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3-Hydroxy-5,6-epoxy-β-ionone isolated from Sargassum horneri, inhibits fine dust induced inflammation through TLR/MyD88 mediated anti-inflammatory pathway and Nrf2/HO-1 mediated antioxidant pathways

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Abstract
In East-Asia region (China, Korea, and Japan) fine dust (FD) have become a major threat of air pollutions and causing negative health effects on human skin, respiratory system, and digestive system. Specifically, the extensive arid or semiarid highlands of northern China and Mongolia (Gobi Desert, Hunshdak Sandy Lands, Loess Plateau, and Taklimakan desert) are considering as the major sources of dust in Asia region. However, coal-burning power plants, rapid developments in industrialization, numerous petroleum vehicles, and large-scale mining operations have contributed to increase the FD concentration in the urban areas located in East-Asia region. Continuous exposure to air pollution such as FD can induce oxidative stress, inflammation, and poses a serious risk to human health. Alveolar macrophages, who lives in lower respiratory tract are capable to phagocyte FD particles reach the lower respiratory. However, depending on the size and particle composition, exposed macrophages may produce inflammatory responses. Recently, several studies reported that, the exposure of FD to macrophages led to inflammatory responses in macrophages including RAW 264.7 cells. According to the recent studies, the exposure of macrophages to FD triggers inflammatory responses in macrophages via altering multiple cell signaling pathways. The endotoxins presented in FD particles found to induce the toll-like receptor-4 mediated inflammation, and reactive oxygen species induce pro-inflammatory cytokine production in macrophages. The continuous/uncontrolled inflammatory activities leading to develop chronic inflammatory responses and end up with the pathogenesis of catastrophic disease conditions like cancer and immunomodulatory diseases. In addition, dust particles inside of the lungs attacked/consumed by alveolar macrophages, and then activated cells removed by lysosomes. Thus, number of healthy macrophages decrease and which might affect to the immune system that further results in less immunity in our body. Therefore, it is very important to down-regulate inflammation induced by FD particles to reduce health concerns associated with FD. Besides the respiratory system, FD also has a possibility to damage digestive system as some part of inhaled dust moved to the digestive system and directly going to the digestive tract with FD contaminated foods. Thus, effect of FD to digestive system cannot neglect and require in-depth studies to expose it effects on a digestive system like inflammation and oxidative stress. According to the statistics cancer incidents reported from colon and rectum were nearly doubled from 1999 to 2012 period, where lung cancer levels were remaining constant in that period. Other than the bad food habits FD also might responsible for this increased levels of colon and rectum cancers. Therefore, author also attempted to evaluate effect of fine dust using digestive tract epithelial cells. CMT-93 is an epithelial cell line separated from a 19 months old male mouse rectum. Recent studies carried out with CMT-93 reported the exposure of CMT-93 to LPS, triggers the inflammatory responses in CMT-93 cells. Therefore, CMT-93 cell model is a promising model to evaluate complication associated with the inflammation in digestive system. However, use of in vitro results for the development of functional materials have limited possibility as the culture cells were maintained in artificial conditions. Therefore, to validate in vitro results it is compulsory to use in vivo research models. Recently, zebrafish (Danio rerio) has been recognized as a promising in vivo model to use in research areas such as cancer, stem cell research and immunology and infectious diseases research due to its morphological and physiological similarity to the mammals, transparency, easy to handle, and less maintenance cost. Other than that, the optical transparency of zebrafish embryos allows for non-destructive and live imaging of the inflammatory responses developed in embryos. Due to these specific morphological and physiological features of zebrafish models provide great opportunities to accelerate the process of drug discovery including target identification, disease modelling, lead discovery, and toxicology. Taken together, during this study author attempted to screen seaweeds with potential anti-inflammatory compounds to develop as a functional ingredient to act against FD induced inflammatory complications. For preliminary screening author used 4 seaweeds (Ecklonia cava, Ishige okamurae, Sargassum horneri, and Porphyra yezoensis). According to the results, Sargassum horneri and Ecklonia cava had strong anti-inflammatory properties against FD-induced inflammation in macrophage cells. Moreover, our results reviled that the treatment of 3-Hydroxy-5,6-epoxy-β-ionone, the active compound isolated from S. horneri blocked the FD-induced inflammation via inhibiting NF-κB, MAPK, and NRF2/HO-1 signal pathways. The evidence in this study providing solid information's to develop functional materials from S. horneri against dust induced inflammation.
Author(s)
Sanjeewa, Kalu Kapuge Asanka
Issued Date
2019
Awarded Date
2019. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000008780
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
전유진
Table Of Contents
Contents i
Summary IX
List of figures XII
List of tables XXII
Background 1
Fine dust 1
What is inflammation? 2
What is Oxidative stress? 3
Importance of seaweeds as a food source to avoid FD-induced inflammation and
oxidative stress 4
Sargassum horneri, the brown seaweed used to isolate pure compounds 5
Selection of suitable extraction method 6
Water extraction methods 6
Enzyme-assisted extraction methods 7
Organic solvent assisted extraction methods 7
Part- 1 8
Initial screening of seaweeds to identify potential candidates for separate antiinflammatory
compounds 8
Abstract 9
Graphical abstract 11
1.1. introduction 12
1.2. Materials and Methods 14
1.2.1. Sample collection 14
1.2.2. Chemicals and regents 14
1.2.3. preparation of crude 80% methanolic extracts from seaweeds 14
1.2.4. Preparation of enzymatic digests from seaweeds 15
1.2.5. Analysis of chemical composition 15
1.2.6. purification of bioactive compounds from S. horneri 17
1.2.7 Apparatus used for the isolation of active compounds 17
1.2.8. HPCPC separation of S. horneri compounds 18
1.2.9. HPCPC separation procedure 18
1.2.10. HPLC analysis 18
1.2.11. Cell culture 19
1.2.12. Cell viability assay 19
1.2.13. Determination of NO production 19
1.2.14. Determination of PGE2, TNF-α, IL-6, and IL-1β production 20
1.2.15. Total RNA extraction and cDNA synthesis 20
1.2.16. Quantitative real-time PCR (qPCR) analysis 20
1.2.17. Western blot analysis 21
1.2.18. Statistical analysis 24
1.3. Results and discussion 25
1.3.1 Extraction efficiency of seaweed samples 25
1.3.3 Anti-inflammatory effects of seaweed extracts against LPS-induced NO production in RAW 264.7 cells 30
1.3.4 PGE2 and Pro-inflammatory cytokine inhibitory effect of 80 % methanolic extracts of E. cava and S. horneri 34
1.3.5 Effects of the seaweed extracts on LPS-induced iNOS and COX2 expression in RAW 264.7 cells 36
1.3.6. Effect of S. horneri extracts on LPS-induced NF-κB protein expressions 38
1.3.7. The effects of S. horneri 80% methanol extract against pro-inflammatory gene expression in LPS-exposed RAW 264.7 cells. 39
1.3.8. fractionation of S. horneri 80% methanol extract for isolate bioactive compounds 42
1.3.9 NO inhibitory effect of three solvent fractions separated from S. horneri against LPS-induced RAW 264.7 cells 42
1.3.10. HPCPC and HPLC spectrums of SHMC after HPCPC chromatography 46
1.3.11. Anti-inflammatory properties and cytoprotective effect of four pure compounds isolated from S. horneri50
1.4. Conclusions 52
1.5. References 53
Part- 2 60
3-Hydroxy-5,6-epoxy-β-ionone isolated from Sargassum horneri protect MH-S mouse
lung cells against fine dust induced inflammation and oxidative stress 60
Abstract 61
Graphical abstract 63
2.1. Introduction 64
2.2. Materials and methods 66
2.2.1. Chemicals and regents 66
2.2.2. Purification and isolation of HEBI from S. horneri 66
2.2.3. Estimation of fine dust particle size by scanning electron microscopy 69
2.2.4. Cell culture 69
2.2.5. Determination of cell viability 69
2.2.6. Evaluation of cell death rates by analysis of lactate dehydrogenase (LDH) levels 70
2.2.7. Determination of NO inhibition effect 70
2.2.8. Determination of PGE2 and pro-inflammatory cytokine production 70
2.2.9. Western blot assay 71
2.2.10. Level of ROS in fine-dust exposed MH-S cells71
2.2.11. Analysis of SOD Activities 72
2.2.12. Effect of HEBI on MAPK pathway related proteins 72
2.2.13. Total RNA extraction and cDNA synthesis 73
2.2.14. Quantitative real-time PCR (qPCR) analysis 73
2.2.15. Statistical analysis 77
2.3. Results and discussion 78
2.3.1-Anti-inflammatory properties of HEBI against fine dust induced inflammation in MH-S cells 78
2.3.1.1. Composition of fine dust 78
2.3.1.2. cell viability and NO production in fine dust exposed MH-S cells 80
2.3.1.3. Cyto-toxic effect of HEBI on MH-S cells 80
2.3.1.4. Protective effect of HEBI against fine dust-induced cell death and NO production in MH-S cells 82
2.3.1.5. Effects of HEBI on fine dust induced PGE2 and pro-inflammatory cytokine secretion (ELISA) 84
2.3.1.6. Effects of HEBI on FD-induced iNOS and COX2 protein production 84
2.3.1.7. Suppressive effect of HEBI in fine dust induced NF-κB phosphorylation and translocation to the nucleus in MH-S macrophages 88
2.3.1.8. HEBI inhibits MAPK protein expression in fine dust exposed MH-S sells 92
2.3.1.9. Effect of HEBI against fine dust induced inflammatory gene expression in MH-S cells 95
2.3.1.10 inhibitory effect of HEBI against fine dust induced TLR activations (RTqPCR). 97
2.3.2-Anti-oxidant properties of HEBI against FD-induced inflammation in MH-S cells 100
2.3.2.1 HEBI inhibits fine dust induced ROS levels in MH-S cells 101
2.3.2.2. Effect of HEBI in fine dust induced SOD and catalase levels in MH-S cells 101
2.3.2.3. HEBI increased the cytosolic antioxidant protein levels in fine dust exposed MH-S cells 104
2.2.2.4 HEBI induced antioxidant mechanism in fine dust exposed MH-S cells through Nrf2/Keap1 mediated antioxidant pathway. 106
2.2.2.5. Fine dust induced NO production and Effect of MAPK inhibitors 109
2.4. Conclusions 111
2.5. References 112
Part- 3 119
Anti-inflammatory and antioxidant mechanisms of 3-Hydroxy-5,6-epoxy-β-ionone isolated from Sargassum horneri on fine dust-exposed CMT-93 mouse epithelial cells (digestive tract) 119
Abstract 120
3.1. Introduction 122
3.2. Materials and methods 125
3.2.1. Chemicals and regents 125
3.2.2. Estimation of fine dust particle size by scanning electron microscopy 125
3.2.3. Culture conditions of CMT-93 cell line 125
3.2.4. Cell viability assay (MTT) and measurement of nitrite by Griess reaction 126
3.2.5. Determination of PGE2 and pro-inflammatory cytokine secretion levels 127
3.2.6. Total RNA extraction and cDNA synthesis 127
3.2.7. Quantitative real-time PCR (qPCR) analysis 127
3.2.8. Western blot analysis 128
3.2.9. Statistical analysis 131
3.3. Results and discussion 132
3.3.1. Composition of fine dust and size distribution 132
3.3.2. cell viability and NO production in fine dust exposed MH-S cells 136
3.3.3 HEBI inhibits fine dust-induced PGE2 and pro-inflammatory cytokine production from CMT-93 cells (ELISA) 136
3.3.4 HEBI inhibits fine dust-induced inflammatory cytokine related gene production from CMT-93 cells (ELISA) 140
3.3.5 Inhibitory effect of HEBI against iNOS and COX2 production from fine dust stimulated CMT-93 cells142
3.3.6 inhibitory effect of HEBI against fine dust induced TLR activations in CMT-93 cells (RT-qPCR144
3.3.7. Effect of HEBI against MyD88 protein expression in fine dust exposed CMT-93 cells 149
3.3.8. Effect of HEBI against fine dust induced NF-κB protein expression in CMT-93 cells 151
3.3.9. Effect of HEBI against fine dust induced MAPK expression in CMT-93 cells 155
3.3.10 HEBI up-regulates anti-oxidant proteins expression in fine dust exposed CMT-93 cells 157
3.3.11 HEBI upregulates anti-oxidant gene expressions in fine dust exposed CMT-93 cells 160
3.4. Conclusions 164
3.5 References 165
Part- 4 173
Abstract 174
Graphical abstract 176
4.1 Introduction 177
4.2. Materials and methods 180
4.2.1. Sample collection and purification 180
4.2.2. Chemicals and regents 182
4.2.3. Estimation of fine dust particle size by scanning electron microscopy 182
4.2.4. In vivo zebrafish experiments 183
4.2.4.1. Origin and maintenance of parental zebrafish 183
4.2.4.2. Measurement of the toxicity of fine dust and HEBI on zebrafish embryo 183
4.2.4.3. Measurement of heart-beating rate of zebrafish 183
4.2.4.4. The toxicity of HEBI on zebrafish embryo by means of the cell death 184
4.2.4.5. Estimation of fine dust induced ROS generation 184
4.2.4.6. Measurement of in vivo NO production 184
4.2.5. Western blot analysis 185
4.2.6 RNA extraction, cDNA synthesis and RT-qPCR analysis 185
4.2.7 Statistical analysis 188
4.3. Results and discussion 189
4.3.1. Composition of fine dust and size distribution analysis of fine dust 189
4.3.2. Fine dust induced toxicity rates in zebrafish embryo 193
4.3.3. Effects of HEBI on fine dust-exposed heart-beating rate of zebrafish model . 196
4.3.4. Protective effect of HEBI against fine dust induced cell deaths in zebrafish embryos 198
4.3.5. protective effect of HEBI on fine dust –induced ROS production in zebrafish embryo 198
4.3.6. Protective effect of HEBI against fine dust induced NO production in zebrafish embryos 199
4.3.7. HEBI down-regulate fine dust-induced iNOS and COX2 expression in zebrafish embryos 203
4.3.8 HEBI protects zebrafish embryo against fine dust induced pro-inflammatory cytokine production 205
4.4. Conclusions 207
4.5. References 208
Acknowledgements 214
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Sanjeewa, Kalu Kapuge Asanka. (2019). 3-Hydroxy-5,6-epoxy-β-ionone isolated from Sargassum horneri, inhibits fine dust induced inflammation through TLR/MyD88 mediated anti-inflammatory pathway and Nrf2/HO-1 mediated antioxidant pathways
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