Effects of Gallic acid-graft-chitosans on osteogenesis and adipogenesis in mouse mesenchymal stem cells
- Alternative Title
- Gallic acid-graft-chitosans이 mouse mesenchymal stem cells에서 골형성 및 지방생성에 미치는 영향
- Abstract
- Chitosan is the second most abundant mucopolysaccharides in nature and has many useful biological properties such as biocompatibility, biodegradability and less toxic nature. Gallic acid (GA) is a natural phenolic antioxidant extractable from plants, especially from green tea and widely used in foods, drugs, and cosmetics. It is possible to reduce intra- and intermolecular hydrogen bond network of chitosan by means of the bulky group of the benzene ring of GA, and to increase antioxidant ability of chitosan by conjugation of GA onto chitosan. In this study, four kinds of GA-g-chitosans with different gallic acid contents were synthesized, and their antioxidant activities, effects on differentiation and formation of osteogenesis and anti-obesity effects were investigated.
The antioxidant activates of the GA-g-chitosans were evaluated using a variety of cellular and non-cellular oxidative systems such as 1,1-diphynyl-2picrylhydrazyl (DPPH) radical scavenging assay, intercellular reactive oxygen species (ROS) by 2’,7’-dichlorofuorencein diacetate (DCFH-DA), inhibition of lipid peroxidation assayed by DPPP fluorescence, and expression levels of antioxidant enzyme by western blot analysis. These results demonstrated that GA-g-chitosans have strong antioxidant activities and potentials for antioxidant materials.
In the osteogenic differentiation in mouse bone marrow cells, GA-g-chitosans enhanced phenotype markers of osteogeneic differentiation such as alkaline phophatase (ALP), type-1 collagen, osteocalcin, osteopontin and mineralization. GA-g-chitosans increased mRNA and protein levels of bone morphogenetic protein -2 and bone morphogenetic protein -4, and also stimulated mRNA and protein expressions lvels of SMAD1/5/8, pSMAD1/5/8, Runx2, Ostrix, Dlx5, p38 and pERK, which are associated BMP/SMADs pathway. Moreover, GA-g-chitosan induced wnt/β-catenin signals, which are belong to another pathway of osteogenic differentiation.
Furthermore, potential inhibitory effects of the GA-g-chitosans on adipocyte differentiation in mouse bone marrow cell were studied to evaluate abilities of anti-obesity. The effects of GA-g-chitosans on liipid accumulation and the expression of several genes and protein related with adipogenesis were examined using reverse transcriptional-polymerase chain reaction (PCR) and western blot analysis. At Oil red O staining assay, treatment of GA-g-chitosan significantly decreased lipid accumulation level during adipocyte differentiation and down-regulated peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhencer-binding proteins α (C/EBPα), sterol regulatory element-binding protein(SREBP). GA-g-chitosan suppressed adipogenetic targets gene related to fatty acid synthase, lipoprotein lipase and leptin. In order to understand the effects of GA-g-chitosan on obesity-induced inflammation, various factors associated with inflammatory response such as TNF-α, IL-6, IL-1β were evaluated using RT-PCR, Western-bot and ELISA. After incubation for 9 days, GA-g-chitosan decreased mRNA and protein expression of these cytokines in a dose-dependent manner. These results suggested that GA-g-chitosan reduced obesity-induced inflammation levels.
In conclusion, our findings prove that GA-g-chitosans have antioxidant effects. In addition, GA-g-chitosan not only stimulates osteoblastic differentiation, but also inhibit adipogenetic differentiation through Wnt/β-catenin pathways.
- Issued Date
- 2010
- Awarded Date
- 2010. 8
- Type
- Dissertation
- Keyword
- osteogenesis adipogensis chitosan
- Publisher
- 부경대학교
- Alternative Author(s)
- Cho, Young-Sook
- Affiliation
- 부경대학교
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract ⅰ
Table of Contents iii
List of Table viii
List of Figures ix
Chapter 1 Preparation, characterization and antioxidant properties of gallic acid-grafted-chitosans 1
1.Introduction 2
2. materials and methods 4
2.1.Materials 4
2.2.Preparation of GA-g-chitosans 4
2.3. Instruments 4
2.4. Determination of gallic acid contents in GA-g-chitosans and estimation of solubility 6
2.5 DPPH scavenging activity 6
2.6. Hydrogen peroxide scavenging activity 7
2.7. Reducing power 7
2.8. Cell culture 7
2.9. Cytotoxicity assay 8
2.10. Determination of intracellular ROS formation using DCFH-DA labeling 8
2.11. Membrane lipid peroxidation assessment by the DPPP fluorescence method 9
2.12 Genomic DNA isolation 9
2.13. Determination of radical mediated DNA damage 10
2.14. Western blot analysis 11
2.15. Statistical analysis 11
3.Results and Discussion 12
3.1. Preparation and characterization of GA-g-chitosans 12
3.2. Determination of GA contents in GA-g-chitosans 15
3.3. DPPH scavenging capacity 17
3.4. Hydrogen peroxide scavenging capacity 20
3.5. Reducing power 21
3.6.Cytotoxicity, intracellular ROS determination and lipid peroxidation inhibition 21
3.7. Inhibition of radical-mediated DNA damage by GA-g-chitosans 30
3.8 Effect on H2O2 induced antioxidant enzyme expressions 30
4. Summary 34
5. References 36
Chapter 2.Effect of GA-g-chitosan on D1 mouse mesenchymal cell during osteoblasic differentiation 41
1. Introduction 42
2. Materials and Methods 49
2.1. Cell culture 49
2.2. Differentiation 49
2.3. Cell viability assay 49
2.4. Alkaline phosphatase (ALP) activity 50
2.5. Analysis of mineralization 51
2.6. Assaying the levels of osteocalcin, osteopontine and BMP-2,4 51
2.7. Measurement of collagen amount 52
2.8. RT-PCR analysis 52
2.9. Western blot analysis 53
2.10. Statistical analysis 53
3.Results and Discussion 55
3.1. Cytotoxocity of GA-g-chitosans 55
3.2. Alkaline Phosphatase activity 55
3.3 Assaying of Mineralization in D1 mouse mesencymal cells and measure collagen content 60
3.4. GA-g-chitosan(I) increase bone phenotype makers (ALP, collagen, mineralization and osteocalcin 64
3.5. Measurement of Bone morptogentic proteins(BMP-2,3,4) level by PCR ,wsternblotting and ELISA 71
3.6. Activations of SMAD1/5/8 by GA-g-chitosan (I) 77
3.7. Activations of osterix dlx5 and Rrnx2 by GA-g-chitosan (I) 80
3.8. Effect of noggin during BMP-2 mediated osteogenesis 83
3.9. Differentiation of effect of GA-g-chitosan through wnt /β-catenin pathway 86
4. Summary 93
5. References 95
Chater 3. Inhibitory effects Adipogenesis and adiopo-realated inflammation in D1 mouse mesenchymal cells 103
1.Introduction 104
2. Materials and Methods 111
2.1. Cell culture 111
2.2. Differentiation 111
2.3. Cell viability assay 111
2.4. Apoptotic assay using Hoechest 33258 staining 112
2.5. Oil Red O staining 112
2.6. RT-PCR analysis 113
2.7 Western blotting analysis 113
2.8. Assaying the levels of proinflammtion cytokines 114
3. Results and Discussion 116
3.1. Effect of GA-g- chitosan derivatives (I to IV) on viability bone marrow induced adipocyte cell 116
3.2. Effect of GA-g- chitosan derivatives (I to IV) on lipid accumulation in bone marrow induced adipocyte cell 116
3.3. Effect of GA-g-cihitosan (I) on mRNA and protein expression of adipogenic factor 120
3.4. Effect of GA-g-cihitosan (I) on mRNA and proetein expression of adipogenic enzymes 123
3.5. Activation of wnt/β-catenin pathway by GA-g-cihitosan (I) in bone marrow derived Adipocyte 126
3.6. Effect of GA-g-chitosan (I) on mRNA and protein expression of inflammatory cytokine 127
4. Summary 135
5. References 137
CONCLUSION 141
- Degree
- Doctor
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