM1269 upregulated the expression of TLR4(p<0.05)and promoted the production of colonic IFN-β in 3-week-old mice through the TLR4-TRIF-dependent pathway.However,CCFM1269 did not affect the levels of TLR4 ligands and receptors(lipopolysaccharide and lipopolysaccharide-binding protein)in the serum.CCFM1269 could enhance intestinal barrier function by upregulating the expression of colonic tight junction proteins(ZOs,Claudins,Mucin2,Occludin,and JAMs)and regulating the microbial barrier by gut microbiota modulation.Therefore,CCFM1269 may activate the colonic TLR4/TRIF signaling pathway to increase the content of colonic IFN-β and regulate the intestinal mechanical and intestinal microbial barriers in growing male and female mice.The mechanism for CCFM1269 regulating bone growth in growing mice was elucidated.The results showed that CCFM1269 increased the lengths of femur in all 3-week-old,4-week-old,and 5-week-old mice(p<0.05),but only increased the length of tibia in 5-week-old mice(p<0.05).Histological staining,TRAP staining,and serum bone metabolism markers indicated that CCFM1269 primarily promoted osteoblast differentiation.In terms of gut microbiota,CCFM1269 affected intestinal metabolites by increasing the relative abundance of Bifidobacterium in 3-week-old mice,whereas it affected the composition of intestinal metabolites in mice at 4-week-old and 5-week-old by modulating the relative abundance of other gut microorganisms.In terms of metabolites,CCFM1269 increased the levels of 5-hydroxyindole-3-acetic acid,xanthuric acid,and isobutyric acid in the intestine.Therefore,CCFM1269 may promoted the levels of IGF-1,GH,and IGFBP3 in the serum by adjusting the metabolism of short-chain fatty acids and tryptophan pathways and regulating GH/IGF axis by activating the expression of genes and proteins in the PI3K/AKT pathway in the femur of all 3-week-old,4-week-old,and 5-week-old mice and the tibia in 5-week-old mice,increasing the content of osteocalcin,and ultimately promoting bone growth.The composition and characterization of infant gut microbiota,breast milk microbiota,and maternal gut microbiota were analyzed,particularly s Ig A-coated bacteria community.The 16 S r RNA gene sequencing results showed significant differences in the total bacterial community in breast milk,maternal gut,and infant gut.The dominant genus in colostrum,transitional milk,and mature milk was Staphylococcus,Streptococcus,and Bifidobacterium,respectively.Additionally,the dominant genus in their corresponding stage of infant gut was Bifidobacterium,while in maternal gut,it was Bacteroides.Moreover,in breast milk,maternal gut,and infant gut,the composition of s Ig A-coated bacterial and-uncoated bacterial were different,but the dominant genus in both s Ig A-coated bacterial and-uncoated bacterial community were Escherichia /Shigella and Pseudomonas.Bifidobacterial gro EL gene sequencing results indicated that the dominant Bifidobacterium in colostrum,transitional milk,and mature milk was B.pseudocatenulatum.Whilst the dominant Bifidobacterium in maternal gut was B.pseudocatenulatum and B.longum subsp.infantis.In addition,the dominant Bifidobacterium in the infant gut was B.longum subsp.infantis.Both B.pseudocatenulatum and B.longum subsp.infantis were the dominant species in both s Ig A-coated and uncoated Bifidobacterium in the maternal intestine,and B.longum subsp.infantis was the dominant s Ig A-coated and uncoated Bifidobacterium in the infant intestine.The high abundance of Bifidobacterium longum subsp.infantis in the infant gut may help enhance its intestinal barrier and regulate bone growth. |
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