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Fucosterol and alginic acid purified from Padina commersonii from the Maldives; inhibit particulate matter induced inflammation through NF-κB, MAPK mediated inflammatory pathway and oxidative stress via Nrf2/HO-1 pathway

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Abstract
Bioactive components from marine algae have provided new insights to the natural product research. The study was concentrated on the marine brown algae Padina commersonii; prominent species in the Laccadive Sea. Particulate matter (PM) was
associated with the study as it's considered to be a significant participant in the air
pollution and hence an inducer in serious health issues. Particulate matter which is airborne due to natural sources is combined with anthropogenic sources such as industrial and traffic emissions. PM causes asthma, lung tissue damage as well as inflammation and oxidative stress.
The first part of the study focuses on the isolation of fucosterol and evaluation of its potential to attenuate inflammatory responses. Fucosterol was isolated through successive open silica columns and was characterized via GC-MS and FT-NMR. The study discovered the resilient anti-inflammatory potential via the down-regulation of nitric oxide (NO) levels in PM stimulated RAW 264.7 macrophages. Continued studies revealed its ability to abate the activity of pro-inflammatory cytokines (IL-1β, IL-6, TNFα) including PGE 2 . Gene expression studies via q-PCR and western blot analysis of the iNOS and COX-2 supported the above. Further, the mediation of the responses was evident to be activated through nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. The Nrf2/HO-1 studies solidified the interconnection between inflammation and oxidative stress. Hence, the first part exposed the ability of fucosterol to successfully down-regulate the inflammatory responses stimulated via PM in RAW 264.7 macrophages.
Alginic acid was purified in the second part of the study. The protective effect of alginate from Padina commersonii (PCA) against PM was evaluated. In between theexternal and internal environments, skin is considered to be more than a physical barrier.
A crucial role of the skin is to provide immune functions. It is suggested to function as a semiautonomous immunological organ. As the keratinocytes are the outermost layer of the skin, it is regularly used to assess the effect of the irritants in the dermatology. These cells participate in immune responses via the production of cytokines against the inflammatory events. It was observed that PM stimulate inflammation in HaCaT keratinocytes and in the dermal fibroblasts (HDF) inducing inflammatory pathways. PCA was evident to act as an active protector of the PM and to down-regulate the proinflammatory cytokines and inflammatory mediators. Further, certain key molecules in the NF-κB and MAPK pathways in the cells were studied. The activation of the above was inhibited via the activity of PCA. The metal ion content of PM was considerably reduced by PCA and thus attributed to its chelation ability. Hence, PCA is a potent protector against PM-induced inflammation in skin damage.
Concisely, results suggest the bioactive components derived from the brown algae Padina commersonii increases the cellular resistance to PM stimulated inflammation and oxidative stress.
Author(s)
Thilina U. Jayawardena
Issued Date
2019
Awarded Date
2019. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009029
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Jeon, You Jin
Table Of Contents
PART Ⅰ . 1Fucosterol isolated from Padina commersonii, inhibits particulate matter-induced inflammation through NF-κB, MAPK mediated inflammatory pathway and oxidative stress via Nrf2/HO-1 pathway in RAW 264.7 macrophages 1
1.1. Introduction 2
1.2. Methods 5
1.2.1 Materials . 5
1.2.2 Collection of samples, extraction and isolation of fucosterol 5
1.2.3 Fucosterol structure elucidation and characterization 8
1.2.4 Fine dust particle size estimation using SEM . 8
1.2.5 Cell culture . 9
1.2.6 Measurement of cell viability . 9
1.2.7 Evaluation of NO inhibition activity 9
1.2.8 Assessing PGE 2 and pro-inflammatory cytokine production . 10
1.2.9 Western blot analysis 10
1.2.10 Extraction of RNA and cDNA synthesis 11
1.2.11 Quantitative real-time PCR (qPCR) analysis . 11
1.2.12 Statistical analysis 12
1.3. Results 14
1.3.1 Characterization of particulate matter 14
1.3.2 Purification and structural characterization of fucosterol 16
1.3.3 Effect of FST against PM induced cell viability and NO production 24
1.3.4 Pro-inflammatory cytokines and PGE 2 regulation in PM-induced macrophages assessed via ELISA, western blotting and gene expression 27
1.3.5 Potential of FST on PM-induced macrophages in oxidative stress related pathway 31
1.4. Discussion 33
1.5. Conclusions 38
Part Ⅱ Alginic acid from Padina commersonii abate PM-induced inflammatory responses inkeratinocytes and dermal fibroblasts 39
2.1. Introduction 40
2.2. Methods 42
2.2.1 Materials . 42
2.2.2 Alginic acid purification from P. commersonii 42
2.2.3 Analysis of proximate composition of P. commersonii . 43
2.2.4 Evaluation of chemical composition of SHA . 44
2.2.5 Functional group analysis of PCA using FTIR 44
2.2.6 FTIR spectra interpretation using computational calculations . 45
2.2.7 Fine dust particle size estimation using SEM . 45
2.2.8 Maintenance of cell lines 46
2.2.9 Analysis of cell viability and intracellular ROS . 46
2.2.10 PGE 2 and pro-inflammatory cytokine production level assessment 47
2.2.11 Western blot analysis 47
2.2.12 Spectroscopic analysis (ICP-OES) . 48
2.2.13 Statistical analysis 48
2.3. Results 50
2.3.1 Proximate composition and chemical composition 50
2.3.2 Structural characterization of PCA . 52
2.3.3 Potential of PCA to reduce PM-stimulated inflammatory responses in keratinocytes and fibroblasts . 55
2.3.4 Potential of PCA to abate PM induced inflammatory responses via NF-κB and MAPK pathways . 61
2.3.5 Keratinocytes stimulated with PM and treated with PCA; compositional analysis 64
2.4. Discussion 67
2.5. Conclusions 72
Acknowledgment 73
References . 74
Degree
Master
Publisher
제주대학교 대학원
Citation
Thilina U. Jayawardena. (2019). Fucosterol and alginic acid purified from Padina commersonii from the Maldives; inhibit particulate matter induced inflammation through NF-κB, MAPK mediated inflammatory pathway and oxidative stress via Nrf2/HO-1 pathway
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General Graduate School > Marine Life Sciences
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