Structure, synthesis and discovery of KKC080096 as a HO-1 inducer. (A) The synthesis scheme and chemical structure of KKC080096. (B and C) BV-2 cells were treated with various concentrations of KKC080096. (B) After 24 h, western blotting was performed for HO-1 using β-actin as a loading control. The number on each gel pictogram represents the fold increase with respect to untreated control, calculated from densitometry and normalized against respective loading control. (C) After 24 h, cell viability was assessed using the ATP assay.|@|~(^,^)~|@|KKC080096 downregulates proinflammatory gene expression, upregulates anti-inflammatory gene expression, and suppresses IKK and MAPK activation in activated microglia. BV-2 cells were stimulated with LPS (0.2 μg/ml) and treated with various concentrations of KKC080096. After 24 h, the nitrite (A) and IL-1β (B) levels were determined using the Griess assay and ELISA, respectively. The data are expressed as percentage of LPS-induced control ± SEM; **P < 0.01. The cells were harvested after 6 h and RT-PCR was performed for (C) iNOS and IL-1β and (D) Arg1, YM1, CD206, IL-10, and TGF-β using GAPDH as an internal control. (E) BV-2 cells were treated with various concentrations of KKC080096 for 1 h before stimulation with 0.2 μg/ml LPS. After 0.5 h, the cells were harvested, and the levels of phosphorylated and total IKK and MAPKs JNK, p38, and ERK were assessed using western blotting. (C-E) The number on each gel pictogram represents the fold change with respect to LPS-treated control, calculated from densitometry and normalized against respective internal control.|@|~(^,^)~|@|KKC080096 suppresses microglial activation and protects dopaminergic neurons in MPTP-treated mice. Immunohistochemistry in mice administered MPTP alone only or co-treated with 30 mg/kg KKC080096. (A) Photomicrographs of the nigral sections showing Iba-1 expression (top) and quantitative analysis of the Iba-1-immunopositive microglia in the SN using densitometry (bottom). Scale bar = 200 μm. (B) Photomicrographs of the nigral (top left) and striatal (Str) (top right) sections immunostained against TH and quantitative analysis of the TH-immunopositive neurons in the SN by counting (bottom left) and TH-immunopositive terminals in the striatum by densitometry (bottom right). Scale bars = 200 μm. The data are expressed as the percentage of vehicle control group ± SEM; *P < 0.05, **P < 0.01 vs MPTP group.|@|~(^,^)~|@|KKC080096 improves motor activities in MPTP-treated mice. Motor deficit analyses of mice administered MPTP alone or co-treated with 30 mg/kg KKC080096. (A) Hindlimb test score. (B) Latent time on rotarod. (C) Hanging time on pole. (D) Time taken to make a turn on the vertical grid. (E) Total time taken on the vertical grid. (F) Percentage of successful steps made on the vertical grid. The data are expressed as mean ± SEM; *P < 0.05, **P < 0.01 vs MPTP group.|@|~(^,^)~|@|KKC080096 activates AMPK, LKB1, and CaMKKβ in microglial cells. BV-2 cells were treated with various concentrations of KKC080096 for 15 min. The cells were harvested and (A) the levels of total and phosphorylated AMPK and (B) phosphorylated LKB1 and CaMKKβ were detected by western blotting with β-actin as a loading control. (C) The cells were pretreated with Compound C for 1 h, treated with KKC080096 (20 μM), and then cultured for additional 24 h. Western blotting was performed against HO-1 using β-actin as loading control. (A-C) The number on each gel pictogram represents the fold change with respect to untreated control (A and B) or KKC080096-treated control, calculated from densitometric value that had been normalized against β-actin. (D) The cells were pretreated with Ara A or Compound C for 1 h, treated with KKC080096 (20 μM) and/or 0.2 μg/ml LPS, and then cultured for additional 24 h. The nitrite level was determined using the Griess assay. The data are expressed as percentage of LPS-induced control ± SEM; ^###P < 0.001 vs LPS-treated; ***P < 0.001 vs (KKC080096 + LPS)-treated.|@|~(^,^)~|@|KKC080096 activates Nrf2 pathway and upregulates the expression of Nrf2 target enzymes in microglial cells. (A) BV-2 cells were treated with various concentrations of KKC080096. After 24 h, the cells were harvested and the cell lysates were subjected to western blotting for Nrf2 using β-actin as an internal control (upper panel). After 6 h, the cells were harvested and the nuclear and cytosolic fractions were subjected to western blotting for Nrf2, lamin B (a nuclear marker used as an internal control), and HSP90 (a cytosolic marker used as a negative control) (lower panel). (B) Surface plasmon resonance was recorded in the presence of KKC080096. The data are expressed as resonance units (RU). (C) The biotinylated Nrf2 ETGE peptide immobilized on streptavidin-agarose beads was incubated with Keap1-Kelch domain in the presence of KKC080096, and the Nrf2-bound Keap1 was detected using western blotting. The data are plotted as percentage of control ± SEM; **P < 0.01, ***P < 0.001. (D) BV-2 cells were treated with various concentrations of KKC080096 for 6 h. The cells were harvested and RT-PCR was performed for NQO1, GCLC, and GCLM. (A and D) The number on each gel pictogram represents the fold increase with respect to untreated control, calculated from densitometry and normalized against respective loading control.|@|~(^,^)~|@|KKC080096 increases Nrf2 and antioxidant enzyme expression in dopaminergic neuronal cells. CATH.a cells were treated with various concentrations of KKC080096. After 24 h, cell lysate was subjected to western blotting to assess the levels of Nrf2 (A) and the antioxidant enzyme proteins (C). After 6 h, total RNA was subjected to RT-PCR to check the expression of antioxidant enzymes (B). The number on each gel pictogram represents the fold increase with respect to untreated control, calculated from densitometry and normalized against respective loading controls.

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