Immune-Enhancing Effects of Red Platycodon grandiflorus Root Extract via p38 MAPK-Mediated NF-κB Activation
<p>Effect of Red <span class="html-italic">Platycodon grandiflorus</span> Root Extract (RPGE) on cell viability in RAW 264.7 cells. (<b>a</b>) RPGE-concentrate (RPGE-C); (<b>b</b>) RPGE-powder (RPGE-P). The results are expressed as relative values to that of the vehicle-treated cells (vehicle control, VC), which is set to 100%. The data are represented as the mean ± SEM. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 when compared with the VC.</p> "> Figure 2
<p>Effect of RPGE on the phagocytic activity of RAW 264.7 cells. (<b>a</b>) RPGE-C; (<b>b</b>) RPGE-P. PI, phagocytosis inhibitor. Values are represented as the mean ± SEM. ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 when compared with the VC treated with an <span class="html-italic">E. coli</span> suspension.</p> "> Figure 3
<p>Effect of RPGE on NO production and related mRNA expression in RAW 264.7 cells. (<b>a</b>–<b>c</b>) RPGE-C; (<b>d</b>–<b>f</b>) RPGE-P. (<b>a</b>,<b>d</b>) NO production; (<b>b</b>,<b>e</b>) <span class="html-italic">Nos2</span> mRNA expression level; (<b>c</b>,<b>f</b>) <span class="html-italic">Ptgs2</span> mRNA expression level. NO, Nitric oxide. All gene expression values were normalized to <span class="html-italic">Actb</span>, and the results were expressed as relative values to that of the VC, which is set to 1. The data are represented as the mean ± SEM. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 when compared with the VC.</p> "> Figure 4
<p>Effect of RPGE on cytokine levels in RAW 264.7 cells. (<b>a</b>–<b>d</b>) RPGE-C; (<b>e</b>–<b>h</b>) RPGE-P. (<b>a</b>,<b>e</b>) <span class="html-italic">Tnf-α</span> mRNA expression level; (<b>b</b>,<b>f</b>) <span class="html-italic">IL-1b</span> mRNA expression level; (<b>c</b>,<b>g</b>) <span class="html-italic">IL-6</span> mRNA expression level. The gene expression values were normalized to <span class="html-italic">Actb</span>, and the results were expressed as relative values to that of the VC, which is set to 1. (<b>d</b>,<b>h</b>) IL-6 protein levels in the culture supernatant. All data are represented as the mean ± SEM. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 when compared with the VC.</p> "> Figure 5
<p>Effect of RPGE on NF-κB activation in RAW 264.7 cells. (<b>a</b>) RPGE-C; (<b>c</b>) RPGE-P. Luciferase activity was expressed as a relative value to that of the VC, which is set to 100%. The figures show the mean ± SEM. * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.001. (<b>b</b>) The cells were treated with 500 μg/mL of RPGE-C for the indicated times or indicated concentrations for 60 min prior to Western blot analysis; (<b>d</b>) The cells were treated with 500 μg/mL of RPGE-P for the indicated times or indicated concentrations prior to Western blot analysis.</p> "> Figure 6
<p>Effect of RPGE on MAPK signaling in RAW 264.7 cells. (<b>a</b>) The cells were treated with 500 μg/mL of RPGE-C for the indicated times or indicated concentrations for 60 min prior to Western blot analysis; (<b>b</b>) The cells were treated with 500 μg/mL of RPGE-P for the indicated times or indicated concentrations for 60 min prior to Western blot analysis; (<b>c</b>) The time–response and concentration–response relationships of RPGE-C were represented as heat maps. (<b>d</b>) The time–response and concentration–response relationships of RPGE-P were represented as heat maps.</p> "> Figure 7
<p>Effect of MAPK signaling on NF-κB activation in RAW 264.7 cells. (<b>a</b>) without RPGEs; (<b>b</b>) RPGE-C; (<b>c</b>) RPGE-P. Luciferase activity was expressed as a relative value to that of the VC (-), which is set to 100%. The figures show the mean ± SEM. * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01 when compared with cells treated with RPGEs alone. SB203580, p38 MAPK inhibitor and SP600125, JNK inhibitor.</p> "> Figure 8
<p>Effect of RPGE on cell viability and secretion of IL-10 in mouse splenocytes. (<b>a</b>) Mouse splenocytes were incubated with Con A (5 μg/mL), LPS (1 μg/mL), or various concentrations of RPGE-C; (<b>c</b>) Mouse splenocytes were incubated with Con A (5 μg/mL), LPS (1 μg/mL), or various concentrations of RPGE-P. Con A, Concanavalin A; LPS, lipopolysaccharide. The results were expressed as a relative value to that of VC, which is set to 100%. The data are represented as the mean ± SEM. ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 as when compared with the VC. (<b>b</b>) IL-10 protein level (RPGE-C); (<b>d</b>) IL-10 protein level (RPGE-P). All data are represented as the mean ± SEM. * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.001 as when compared with the VC.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Red Platycodon grandiflorus Root Extract (RPGE) Preparation
2.2. Cell Culture and Reagents
2.3. Splenocyte Isolation
2.4. Cell Viability Assay
2.5. Phagocytosis Assay
2.6. Measurement of Nitric Oxide (NO) Production
2.7. RNA Isolation and Real-Time Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
2.8. Quantification of Cytokine Levels
2.9. Luciferase Assay
2.10. Western Blot Analysis
2.11. Statistical Analysis
3. Results
3.1. RPGE Increases Phagocytic Activity in RAW 264.7 Cells
3.2. RPGE Enhances NO Production in RAW 264.7 Cells
3.3. RPGE Increases Cytokine Levels in RAW 264.7 Cells
3.4. RPGE Activates NF-κB and MAPK Signaling in RAW 264.7 Cells
3.5. RPGE-Induced NF-κB Activation Is Associated with p38 MAPK in RAW 264.7 Cells
3.6. RPGE Induces Cell Proliferation and Increases IL-10 Expression Levels in Mouse Splenocytes
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Genes | Forward Sequence | Reverse Sequence | Ref. |
---|---|---|---|
Nos2 | GCGAAAGGTCATGGCTTCAC | CTGGTCCATGCAGACAACCT | This study |
Ptgs2 | CATCCCCTTCCTGCGAAGTT | GGCCCTGGTGTAGTAGGAGA | This study |
Tnf-α | TGTCCCTTTCACTCACTGGC | CATCTTTTGGGGGAGTGCCT | [13] |
IL-1b | AACTGTTCCTGAACTCAACTGT | GAGATTTGAAGCTGGATGCTCT | [14] |
IL-6 | GGGACTGATGCTGGTGACAA | TCCACGATTTCCCAGAGAACA | [13] |
Actb | GACGTTGACATCCGTAAAG | CAGTAACAGTCCGCCT | [2] |
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Park, E.-J.; Lee, Y.-S.; Kim, S.M.; Jung, A.J.; Yoo, J.-H.; Lee, S.-H.; Jeong, H.C.; Lee, H.-J. Immune-Enhancing Effects of Red Platycodon grandiflorus Root Extract via p38 MAPK-Mediated NF-κB Activation. Appl. Sci. 2020, 10, 5457. https://doi.org/10.3390/app10165457
Park E-J, Lee Y-S, Kim SM, Jung AJ, Yoo J-H, Lee S-H, Jeong HC, Lee H-J. Immune-Enhancing Effects of Red Platycodon grandiflorus Root Extract via p38 MAPK-Mediated NF-κB Activation. Applied Sciences. 2020; 10(16):5457. https://doi.org/10.3390/app10165457
Chicago/Turabian StylePark, Eun-Jung, You-Suk Lee, Sung Min Kim, Ah Jin Jung, Jeong-Hyun Yoo, Sung-Hyen Lee, Hyun Cheol Jeong, and Hae-Jeung Lee. 2020. "Immune-Enhancing Effects of Red Platycodon grandiflorus Root Extract via p38 MAPK-Mediated NF-κB Activation" Applied Sciences 10, no. 16: 5457. https://doi.org/10.3390/app10165457