Title Page
Contents
List of abbreviations 14
Abstract 16
Chapter 1. Literature review 18
1.1. Sarcopenia 18
1.1.1. Definition of sarcopenia 18
1.1.2. Cause and mechanism of sarcopenia 19
1.2. Probiotics 20
1.2.1. Definition of probiotics 20
1.2.2. Effect of Lactobacillus as a probiotics 22
1.2.3. Postbiotics 22
1.3. Caenorhabditis elegans 23
1.3.1. in vivo model C. elegans 23
Chapter 2. Screening of potential probiotics isolated from Kimchi 25
2.1. Materials and methods 25
2.1.1. Isolation of lactobacilli from kimchi 25
2.1.2. Acid tolerance and bile acid tolerance 25
2.1.3. Adhesion capacity assay using HT-29 cells 26
2.1.4. Measurement of GFP and Life span assay 27
2.1.5. Movement measurement according to aging of PD4251 28
2.1.6. Statistical analyses 28
2.2. Results and discussion 29
2.2.1. Sample collection and isolation of lactic acid bacteria 29
2.2.2. Evaluation of potential probiotics properties of kimchi-derived LAB 31
2.2.3. Identification of isolated strains 36
2.2.4. Screening of probiotics through the expression of pmk-1 :: GFP intensity in C. elegans. 38
2.2.5. Evaluation of lifespan extension of probiotics using C. elegans. 41
2.2.6. Screening through expression of myo-3 :: GFP intensity in C. elegans. 43
2.2.7. Evaluation of lifespan extension of probiotics using PD4251 45
2.2.8. Movement measurement of C. elegans PD4251 according to probiotics treatment 47
Chapter 3. Preventing effect on Dexamethasone-induced muscle atrophy in vitro 51
3.1. Materials and methods 51
3.1.1. Cell culture and differentiation 51
3.1.2. Manufacture of cell extract or conditioned media using L. rhamnosus JY02 52
3.1.3. Measurement of cell viability 53
3.1.4. Pas stain and Giemsa stain visualization in the Myotubes 53
3.1.5. Immunofluorescence and fusion index determination 54
3.1.6. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in C2C12 56
3.1.7. Western blotting analysis 58
3.1.8. Statistical analysis 58
3.2. Results and discussion 59
3.2.1. Evaluation of cell viability in C2C12 cells 59
3.2.2. Evaluation of efficacy of JY02 in relieving Dex-induced C2C12 atrophy myotube diameter 61
3.2.3. Immunofluorescence and determination of C2C12 myotube fusion index 67
3.2.4. mRNA expression by RT-qPCR in Dex-induced myotubes 70
3.2.5. Protein expression by Western blot in Dex-induced myotubes 73
Chapter 4. Preventing effect on Dexamethasone-induced muscle atrophy in vivo 75
4.1. Materials and methods 75
4.1.1. Animal experiments 75
4.1.2. Assessment of body composition and grip strength 76
4.1.3. Statistical analysis 76
4.2. Results and discussion 77
4.2.1. Histological analysis 77
4.2.2. Body composition and Exercise capacity testing 77
4.2.3. Body composition and Exercise capacity testing 80
4.2.4. H&E staining of mouse muscle fibers under JY02 treatment 83
4.3. Conclusion 88
Reference 89
Summary in Korean 101
Table 1. List of currently used probiotic strains. 21
Table 2. Potentially probiotic lactic acid bacteria strains isolated from Kimchi. 30
Table 3. List of 44 potential probiotics isolated from kimchi identified by 16S rRNA sequencing. 37
Table 4. Nucleotide sequences of 16S rRNA of LFR20-030. 49
Table 5. Primer sequences used in this study. 57
Figure 1. Evaluation of growth in the presence of acid. 33
Figure 2. Evaluation of growth in the presence of bile acid. 34
Figure 3. Adhesion capacity of isolates to HT-29 cell line. 35
Figure 4. Induction of pmk-1::GFP exposed to LAB strains and E. coli OP50 for 24h. 40
Figure 5. Lifespan analysis of C. elegans exposed to probiotic lactic acid bacteria. 42
Figure 6. Induction of myo-3::GFP exposed to LAB strains and E. coli OP50 for days 7. 44
Figure 7. Lifespan analysis of PD4251 exposed to lactic acid bacteria. 46
Figure 8. Movement measurement analysis by date of C. elegans PD4251. 48
Figure 9. The phylogenetic position of LFR20-030. 50
Figure 10. Effect of Cell extract (CE) or Conditioned media (CM) on cell viability of C2C12 myoblasts and myotubes. 60
Figure 11. Cell extract of JY02 increases the diameter of C2C12 myotubes in Pas staining. 63
Figure 12. Conditioned media of JY02 increases the diameter of C2C12 myotubes in Pas staining. 64
Figure 13. Cell extract of JY02 increases the diameter of C2C12 myotubes in Giemsa staining. 65
Figure 14. Conditioned media of JY02 increases the diameter of C2C12 myotubes in Giemsa staining. 66
Figure 15. Immunofluorescence and determination of cell extract treated C2C12 myotube fusion index. 68
Figure 16. Immunofluorescence and determination of conditioned media treated C2C12 myotube fusion index. 69
Figure 17. The RT-qPCR analysis and quantification of MuRF1 and atrogin-1 in Dex-induced myotube. 71
Figure 18. The RT-qPCR analysis and quantification of MyoD in Dex-induced myotube. 72
Figure 19. Measurement of MuRF1 protein expression in myotubes. 74
Figure 20. Schematic of experimental schedules. 78
Figure 21. Body weight change during the mouse experiment. 79
Figure 22. Changes in the muscle weight in mice with dexamethasone-induced muscle atrophy. 82
Figure 23. Cross-sectional areas (CSA) of the quadriceps (QD), muscle fibers. 85
Figure 24. Cross-sectional areas (CSA) of the gastrocnemius (GC), muscle fibers. 86
Figure 25. Cross-sectional areas (CSA) of the tibialis anterior muscle (TA), muscle fibers. 87