FIGURE 6
CK inhibited NLRP3 inflammasome activation and mitochondrial damage in cultured macrophages. Murine J774A.1 macrophages were incubated for 30 min with or without CK, then for 5.5 h with or without 1 µg/mL of LPS, and with or without (A) 5 mM ATP (30 min), (B) 500 μg/mL of alum (24 h) or (C) 100 μg/mL of MSU (24 h) for IL-1β levels in culture media measured using an ELISA. The cells were incubated for 30 min with or without CK, followed by incubation for 6 h with or without 1 µg/mL of LPS, and then with or without 5 mM ATP for (D) IL-1β, (E) caspase-1 and (F) NLRP3 determined by western blot analysis. The analysis of IL-1β and caspase-1 released into the culture medium was performed on the extracted supernatants. J-Blue cells were incubated for 30 min with or without CK, and 24 h with or without 1 µg/mL of LPS for (G) the activation levels of NF-κB determined by an NF-κB reporter assay. (H) Use of MCC950 (1 µM), an NLRP3 inhibitor, for IL-1β and caspase-1 by ELISA or western blot analysis. Macrophages were incubated for 30 min with or without CK, 5.5 h with or without 1 µg/mL of LPS, 15 min with or without 5 mM ATP, and 15 min with 25 nM MitoTracker Deep Red and MitoTracker Green for (I) the mitochondrial inner transmembrane potential by flow cytometry. Macrophages were incubated for 30 min with or without CK, 5.5 h with or without 1 µg/mL of LPS, 15 min 5 μM MitoSOX, and 10 min with or without 5 mM ATP for (J) mitochondrial ROS generation in the cells by flow cytometry. The cells were incubated for 30 min with or without CK or Mito-TEMPO, 5.5 h with or without 1 µg/mL of LPS, and 30 min with or without 5 mM ATP for (K) mtDNA copy number by real-time PCR. The data represent three separate experiments. Alum, alum crystals; MCC950, N-(1, 2, 3, 5, 6, 7-Hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide, an NLRP3 inhibitor; MFI, mean fluorescence intensity; MSU, monosodium urate; mtDNA, mitochondrial DNA; Mito-TEMPO, mitochondria-targeted antioxidant; NAC, N-acetyl-L-cysteine. #Not detectable. *P < 0.05, **P < 0.01, ***P < 0.005.

CK inhibited NLRP3 inflammasome activation and mitochondrial damage in cultured macrophages. Murine J774A.1 macrophages were incubated for 30 min with or without CK, then for 5.5 h with or without 1 µg/mL of LPS, and with or without (A) 5 mM ATP (30 min), (B) 500 μg/mL of alum (24 h) or (C) 100 μg/mL of MSU (24 h) for IL-1β levels in culture media measured using an ELISA. The cells were incubated for 30 min with or without CK, followed by incubation for 6 h with or without 1 µg/mL of LPS, and then with or without 5 mM ATP for (D) IL-1β, (E) caspase-1 and (F) NLRP3 determined by western blot analysis. The analysis of IL-1β and caspase-1 released into the culture medium was performed on the extracted supernatants. J-Blue cells were incubated for 30 min with or without CK, and 24 h with or without 1 µg/mL of LPS for (G) the activation levels of NF-κB determined by an NF-κB reporter assay. (H) Use of MCC950 (1 µM), an NLRP3 inhibitor, for IL-1β and caspase-1 by ELISA or western blot analysis. Macrophages were incubated for 30 min with or without CK, 5.5 h with or without 1 µg/mL of LPS, 15 min with or without 5 mM ATP, and 15 min with 25 nM MitoTracker Deep Red and MitoTracker Green for (I) the mitochondrial inner transmembrane potential by flow cytometry. Macrophages were incubated for 30 min with or without CK, 5.5 h with or without 1 µg/mL of LPS, 15 min 5 μM MitoSOX, and 10 min with or without 5 mM ATP for (J) mitochondrial ROS generation in the cells by flow cytometry. The cells were incubated for 30 min with or without CK or Mito-TEMPO, 5.5 h with or without 1 µg/mL of LPS, and 30 min with or without 5 mM ATP for (K) mtDNA copy number by real-time PCR. The data represent three separate experiments. Alum, alum crystals; MCC950, N-(1, 2, 3, 5, 6, 7-Hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide, an NLRP3 inhibitor; MFI, mean fluorescence intensity; MSU, monosodium urate; mtDNA, mitochondrial DNA; Mito-TEMPO, mitochondria-targeted antioxidant; NAC, N-acetyl-L-cysteine. #Not detectable. *P < 0.05, **P < 0.01, ***P < 0.005.

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