Specific Heat-Killed Lactic Acid Bacteria Enhance Mucosal Aminopeptidase N Activity in the Small Intestine of Aged Mice
Abstract
1. Introduction
2. Results
2.1. Experiment 1
2.1.1. Body Weight, Skeletal Muscle and Adipose Tissue Weights, and Feed Intake
2.1.2. Total Length, Villous Height, and Mucosal APN Activity of Small Intestine
2.1.3. Serum L-Amino Acid and Total Protein Levels
2.1.4. BMD of Femur
2.1.5. Serum Levels of Bone Metabolism Markers and Calcium
2.2. Experiment 2
2.2.1. APN Activity of IEC-6 Cells Co-Cultured with Heat-Killed LAB
2.2.2. APN Activity in IEC-6 Cells Co-Cultured with RNase-, DNase-, or Lysozyme-Treated LP
2.2.3. APN Activity of IEC-6 Cells Treated with TLR2 Agonists
2.2.4. TLR2 Neutralization Study
3. Discussion
4. Materials and Methods
4.1. Experiment 1
4.1.1. Preparation of Heat-Killed Lactic Acid Bacteria and Diet
4.1.2. Animal Experiment
4.1.3. APN Activity in Small Intestinal Mucosa
4.1.4. Measurement of Small Intestinal Villus Height
4.1.5. Micro-X-Ray-Computed Tomography Analysis of Femur
4.1.6. Serum Biochemical Analysis
4.2. Experiment 2
4.2.1. Cell Line
4.2.2. APN Activity in IEC-6 Cells Co-Cultured with Heat-Killed LAB
4.2.3. APN Activity in IEC-6 Cells Co-Cultured with RNase-, DNase-, or Lysozyme-Treated LP
4.2.4. APN Activity in IEC-6 Cells Treated with Toll-Like Receptor 2 Agonists
4.2.5. TLR2 Neutralization Study
4.2.6. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
APN | Aminopeptidase N |
ANOVA | Analysis of variance |
BCA | Bicinchoninic acid |
BMD | Bone mineral density |
CCK-8 | Cell counting kit-8 |
CTX-1 | C-terminal telopeptides of type I collagen |
EF | Enterococcus faecalis |
FBS | Fetal bovine serum |
FSL1 | Fibroblast-stimulating lipopeptide1 |
GlcNAc | N-acetylglucosamine |
HE | Hematoxylin and eosin |
IEC-6 | Intestinal epithelial cell 6 |
LAB | Lactic acid bacteria |
LM | Leuconostoc mesenteroides |
LP | Lactiplantibacillus plantarum |
MurNAc | N-acetylmuramic acid |
NEFA | Non-esterified fatty acids |
NTX | N-terminal telopeptide of type I collagen |
Pam3CSK4 | Pam3Cys-Ser-Lys4 |
PGN | Peptidoglycan |
pNA | p-nitroaniline |
STING | Stimulator of interferon genes |
TLR | Toll-like receptor |
TNF-α | Tumor necrosis factor-α |
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YC | Aged Mice | ||||
---|---|---|---|---|---|
AC | EF | LM | LP | ||
Final body weight (g) | 41.17 ± 1.42 | 46.68 ± 3.23 | 45.63 ± 2.40 | 46.87 ± 2.85 | 45.48 ± 1.94 |
Gastrocnemius muscle | |||||
Weight (g) | 0.18 ± 0.00 | 0.15 ± 0.03 | 0.18 ± 0.00 | 0.17 ± 0.01 | 0.16 ± 0.01 |
Ratio in BW (%) | 0.44 ± 0.01 a | 0.31 ± 0.06 b | 0.40 ± 0.02 ab | 0.36 ± 0.01 b | 0.35 ± 0.02 b |
Epididymal adipose tissue | |||||
Weight (g) | 1.07 ± 0.07 | 1.25 ± 0.20 | 1.38 ± 0.15 | 1.23 ± 0.15 | 1.30 ± 0.07 |
Ratio in BW (%) | 2.61 ± 0.14 | 2.60 ± 0.31 | 3.01 ± 0.28 | 2.58 ± 0.24 | 2.88 ± 0.20 |
Feed intake (g/mouse/day) | 2.26 ± 0.19 | 2.14 ± 0.23 | 2.27 ± 0.17 | 2.56 ± 0.24 | 2.51 ± 0.21 |
Ingredients (g/kg Diet) | Control Diet | LAB-Supplemented Diet |
---|---|---|
Maize starch | 519.486 | 517.486 |
α- Maize starch | 10 | 10 |
Sucrose | 100 | 100 |
Casein | 200 | 200 |
Soybean oil | 70 | 70 |
Cellulose | 50 | 50 |
AIN-93 Vitamin mix | 10 | 10 |
AIN-93 Mineral mix | 35 | 35 |
L-Cystine | 3 | 3 |
Choline bitartrate | 2.5 | 2.5 |
Tert-butylhydroquinone | 0.014 | 0.014 |
Heat-killed LAB | - | 2 |
Total | 1000 | 1000 |
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Tsuruta, T.; Wakisaka, M.; Watanabe, T.; Nishijima, A.; Ikeda, A.; Teraoka, M.; Wang, T.; Chen, K.; Nishino, N. Specific Heat-Killed Lactic Acid Bacteria Enhance Mucosal Aminopeptidase N Activity in the Small Intestine of Aged Mice. Int. J. Mol. Sci. 2025, 26, 5742. https://doi.org/10.3390/ijms26125742
Tsuruta T, Wakisaka M, Watanabe T, Nishijima A, Ikeda A, Teraoka M, Wang T, Chen K, Nishino N. Specific Heat-Killed Lactic Acid Bacteria Enhance Mucosal Aminopeptidase N Activity in the Small Intestine of Aged Mice. International Journal of Molecular Sciences. 2025; 26(12):5742. https://doi.org/10.3390/ijms26125742
Chicago/Turabian StyleTsuruta, Takeshi, Mami Wakisaka, Takumi Watanabe, Aoi Nishijima, Akihito Ikeda, Mao Teraoka, Tianyang Wang, Kuiyi Chen, and Naoki Nishino. 2025. "Specific Heat-Killed Lactic Acid Bacteria Enhance Mucosal Aminopeptidase N Activity in the Small Intestine of Aged Mice" International Journal of Molecular Sciences 26, no. 12: 5742. https://doi.org/10.3390/ijms26125742
APA StyleTsuruta, T., Wakisaka, M., Watanabe, T., Nishijima, A., Ikeda, A., Teraoka, M., Wang, T., Chen, K., & Nishino, N. (2025). Specific Heat-Killed Lactic Acid Bacteria Enhance Mucosal Aminopeptidase N Activity in the Small Intestine of Aged Mice. International Journal of Molecular Sciences, 26(12), 5742. https://doi.org/10.3390/ijms26125742