3.

Crocetin inhibits MEK-ERK1/2 signalling in vitro and in vivo. (A) The dose and time courses of crocetin in the generation of ROS induced by Ang II. Cardiac myocytes were pre-treated with different concentrations of crocetin for 30 min. and subsequently incubated with 1 μM Ang II for 60 min. Four parallel experiments were indicated. *P < 0.05 versus exposed to control. (B) Crocetin inhibits pressure overload-induced increase of signal decay rate. *P < 0.05 versus sham/ vehicle. (C) Representative blots of MEK1/2, ERK1/2, p38 and JNK phos-phorylation and their total protein expression in the indicated group's mice. (D) Representative blots of MEK1/2 and ERK1/2 phosphorylation and their total protein expression in cultured cardiac myocytes. (E) The effect of ERK1/2 activation on [³H]-leucine incorporation and ANP promoter activity induced by Ang II. Cardiac myocytes were pre-treated with crocetin for 60 min. and treated with Ang II for 48 hrs. The results were reproducible in three separate experiments as mean ± S.E.M. *P < 0.01 was obtained for the PBS-treated control group. (F) The effect of NAC on MEK1/2 and ERK1/2 activation as well as on ANP and BNP protein expressions induced by Ang II in cultured myocytes. Cardiac myocytes were pre-treated with 10 mM NAC for 30 min. and incubated with Ang II for 120 min. (G) Crocetin inhibits pressure overload-induced GATA-4 DNA-binding activity. (H) Inhibition of ERK1/2 by treatment of U0126 (5 μM) or infection with Ad-dnERK1/2-blocked GATA-4 activation induced by Ang II.