Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes
<p>Effect of grape/blueberry anthocyanins and their gut-derived metabolites on THP-1 cell viability. THP-1 monocytes were incubated with the indicated concentrations of (<b>a</b>) the GBE, (<b>b</b>) HVA, or (<b>c</b>) THBA for 24 h before cell viability was measured by flow cytometry. Data are presented as mean ± SD (<span class="html-italic">n</span> = 3). Significant differences compared to the untreated control were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 2
<p>Detection of ASC speck formation in THP-1 cells using flow cytometry. THP-1 monocytes were either left untreated or primed with LPS followed by activation of the NLRP3 inflammasome with nigericin as mentioned in the methods section. Flow analysis was performed and the percentage of ASC speck-positive cells was quantified. Data are presented as (<b>a</b>) representative dot plots and (<b>b</b>) column bars with mean ± SD (<span class="html-italic">n</span> = 3). Significant differences compared to the untreated control were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>Effect of grape/blueberry anthocyanins and their gut-derived metabolites on ASC speck formation in THP-1 cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. Cells were flow cytometrically analyzed and the percentage of ASC speck-positive cells was quantified. Data are presented as mean ± SD (<span class="html-italic">n</span> = 3). Significant differences compared to (<b>a</b>,<b>c</b>) LPS- and nigericin-stimulated cells or (<b>b</b>,<b>d</b>) cells treated only with nigericin were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 4
<p>Effect of grape/blueberry anthocyanins and their gut-derived metabolites on caspase-1 activity in THP-cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. Caspase-1 activity was measured by using the Caspase-Glo<sup>®</sup> 1 Inflammasome Assay and luminescence was measured. Data are presented as mean ± SD (<span class="html-italic">n</span> = 3). Significant differences compared to (<b>a</b>,<b>c</b>) LPS- and nigericin-stimulated cells or (<b>b</b>,<b>d</b>) cells treated only with nigericin were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. * <span class="html-italic">p</span> < 0.05, **** <span class="html-italic">p</span> < 0.0001. RLU, relative light unit.</p> "> Figure 5
<p>Effect of grape/blueberry anthocyanins and their gut-derived metabolites on inflammatory cytokine secretion in THP-1 cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. The release of (<b>a</b>,<b>c</b>) IL-1β and (<b>b</b>,<b>d</b>) IL-18 into the cell-culture supernatant was measured using ELISA. Data are presented as mean ± SD of at least three replicated experiments. Significant differences compared to LPS- and nigericin-stimulated cells were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation and Characterization of the Powdered Anthocyanin-Rich Grape/Blueberry Extract
2.2. THP-1 Cell Culture
2.3. Cell Viability and Cytotoxicity
2.4. NLRP3 Inflammasome Activation
2.5. Flow Cytometry
2.6. Caspase-Glo® 1 Inflammasome Assay
2.7. Enzyme-Linked Immunosorbent Assays (ELISA)
2.8. Statistical Analyses
3. Results
3.1. Composition of the Powdered Anthocyanin-Rich Grape/Blueberry Extract
3.2. Effect of Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites on THP-1 Cell Viability
3.3. Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Have No Effect on ASC and NLRP3 Protein Expression in THP-1 Monocytes
3.4. Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate ASC Speck Formation in THP-1 Monocytes
3.5. Effect of Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites on Caspase-1 Activity in THP-1 Monocytes
3.6. Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Ameliorate Inflammatory Cytokine Secretion after NLRP3 Inflammasome Activation in THP-1 Monocytes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Anthocyanins 1 | Rt [min] | [M+H]+ | λmax [nm] | mg/g | [%] |
---|---|---|---|---|---|
Delphinidin-3,5-diglucoside | 7.10 | 627, 465, 303 | 520 | 0.3 | 0.4 |
Cyanidin-3,5-diglucoside | 8.65 | 611, 449, 287 | 514 | 1.3 | 2.0 |
Delphinidin-3-galactoside | 9.57 | 465, 303 | 522 | 3.2 | 5.2 |
Delphinidin-3-glucoside | 10.41 | 465, 303 | 522 | 3.7 | 6.0 |
Peonidin-3,5-diglucoside | 11.13 | 625, 463, 301 | 513 | 23.4 | 38.1 |
Delphinidin-3-arabinoside | 11.84 | 435, 303 | 522 | 6.7 | 10.9 |
Malvidin-3,5-diglucoside | 12.06 | 655, 493, 331 | 521 | 6.7 | 10.9 |
Petunidin-3-galactoside | 12.72 | 479, 317 | 524 | 0.8 | 1.3 |
Cyanidin-3-arabinoside | 13.03 | 419, 287 | 517 | 1.5 | 2.5 |
Petunidin-3-glucoside | 13.52 | 479, 317 | 521 | 2.6 | 4.3 |
Peonidin-3-galactoside | 14.10 | 463, 301 | 515 | 0.2 | 0.4 |
Petunidin-3-arabinoside | 14.59 | 449, 317 | 524 | 0.5 | 0.9 |
Peonidin-3-glucoside | 15.00 | 463, 301 | 516 | 5.1 | 8.3 |
Malvidin-3-glucoside | 15.95 | 493, 331 | 524 | 3.9 | 6.4 |
Malvidin-3-arabinoside | 16.99 | 463, 331 | 526 | 0.3 | 0.5 |
Malvidin-3-(6″-coumaryl)-5-diglucoside | 21.69 | 801, 639, 493, 331 | 524 | 1.2 | 1.9 |
∑ | 61.4 | 100 |
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Behrendt, I.; Röder, I.; Will, F.; Michel, G.; Friedrich, E.; Grote, D.; Martin, Z.; Dötzer, H.P.; Fasshauer, M.; Speckmann, M.; et al. Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes. Metabolites 2024, 14, 203. https://doi.org/10.3390/metabo14040203
Behrendt I, Röder I, Will F, Michel G, Friedrich E, Grote D, Martin Z, Dötzer HP, Fasshauer M, Speckmann M, et al. Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes. Metabolites. 2024; 14(4):203. https://doi.org/10.3390/metabo14040203
Chicago/Turabian StyleBehrendt, Inken, Isabella Röder, Frank Will, Gabriela Michel, Elvira Friedrich, Daniela Grote, Zoe Martin, Hanna Pauline Dötzer, Mathias Fasshauer, Martin Speckmann, and et al. 2024. "Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes" Metabolites 14, no. 4: 203. https://doi.org/10.3390/metabo14040203