Anti-Inflammatory Efficacy of Resveratrol-Enriched Rice Callus Extract on Lipopolysaccharide-Stimulated RAW264.7 Macrophages
Abstract
:1. Introduction
2. Materials and Methods
2.1. Callus Culture
2.2. Extraction of Resveratrol-Enrich Compound from Rice Callus
2.3. Quantification of Piceid and Resveratrol Content Using High-Performance Liquid Chromatography (HPLC) Analysis
2.4. RAW264.7 Cell Culture
2.5. Cell Viability and Nitric Oxide Production Analysis
2.6. RNA Isolation, RNA Quantification, and cDNA Synthesis
2.7. Measurement of mRNA from Inflammatory Genes by RT-qPCR
2.8. Prostaglandin E2 (PGE2) Production
2.9. Western Blot Analysis
2.10. Statistical Analysis
3. Results
3.1. Piceid and Resveratrol Contents in Rice Callus Extracts
3.2. Effects of Resveratrol-Enriched Rice Callus Extract on the Viability of LPS-Stimulated RAW264.7 Cells
3.3. Resveratrol-Enriched Rice Callus Extract Inhibited LPS-Induced NO Production by RAW264.7 Cells
3.4. Resveratrol-Enriched Rice Callus Extract Suppressed LPS-Induced Upregulation of Multiple Proinflammatory Genes in RAW264.7 Cells
3.5. Resveratrol-Enriched Rice Callus Extract Reduced the Production of PGE2 by LPS-Stimulated RAW264.7 Cells
3.6. Resveratrol-Enriched Rice Callus Extract Inhibited MAPK and NF-κB Pathway Activation in LPS-Stimulated RAW264.7 Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Treatment | Concentration | A260:A280 | A260:A230 | RNA Concentration (ng/µL) | CV * |
---|---|---|---|---|---|
Nontreatment | − | 1.888 | 1.908 | 394.05 ± 9.55 | 2.42 |
DMSO | 0.1% | 1.966 | 1.877 | 547.81 ± 8.23 | 1.50 |
DJ | 10 µg/mL | 1.927 | 1.839 | 453.12 ± 11.64 | 2.57 |
DJ | 25 µg/mL | 1.997 | 1.955 | 521.39 ± 10.57 | 2.03 |
DJ | 50 µg/mL | 1.980 | 1.925 | 537.57 ± 12.12 | 2.26 |
DJ | 100 µg/mL | 1.912 | 1.990 | 1033.92 ± 44.85 | 4.34 |
DJ526 | 10 µg/mL | 1.948 | 1.899 | 497.55 ± 11.60 | 2.33 |
DJ526 | 25 µg/mL | 1.927 | 1.813 | 450.40 ± 12.55 | 2.79 |
DJ526 | 50 µg/mL | 1.882 | 1.956 | 551.36 ± 19.13 | 3.47 |
DJ526 | 100 µg/mL | 1.876 | 1.893 | 441.44 ± 14.03 | 3.18 |
Aspirin | 200 µg/mL | 1.992 | 1.930 | 548.85 ± 13.80 | 2.51 |
Gene | Nucleotide Sequence (5′−3′) | Accession Number | Target Size (bp) |
---|---|---|---|
COX-2 | Forward primer: 1409-AGAAGGAAATGGCTGCAGAA-1428 Reverse primer: 1602-GCTCGGCTTCCAGTATTGAG-1583 | NM_011198.5 | 194 |
iNOS | Forward primer: 185-TTCCAGAATCCCTGGACAAG-204 Reverse primer: 364-TGGTCAAACTCTTGGGGTTC-345 | BC062378.1 | 180 |
IL-1β | Forward primer: 531-GGGCCTCAAAGGAAAGAATC-550 Reverse primer: 713-TACCAGTTGGGGAACTCTGC-694 | NM_008361.4 | 183 |
IL-6 | Forward primer: 33-AGTTGCCTTCTTGGGACTGA-52 Reverse primer: 223-CAGAATTGCCATTGCACAAC-204 | NM_031168.2 | 191 |
TNF-α | Forward primer: 1-ATGAGCACAGAAAGCATGATC-21 Reverse primer: 276-TACAGGCTTGTCACTCGAATT-256 | D84199.2 | 276 |
TLR-4 | Forward primer: 2281-CGCTCTGGCATCATCTTCAT-2300 Reverse primer: 2498-GTTGCCGTTTCTTGTTCTTCC-2478 | NM_021297.3 | 218 |
β-actin | Forward primer: 605-CCACAGCTGAGAGGGAAATC-624 Reverse primer: 797-AAGGAAGGCTGGAAAAGAGC-778 | NM_007393.5 | 193 |
Process | Temperature | Time | Cycle |
---|---|---|---|
Predenaturation | 95 °C | 10 min | 1 cycle |
Denaturation | 95 °C | 20 s | 40 cycles |
Annealing | 60 °C | 20 s | |
Extension | 72 °C | 30 s | |
Final extension | 72 °C | 5 min | 1 cycle |
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Monmai, C.; Kim, J.-S.; Baek, S.-H. Anti-Inflammatory Efficacy of Resveratrol-Enriched Rice Callus Extract on Lipopolysaccharide-Stimulated RAW264.7 Macrophages. Immuno 2024, 4, 131-146. https://doi.org/10.3390/immuno4020009
Monmai C, Kim J-S, Baek S-H. Anti-Inflammatory Efficacy of Resveratrol-Enriched Rice Callus Extract on Lipopolysaccharide-Stimulated RAW264.7 Macrophages. Immuno. 2024; 4(2):131-146. https://doi.org/10.3390/immuno4020009
Chicago/Turabian StyleMonmai, Chaiwat, Jin-Suk Kim, and So-Hyeon Baek. 2024. "Anti-Inflammatory Efficacy of Resveratrol-Enriched Rice Callus Extract on Lipopolysaccharide-Stimulated RAW264.7 Macrophages" Immuno 4, no. 2: 131-146. https://doi.org/10.3390/immuno4020009