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Gut Microbes Volume 16, 2024 - Issue 1
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Research Paper

Human breastmilk-derived Bifidobacterium longum subsp. infantis CCFM1269 regulates bone formation by the GH/IGF axis through PI3K/AKT pathway

Mengfan Dinga State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, ChinaORCID Iconhttps://orcid.org/0000-0002-5820-6736View further author information
ORCID Icon,
Bowen Lia State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, ChinaORCID Iconhttps://orcid.org/0000-0002-4199-7164View further author information
ORCID Icon,
Haiqin Chena State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, ChinaView further author information
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Dong Liangc Department of Applied Nutrition I, China National Center for Food Safety Risk Assessment, Beijing, ChinaCorrespondence[email protected]
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R. Paul Rossd International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China;e APC Microbiome Ireland, University College Cork, Cork, IrelandView further author information
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Catherine Stantond International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China;e APC Microbiome Ireland, University College Cork, Cork, Ireland;f Food Biosciences, Teagasc Food Research Centre, Fermoy, Cork, IrelandView further author information
,
Jianxin Zhaoa State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China;g National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu, ChinaView further author information
,
Wei Chena State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China;g National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu, ChinaView further author information
&
Bo Yanga State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China;b School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China;d International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, ChinaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-1347-3718View further author information
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Article: 2290344 | Received 14 Aug 2023, Accepted 27 Nov 2023, Published online: 20 Dec 2023

Figures & data

Figure 1. Experimental design and change of body weight and bone length.

(a) Experimental design. Neonatal mice were born after a 3-week gestation period and kept with their mothers until weaning at 3 weeks old. Male and female newborn mice were gavaged with normal saline and one of two strains of B. longum subsp. infantis (CCFM1269 or I5TI) from one week through three, four, and five weeks of age (8 male or female mice per group). The newborn mice were subsequently put to death. (b) Body weight of three, four, and five weeks of age. (c) Femur length of three weeks of age. (d) Femur length of four weeks of age. (e) Femur length of five weeks of age. (f) Tibia length of three weeks of age. g, tibia length of four weeks of age. h, tibia length of five weeks of age.
Figure 1. Experimental design and change of body weight and bone length.

Figure 2. Alizarin Red and TRAP staining of femur and tibia.

(a) Alizarin Red of femur, magnification: 180 × .(b) TRAP staining of the femur, magnification: 180 × .(c) Alizarin Red of the tibia, magnification: 140 × .(d) TRAP staining of the tibia, magnification: 140 × .
Figure 2. Alizarin Red and TRAP staining of femur and tibia.

Figure 3. Effects of B. longum subsp. infantis on the serum osteogenic factors.

(a) 3-week-old OPG. (b) 4-week-old OPG. (c) 5-week-old OPG. (d) 3-week-old OT/BGP. (e) 4-week-old OT/BGP. (f) 5-week-old OT/BGP. (g) 3-week-old PINP. (h) 4-week-old PINP. (I) 5-week-old PINP. (j) 3-week-old CTX-I. (k) 4-week-old CTX-I. (l) 5-week-old CTX-I. Data was shown in mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group. OPG, osteoprotegerin; CTX-1, c-telopeptide of type 1 collagen; OT/BGP, osteocalcin/bone-γ-carboxyglutamic acid-containing protein; PINP, procollagen I N-terminal tripeptide
Figure 3. Effects of B. longum subsp. infantis on the serum osteogenic factors.

Figure 4. Effects of B. longum subsp. infantis on the gut microbiota of 3-week-old mice.

(a) Chao1 index. (b) Shannon index. (c) Female β-diversity, (d) Male β-diversity, (e) Female top30 genus stacking plot. (f), male top 30 genus stacking plot. (g) significant changes in the relative abundance of genera in female mice. (h) Significant changes in the relative abundance of genera in male mice. (i) Bifidobacterial observed OTUs. (j) Bifidobacterial Shannon index. (k) Female bifidobacterial β-diversity. (l) Male bifidobacterial β-diversity. (m) Female and male Bifidobacterium species level stacking plot. (n) Significant changes in the relative abundance of Bifidobacterium species in female mice. (o) Significant changes in the relative abundance of Bifidobacterium species in male mice. Data were shown as mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group.
Figure 4. Effects of B. longum subsp. infantis on the gut microbiota of 3-week-old mice.

Figure 5. Effects of B. longum subsp. infantis on the gut microbiota of 4-week-old mice.

(a) Chao1 index. (b) Shannon index. (c) Female β-diversity, (d) Male β-diversity, (e) Female top30 genus stacking plot. (f) Male top 30 genus stacking plot. (g) Significant changes in the relative abundance of genera in female mice. (h) Significant changes in the relative abundance of genera in male mice. (i) Bifidobacterial observed OTUs. (j) Bifidobacterial Shannon index. (k) female bifidobacterial β-diversity. (l) Male bifidobacterial β-diversity. (m) Female and male Bifidobacterium species level stacking plot. Data were shown as mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group.
Figure 5. Effects of B. longum subsp. infantis on the gut microbiota of 4-week-old mice.

Figure 6. Effects of B. longum subsp. infantis on the gut microbiota of 5-week-old mice.

(a) Chao1 index. (b) Shannon index. (c) Female β-diversity, (d) Male β-diversity, (e) Female top30 genus stacking plot. (f) Male top 30 genus stacking plot. (g) Significant changes in the relative abundance of genera in female mice. (h) Significant changes in the relative abundance of genera in male mice. (i), Bifidobacterial observed OTUs. (j), Bifidobacterial Shannon index. (K) Female bifidobacterial β-diversity. (l) Male bifidobacterial β-diversity. (m), Female and male Bifidobacterium species level stacking plot, (n) Significant changes in the relative abundance of Bifidobacterium species in female mice. (o) Significant changes in the relative abundance of Bifidobacterium species in male mice. Data were shown as mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group.
Figure 6. Effects of B. longum subsp. infantis on the gut microbiota of 5-week-old mice.

Figure 7. Effects of B. longum subsp. infantis CCFM1269 on fecal metabolites.

(a) 3-week-old female differential metabolites. (b) 4-week-old female differential metabolites. (c) 5-week-old female differential metabolites, (d) 3-week-old male differential metabolites. (e) 4-week-old male differential metabolites. (f) 5-week-old male differential metabolites. (g) RDA analysis of differential genus and metabolites of 3-week-old females. (h) RDA analysis of differential genus and metabolites of 4-week-old females. (i) RDA analysis of differential genus and metabolites of 5-week-old females. (j) RDA analysis of differential genus and metabolites of 3-week-old males. (k) RDA analysis of differential genus and metabolites of 4-week-old males. (l) RDA analysis of differential genus and metabolites of 5-week-old males.
The length of the arrow in the RDA analysis implies the magnitude of the effect of the different genera on the different metabolites.
Figure 7. Effects of B. longum subsp. infantis CCFM1269 on fecal metabolites.

Figure 8. Co-existing metabolites of different age groups and RDA analysis between differential metabolites and OPG, OT/BGP, PINP, femur length, and tibia length.

(a) Co-existing metabolites of different age groups in females. (b) Co-existing metabolites of different age groups in males. (c) 3-week-old females RDA analysis. (d) 4-week-old females RDA analysis. (e) 5-week-old females RDA analysis. (f) 3-week-old males RDA analysis. (g) 4-week-old males RDA analysis. (h) 5-week-old males RDA analysis.
The length of the arrow in the RDA analysis implies the magnitude of the effect of the different metabolites on the level of OPG, OT/BGP, PINP, femur length, and tibia length.
Figure 8. Co-existing metabolites of different age groups and RDA analysis between differential metabolites and OPG, OT/BGP, PINP, femur length, and tibia length.

Figure 9. Effects of B. longum subsp. infantis on serum growth factors.

(a) 3-week-old GH. (b) 4-week-old GH. (c) 5-week-old GH. (d) 3-week-old IGF-1. (e) 4-week-old IGF-1. (f) 5-week-old IGF-1. (g) 3-week-old IGFBP3. (h) 4-week-old IGFBP3. (i) 5-week-old IGFBP3. (j) 3-week-old 1,25(OH)2D3. (k) 4-week-old 1,25(OH)2D3. (l) 5-week-old 1,25(OH)2D3. Data was shown in mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group. GH, growth hormone; IGF-1, insulin-like growth factors -1. IGFBP3, insulin-like growth factors -1 binding protein 3.
Figure 9. Effects of B. longum subsp. infantis on serum growth factors.

Figure 10. Effects of B. longum subsp. infantis on the PI3K/AKT pathway at the transcript level.

The relative abundance of GHR, IGFR, IGFBP3, IRS1, mTOR, RUNX2, Grb2, and Grb10 mRNA.
Figure 10. Effects of B. longum subsp. infantis on the PI3K/AKT pathway at the transcript level.

Figure 11. Effects of B. longum subsp. infantis on the PI3K/AKT pathway at the translation level of the femur.

(a) Femur protein band of 3-week-old mice. (b) Femur IGF-1 R expression in 3-week-old females. (c) Femur pPI3K expression in expression in 3-week-old females. (d) Femur pAKT expression in 3-week-old mice. (e) Femur RUNX2 expression in 3-week-old mice. (f) Femur osteocalcin expression in 3-week-old mice. (g) A femur protein band of 4-week-old mice. (h) Femur IGF-1 R expression in 4-week-old mice. (i) Femur pPI3K expression in expression in 4-week-old mice. (j) Femur pAKT expression in 4-week-old female mice. (k) Femur RUNX2 expression in 4-week-old mice. (l) Femur osteocalcin expression in 4-week-old mice. (m) A femur protein band of 5-week-old mice. (n) Femur IGF-1 R expression in 5-week-old mice. (o) Femur pPI3K expression in expression in 5-week-old mice. (p) Femur pAKT expression in 5-week-old mice. (q) Femur RUNX2 expression in 5-week-old mice. (r) Femur osteocalcin expression in 5-week-old mice.
Data was shown in mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group.
Figure 11. Effects of B. longum subsp. infantis on the PI3K/AKT pathway at the translation level of the femur.

Figure 12. Effects of B. longum subsp. infantis on PI3K/AKT pathway at translation level of tibia.

(a) Tibia protein band of 5-week-old band. (b) Tibia IGF-1 R expression in 5-week-old mice. (c) Tibia pPI3K expression in expression in 5-week-old mice. (d) Tibia pAKT expression in 5-week-old mice. (e) Tibia RUNX2 expression in 5-week-old mice. (f) Tibia osteocalcin expression in 5-week-old mice. Data was shown in mean ± SEM (n = 8 per group). *, p < 0.05, compared to the corresponding control group.
Figure 12. Effects of B. longum subsp. infantis on PI3K/AKT pathway at translation level of tibia.
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