Increased Expression Ratio of Bcl‐2/Bax Is Associated with Crocin‐Mediated Apoptosis in Bovine Aortic Endothelial Cells

Abstract

Crocin, extracted and purified from Gardenia jasminoids Ellis in our laboratory, has been reported to have antioxidative, hypolipidaemic and anti-atherosclerorotic effects. However, the underlying molecular mechanisms by which crocin acts as a cytoprotective agent remain to be elucidated. In the present study, we examined the mechanisms of crocin, the digentiobiosyl ester of crocetin, on bovine aortic endothelial cell apoptosis induced by hydrogen peroxide (H2O2). The cells were obtained from the thoracic aorta of newborn calves, and apoptosis was induced by 200 microM H2O2. Before addition of H2O2, the cells were pretreated with different concentrations of crocin for 6 hr. After incubation of the cells with H2O2, a comparative reverse transcriptase-polymerase chain reaction-based repeated amplification protocol assay was used to determine the ratio of bcl-2/bax mRNA expression, and cells loaded with fluo-3/AM were subjected to laser scanning confocal microscopy for detection of intracellular calcium ([Ca2+]i) levels. Treatment of the cells with H2O2 alone decreased the ratio of bcl-2/bax expression to almost twice of those of untreated cells (from 0.33+/-0.05 to 0.16+/-0.02). In the presence of 1 and 10 microM crocin, the ratios were enhanced when compared with H2O2 alone respectively (from 0.16+/-0.02 to 0.58+/-0.04, 1.18+/-0.13). The treatment of cells with crocin alone had little effect on the value of this ratio. In the presence or absence of extracellular Ca2+, H2O2 could induce intracellular calcium elevation not only in the elevation presence of extracellular Ca2+ (Hanks), but also without extracellular calcium present (D-Hanks). But the extent of [Ca2+]i under conditions lacking extracellular calcium is less. Crocin concentration dependently inhibited the [Ca2+]i elevation induced by H2O2 under these two conditions. Our data suggest that crocin may exert anti-atherosclerotic effects by increasing the expression ratio of bcl-2/bax, as a result, inhibiting the bovine aortic endothelial cell apoptosis that plays an important role in the initiation and progression of atherosclerosis.