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Api inhibits both in vivo and in vitro activation of <t>NLRP3</t> inflammasome. (A) Relative mRNA expression of Nlrp3 in colonic tissues from Control, DSS, and DSS + Api groups (qPCR; n = 5). (B) Western-blot analysis of inflammasome-related proteins in colonic tissue: cleaved and pro-IL-1β, cleaved and pro-caspase-1, NLRP3, ASC, GSDMD; β-actin was used as a loading control. (C) ELISA quantification of IL-1β in BMDM supernatants after LPS + nigericin stimulation with or without Api ( n = 3). (D) qPCR analysis of IL-1β , Nlrp3 , and GSDMD transcripts in BMDMs under the same conditions as in (C) ( n = 3). (E) SPR sensorgram confirming the direct interaction between Api and recombinant NLRP3. (F) Molecular-docking model showing the predicted binding of Api to the NACHT domain of NLRP3 (PDB: 7ALV); calculated binding energy, −8.6 kcal/mol. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS.
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Api inhibits both in vivo and in vitro activation of NLRP3 inflammasome. (A) Relative mRNA expression of Nlrp3 in colonic tissues from Control, DSS, and DSS + Api groups (qPCR; n = 5). (B) Western-blot analysis of inflammasome-related proteins in colonic tissue: cleaved and pro-IL-1β, cleaved and pro-caspase-1, NLRP3, ASC, GSDMD; β-actin was used as a loading control. (C) ELISA quantification of IL-1β in BMDM supernatants after LPS + nigericin stimulation with or without Api ( n = 3). (D) qPCR analysis of IL-1β , Nlrp3 , and GSDMD transcripts in BMDMs under the same conditions as in (C) ( n = 3). (E) SPR sensorgram confirming the direct interaction between Api and recombinant NLRP3. (F) Molecular-docking model showing the predicted binding of Api to the NACHT domain of NLRP3 (PDB: 7ALV); calculated binding energy, −8.6 kcal/mol. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS.

Journal: Journal of Agricultural and Food Chemistry

Article Title: Natural Dietary Flavonoid Apigenin Mitigates Ulcerative Colitis via Modulating the AMPK/NF-κB/NLRP3 Signaling Axis

doi: 10.1021/acs.jafc.5c15405

Figure Lengend Snippet: Api inhibits both in vivo and in vitro activation of NLRP3 inflammasome. (A) Relative mRNA expression of Nlrp3 in colonic tissues from Control, DSS, and DSS + Api groups (qPCR; n = 5). (B) Western-blot analysis of inflammasome-related proteins in colonic tissue: cleaved and pro-IL-1β, cleaved and pro-caspase-1, NLRP3, ASC, GSDMD; β-actin was used as a loading control. (C) ELISA quantification of IL-1β in BMDM supernatants after LPS + nigericin stimulation with or without Api ( n = 3). (D) qPCR analysis of IL-1β , Nlrp3 , and GSDMD transcripts in BMDMs under the same conditions as in (C) ( n = 3). (E) SPR sensorgram confirming the direct interaction between Api and recombinant NLRP3. (F) Molecular-docking model showing the predicted binding of Api to the NACHT domain of NLRP3 (PDB: 7ALV); calculated binding energy, −8.6 kcal/mol. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS.

Article Snippet: Commercially available recombinant human NLRP3 protein (CSB-EP822275HU7) and AMPK α1/β2/γ1 heterotrimer protein (HY- P76143 , MCE) were immobilized (∼3,000 RU) on a CM5 Chip (GE Healthcare, Piscataway, NJ, USA) according to a standard amine coupling procedure.

Techniques: In Vivo, In Vitro, Activation Assay, Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay

Api alleviates colitis by coordinately modulating AMPK and NLRP3. (A-B) ELISA quantification of TNF-α, IL-6 (A), and IL-1β (B) in BMDM supernatants following LPS + nigericin stimulation with or without Api, the AMPK agonist AICAR, or the AMPK inhibitor BAY-3827 ( n = 4). (C) Effect of AMPKα1 knockdown on the mRNA expression of Prkaa1 , TNF-α , and Nlrp3 in BMDMs subjected to LPS + nigericin stimulation with or without Api treatment ( n = 6). (D) SPR sensorgram confirming the direct interaction between Api and the recombinant AMPK α1/β2/γ1 complex. (E) Molecular-docking models depicting Api bound to multiple reported AMPK allosteric or activator-related pockets, including AMPKα1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 4QFR), and AMPKα2β1γ1 (PDB: 4CFF).

Journal: Journal of Agricultural and Food Chemistry

Article Title: Natural Dietary Flavonoid Apigenin Mitigates Ulcerative Colitis via Modulating the AMPK/NF-κB/NLRP3 Signaling Axis

doi: 10.1021/acs.jafc.5c15405

Figure Lengend Snippet: Api alleviates colitis by coordinately modulating AMPK and NLRP3. (A-B) ELISA quantification of TNF-α, IL-6 (A), and IL-1β (B) in BMDM supernatants following LPS + nigericin stimulation with or without Api, the AMPK agonist AICAR, or the AMPK inhibitor BAY-3827 ( n = 4). (C) Effect of AMPKα1 knockdown on the mRNA expression of Prkaa1 , TNF-α , and Nlrp3 in BMDMs subjected to LPS + nigericin stimulation with or without Api treatment ( n = 6). (D) SPR sensorgram confirming the direct interaction between Api and the recombinant AMPK α1/β2/γ1 complex. (E) Molecular-docking models depicting Api bound to multiple reported AMPK allosteric or activator-related pockets, including AMPKα1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 4QFR), and AMPKα2β1γ1 (PDB: 4CFF).

Article Snippet: Commercially available recombinant human NLRP3 protein (CSB-EP822275HU7) and AMPK α1/β2/γ1 heterotrimer protein (HY- P76143 , MCE) were immobilized (∼3,000 RU) on a CM5 Chip (GE Healthcare, Piscataway, NJ, USA) according to a standard amine coupling procedure.

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, Expressing, Recombinant