Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4
Figure 5
Induction of HO-1 expression via elevation of intracellular Ca2+ levels. (a) RAW 264.7 cells were incubated with BAPTA/AM (B/A, 5 μM), calmidazolium chloride (CC, 5 μM), and nifedipine (Nif, 5 μM) for 1 h and then treated with crocin (500 μM) for 6 h. Equal amounts of cytosolic proteins were subsequently analyzed by Western blotting with HO-1 antibody. Tubulin was used as a loading control. (b) Cells were transfected with HO-1 promoter-luciferase construct and then treated as described above, after which equal amounts of cell extracts were assayed for dual luciferase activity. versus the group treated with crocin alone. (c) Intracellular free Ca2+ levels were assessed by flow cytometry. Cells were treated with vehicle or crocin (500 μM) for 10 min in either Ca2+-containing medium (left) or Ca2+-free medium (right). (d) The changes of intracellular Ca2+ levels with time were represented as percentages of responding cells. (e) Cells were treated with crocin (500 μM) in the absence or presence of BAPTA/AM (B/A, 5 μM), or nifedipine (Nif, 5 μM) for 10 min, after which intracellular free Ca2+ levels (represented as percentage of responding cells) were evaluated by flow cytometry. versus the group treated with PBS (vehicle); versus the group treated with crocin alone in both Ca2+-containing and Ca2+-free medium.
