Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction
Abstract
:1. Introduction
2. Results
2.1. Chemical Composition of Algae Material and Purified Components
2.2. Structural Characterization via FTIR and NMR Spectroscopy
2.3. Effect of Column Fractions on NO Production and Cell Viability in LPS-Induced Macrophages
2.4. Assessed Inflammatory Mediators
2.4.1. Potential of F4 to Inhibit PGE2 and Pro-Inflammatory Cytokine Secretion
2.4.2. iNOS and COX-2 Expression Suppression and NF-κB Translocation Inhibition Ability of F4
2.4.3. F4 Inhibits MAPK Phosphorylation Induced via LPS
2.4.4. Potential of F4 to Modulate the Activity of TLR-2/4 and MyD88 Dependent NF-κB Expression
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Extraction of Crude Fucoidan
4.3. Purification of SWP by Anion-Exchange Chromatography
4.4. Chemical Analysis
4.5. Structural Characterization
4.5.1. FTIR Spectroscopic Characterization
4.5.2. NMR Analysis
4.5.3. Monosaccharide Analysis
4.6. Cell Culture
4.6.1. Maintenance of Cell Line
4.6.2. Cytotoxicity and Cell Viability Assessment
4.6.3. Nitric Oxide Production Inhibition Potential
4.7. Evaluation of Mediators in the Inflammatory Pathway
4.7.1. Assessing Pro-Inflammatory Cytokines
4.7.2. Western Blot Analysis
4.7.3. Nuclear Localization of NF-κB via Immunofluorescence
4.8. Analysis of Gene Expressions Related To Inflammatory Pathway
4.8.1. RNA Extraction and cDNA Preparation
4.8.2. Real-Time Quantitative PCR
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Component | Composition (%) |
---|---|
Ash | 17.74 ± 1.24 |
Moisture | 4.82 ± 0.84 |
Protein | 8.69 ± 0.46 |
Lipid | 0.81 ± 0.17 |
Carbohydrate | 62.78 ± 1.21 |
Polysaccharide Content (%) | Sulfate Content (%) | Protein Content (%) | Polyphenol Content (%) | |
---|---|---|---|---|
Crude fucoidan | 66.61 ± 4.32 | 20.83 ± 0.47 | 2.57 ± 1.44 | 1.66 ± 0.71 |
F1 | 81.05 ± 4.48 | 13.48 ± 0.72 | 0.79 ± 0.36 | 0.66 ± 0.21 |
F2 | 70.95 ± 1.83 | 21.86 ± 0.26 | 0.53 ± 0.36 | 0.41 ± 0.16 |
F3 | 65.91 ± 1.99 | 25.89 ± 0.42 | 0.66 ± 0.18 | 0.35 ± 0.12 |
F4 | 60.98 ± 0.66 | 33.99 ± 0.17 | 0.41 ± 0.18 | 0.32 ± 0.15 |
F1 | F2 | F3 | F4 | |
---|---|---|---|---|
Fucose | 23.86 | 69.37 | 74.05 | 82.46 |
Rhamnose | 2.72 | ND | 0.74 | ND |
Galactose | 20.59 | 4.14 | 2.65 | 3.19 |
Glucose | 16.37 | 2.67 | 1.51 | 1.35 |
Xylose | 18.06 | 3.82 | 1.79 | ND |
Others | 18.40 | 20.00 | 19.26 | 13.00 |
Gene | Primer Sequence |
---|---|
GAPDH | antisense; 5′-AAGGGTCATCATCTCTGCCC-3′ and sense, 5′-GTGATGGCATGGACTGTGGT-3′ |
TLR2 | antisense; 5′-CAGCTGGAGAACTCTGACCC-3′ and sense, 5′-CAAAGAGCCTGAAGTGGGAG-3′ |
TLR4 | antisense; 5′-CAACATCATCCAGGAAGGC-3′ and sense, 5′-GAAGGCGATACAATTCCACC-3′ |
IL-1β | antisense; 5′-CAGGATGAGGACATGAGCACC-3′ and sense, 5′-CTCTGCAGACTCAAACTCCAC-3′ |
IL-6 | antisense; 5′-GTACTCCAGAAGACCAGAGG-3′ and sense, 5′-TGCTGGTGACAACCACGGCC-3′ |
TNF-α | antisense; 5′-TTGACCTCAGCGCTGAGTTG-3′ and sense, 5′-CCTGTAGCCCACGTCGTAGC-3′ |
iNOS | antisense; 5′-ATGTCCGAAGCAAACATCAC-3′ and sense, 5′-TAATGTCCAGGAAGTAGGTG-3′ |
COX2 | antisense; 5′-CAGCAAATCCTTGCTGTTCC-3′ and sense, 5′-TGGGCAAAGAATGCAAACATC-3′ |
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Jayawardena, T.U.; Sanjeewa, K.K.A.; Nagahawatta, D.P.; Lee, H.-G.; Lu, Y.-A.; Vaas, A.P.J.P.; Abeytunga, D.T.U.; Nanayakkara, C.M.; Lee, D.-S.; Jeon, Y.-J. Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction. Mar. Drugs 2020, 18, 601. https://doi.org/10.3390/md18120601
Jayawardena TU, Sanjeewa KKA, Nagahawatta DP, Lee H-G, Lu Y-A, Vaas APJP, Abeytunga DTU, Nanayakkara CM, Lee D-S, Jeon Y-J. Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction. Marine Drugs. 2020; 18(12):601. https://doi.org/10.3390/md18120601
Chicago/Turabian StyleJayawardena, Thilina U., K. K. Asanka Sanjeewa, D. P. Nagahawatta, Hyo-Geun Lee, Yu-An Lu, A. P. J. P. Vaas, D. T. U. Abeytunga, C. M. Nanayakkara, Dae-Sung Lee, and You-Jin Jeon. 2020. "Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction" Marine Drugs 18, no. 12: 601. https://doi.org/10.3390/md18120601