Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4
Figure 8
Akt acts as a downstream regulator of CAMK4 in crocin-mediated HO-1 expression. (a) RAW 264.7 cells were incubated with LY294002 (20 μM), SP600125 (20 μM), PD98059 (20 μM), and SB203580 (20 μM) for 1 h and then treated with crocin (500 μM) for 6 h. Equal amounts of cytosolic and nuclear extract were subsequently analyzed by Western blotting with HO-1 and Nrf2 antibody, respectively. Tubulin and TBP were used as loading controls. (b) Cells were incubated with crocin (500 μM) for the indicated times. (c) Cells were transfected with CAMK4 siRNA or control siRNA and then treated with crocin (500 μM) for 1 h. (d) Cells were transfected with pcDNA3.1 (mock, M), CAMK4 constitutive active (dCT), or kinase dead (K75E) construct and then treated with crocin (500 μM) for 1 h, after which equal amounts of cytosolic extract were analyzed by Western blotting. (e) Cells were cotransfected with HO-1-luciferase construct and pcDNA3.1, or dCT and then treated with LY294002 (20 μM). Equal amounts of cell extracts were then assayed for dual luciferase activity. versus the dCT-transfected (no LY294002) group.
