Anti-allergic effect of chitooligosaccharides on mast cell-mediated allergy model
- Alternative Title
- RBL-2H3 비만세포에서의 키토 올리고당의 항 Allergy 효과
- Abstract
- The discovery of drugs for the treatment of inflammatory allergic diseases is an important subject in human healthy. Chitoologosaccharides (COSs), depolymerized products of chitosan, with low molecular weight (1-10 kDa), are known to have various bioactivities. The aim of the present study was to determine inhibitory effects of COSs on in vitro allergic reactions in RBL-2H3 mast cells. COSs were found to be effective in suppressing the releases of histamine and β-hexosaminidase from RBL-2H3 cells stimulated with calcium ionophore A23187 or immunoglobulin E (IgE)-antigen complexes. The extent of inhibition of COSs was accompanied by intracellular Ca2+ levels. In addition, COSs inhibited the A23187 plus PMA (phorbol 12-myristate 13-acetate)-induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-6 secretion and expression. Moreover, RT-PCR and Western blot analysis revealed that COSs reduced the expression of antigen-stimulated FcεRI, the high affinity IgE receptor through downregulation of ERK phosphorylation. COS with molecular weight of 1-3 kDa was the most effective inhibition in all assays. Taken together, the in vitro anti-allergic effects of COSs suggest possible therapeutic applications for this agent in allergic diseases through inhibition of degranulation depend on the decrease of intracellular Ca2+ levels, reduction of inflammatory cytokines secretion and expression via suppression of MAPKs signaling pathway, and downregulation on the high affinity IgE receptor expression via attenuation of ERK phosphorylation in RBL-2H3 cells.
- Issued Date
- 2010
- Awarded Date
- 2010. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- Department of Chemistry, Graduate School Pukyong National University
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of contents ii
List of Tables v
List of figures vi
List of abbreviations vii
Introduction 1
1. Allergy signs and symptoms 1
2. Allergic causes 4
2.1. Environmental factors 4
2.2. Genetic basis 4
3. The allergic reactions 5
3.1. Allergen sensitization 5
3.2. Early-phase reactions 5
3.3. Late-phase reactions 7
3.4. Chronic allergic inflammation 8
4. Important components in allergic reaction 8
4.1. Allergens 8
4.2. IgE antibody and its receptors 9
4.2.1. Immunoglobulin E 9
4.2.2. IgE pharmacokinetics 9
4.2.3. IgE receptors 10
4.3. Mast Cells and Basophils 12
4.4. Histamine and cytokines 13
4.5. Th1 and Th2 cells 14
5. Therapeutic strategies for allergic diseases 15
5.1. Corticosteroids 15
5.2. Antihistamines 16
5.3. Cytokine modulators 16
5.4. Anti-IgE 16
Materials and methods 19
1. Reagents 19
2. Preparation of chitosan oligosaccharides (COSs) using an UF membrane bioreaction 19
3. Sample preparation for cell treatment 20
4. Cell culture 20
5. MTT assay 20
6. Histamine release assay using A23187 or antigen DNP-BSA stimulation 20
7. β-hexosaminidase release assay using A23187 or antigen DNP-BSA stimulation 21
8. Measurement of intracellular calcium levels 22
9. Microscope images of RBL-2H3 cells after degranulation 22
10. Measurement of cytokine secretion 22
11. Reverse transcription-polymerase chain reaction (RT-PCR) analysis 23
12. Western blot analysis 23
13. Statistical analysis 24
Results 26
1. Cytotoxic effects of COSs on RBL-2H3 cells 26
2. Effects of COSs on the degranulation in A23187 or antigen DNP-BSA-stimulated RBL-2H3 cells 26
2.1. Inhibitory effect on histamine and β-hexosaminidase release in A23187-stimulated RBL-2H3 cells 26
2.2. Inhibitory effect on histamine and β-hexosaminidase release in antigen DNP-BSA-stimulated RBL-2H3 cells 26
3. Inhibitory effect of COSs on intracellular calcium elevation in A23187 or antigen DNP-BSA-stimulated RBL-2H3 cells 27
4. Microscope images of RBL-2H3 cells after degranulation 27
5. Inhibitory effect of COSs on FcεRI expression 34
5.1. Inhibitory effect on FcεRI gene expression in antigen DNP-BSA-induce RBL-2H3 cells 34
5.2. Inhibitory effect on FcεRI protein expression in antigen DNP-BSA-induce RBL-2H3 cells 34
6. Inhibitory effect of COSs on degranulation and FcεRI expression via inhibition on the phosphorylation of ERK signaling pathway in antigen DNP-BSA-induced RBL-2H3 cells 34
7. Effects of COSs on the cytokine secretion and expression in A23187 plus PMA-induced RBL-2H3 cells 38
7.1. Inhibitory effect on the cytokine secretion in A23187 plus PMA-induced RBL-2H3 cells 38
7.2. Inhibitory effect on the cytokine expression in A23187 plus PMA-induced RBL-2H3 cells 38
8. Inhibitory effect of COSs on the phosphorylation of MAPKs signaling pathways in A23187 plus PMA-induced RBL-2H3 cells 41
Discussion 43
Conclusion 50
References 51
Acknowledgements 62
- Degree
- Master
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