Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells
"> Figure 1
<p>The structures of compounds <b>1</b>–<b>7</b>.</p> "> Figure 2
<p>The effects of compounds <b>1</b>–<b>7</b> on nitrite production in BV2 microglial cells stimulated with LPS. The cells were pre-treated for 3 h with the indicated concentrations of compounds <b>1</b>–<b>7</b> and then stimulated for 24 h with LPS (1 μg/mL). The concentrations of nitrite were determined as described in the Materials and Methods section. The data represent the mean values ± SD of three experiments. * <span class="html-italic">p</span> < 0.05, as compared with cells treated with LPS only.</p> "> Figure 3
<p>The effects of cudraflavanone D (<b>1</b>) on TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-12 (<b>C</b>), and IL-6 (<b>D</b>) mRNA expression in BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D (<b>1</b>) and then stimulated for 12 h with LPS (1 μg/mL). The concentrations of TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-12 (<b>C</b>), and IL-6 (<b>D</b>) were determined as described in Materials and Methods. RNA quantification was performed as described in Materials and Methods and representative blots of three independent experiments are shown. The data represent the mean values of three experiments ± SD. * <span class="html-italic">p</span> < 0.05, as compared with the cells treated with LPS only.</p> "> Figure 3 Cont.
<p>The effects of cudraflavanone D (<b>1</b>) on TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-12 (<b>C</b>), and IL-6 (<b>D</b>) mRNA expression in BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D (<b>1</b>) and then stimulated for 12 h with LPS (1 μg/mL). The concentrations of TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-12 (<b>C</b>), and IL-6 (<b>D</b>) were determined as described in Materials and Methods. RNA quantification was performed as described in Materials and Methods and representative blots of three independent experiments are shown. The data represent the mean values of three experiments ± SD. * <span class="html-italic">p</span> < 0.05, as compared with the cells treated with LPS only.</p> "> Figure 4
<p>(<b>A</b>) The effects of cudraflavanone D (<b>1</b>) on protein expression of iNOS and COX-2 (<b>B</b>) in BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D (<b>1</b>) and then stimulated for 24 h with LPS (1 μg/mL). The concentrations of iNOS and COX-2 (<b>B</b>) were determined as described in Materials and Methods. Western blot analyses were performed as described in Materials and Methods and representative blots of three independent experiments are shown. Band intensity was quantified by densitometry and normalized to β-actin; the values are presented below each band. Relative data represent the mean values of three experiments ± SD. * <span class="html-italic">p</span> < 0.05, as compared to the cells treated with LPS only.</p> "> Figure 5
<p>The effects of cudraflavanone D (<b>1</b>) on IκB-α phosphorylation and degradation (<b>A</b>), NF-κB activation (<b>B</b>,<b>C</b>), NF-κB localization (<b>D</b>), and NF-κB DNA binding activity (<b>E</b>) in BV2 microglial cells. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D (<b>1</b>), and then stimulated for 1 h with LPS (1 μg/mL). Western blot analyses of IκB-α and phosphorylated (p)-IκB-α in the cytoplasm (<b>A</b>), and NF-κB in the cytoplasm (<b>B</b>) and nucleus (<b>C</b>), and immunofluorescent analysis (<b>E</b>), were performed as described in Materials and Methods. Band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented below each band. Relative data represent the mean values of three experiments ± SD. * <span class="html-italic">p</span> < 0.05, as compared to the cells treated with LPS only.</p> "> Figure 6
<p>The effects of cudraflavanone D (<b>1</b>) on ERK, JNK, and p38 MAPK protein expression and phosphorylation. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D (<b>1</b>) and stimulated for 1 h with LPS (1 μg/mL) (<b>A</b>–<b>C</b>). The levels of (<b>A</b>) phosphorylated-ERK (p-ERK), (<b>B</b>) phosphorylated-JNK (p-JNK), and (<b>C</b>) phosphorylated-p38 MAPK (p-p38 MAPK) were determined by Western blotting. Representative blots from three independent experiments are shown. Band intensity was quantified by densitometry and normalized to β-actin; the values are presented below each band. Relative data represent the mean values of three experiments.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Structures of Compounds 1–7 and Cell Viability in BV2 Microglial Cells
2.2. Effects of Compounds 1–7 on NO Production in LPS-Stimulated BV2 Microglial Cells
2.3. Effects of Cudraflavanone D (1) on TNF-α, IL-1β, IL-12, and IL-6 mRNA Expression in LPS-Stimulated BV2 Microglial Cells
2.4. Effects of Cudraflavanone D (1) on PGE2 Production and iNOS and COX-2 Protein Expression in LPS-Stimulated BV2 Microglial Cells
2.5. Effects of Cudraflavanone D (1) on IκB-α Levels, NF-κB Nuclear Translocation, and NF-κB DNA Binding Activity in LPS-Stimulated BV2 Microglial Cells
2.6. Effects of Cudraflavanone D (1) on MAPK Phosphorylation in LPS-Stimulated BV2 Microglial Cells
3. Discussion
4. Experimental Section
4.1. Plant Materials
4.2. Chemicals and Reagents
4.3. Cell Culture and Viability Assay
4.4. Quantitative Reverse-Transcription Polymerase Chain Reaction (qPCR)
4.5. DNA Binding Activity of NF-κB
4.6. Preparation of Cytosolic and Nuclear Fractions
4.7. Nitrite Determination
4.8. Western Blot Analysis
4.9. NF-κB Localization and Immunofluorescence
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Kim, D.-C.; Yoon, C.-S.; Quang, T.H.; Ko, W.; Kim, J.-S.; Oh, H.; Kim, Y.-C. Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells. Int. J. Mol. Sci. 2016, 17, 255. https://doi.org/10.3390/ijms17020255
Kim D-C, Yoon C-S, Quang TH, Ko W, Kim J-S, Oh H, Kim Y-C. Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells. International Journal of Molecular Sciences. 2016; 17(2):255. https://doi.org/10.3390/ijms17020255
Chicago/Turabian StyleKim, Dong-Cheol, Chi-Su Yoon, Tran Hong Quang, Wonmin Ko, Jong-Su Kim, Hyuncheol Oh, and Youn-Chul Kim. 2016. "Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells" International Journal of Molecular Sciences 17, no. 2: 255. https://doi.org/10.3390/ijms17020255