Synergistic efficacy of concurrent treatment with cilostazol and probucol on the suppression of reactive oxygen species and inflammatory markers in cultured human coronary artery endothelial cells

Abstract

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3~3 microM) with probucol (0.03~0.3 microM) significantly suppressed TNF-alpha-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-alpha release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3~3 microM) with probucol (0.1~0.3 microM) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.

Keywords: Cilostazol; Endothelial cells; MCP-1; Monocyte adhesion; Probucol; Synergism; VCAM-1.

Figures

Fig. 1

Effects of cilostazol alone and in combination of cilostazol with probucol on the NAD(P)H-dependent superoxide production in the HCAECs stimulated by TNF-α (50 ng/ml). The significant effect was evident by cilostazol in combination with probucol. Results are expressed as mean±S.E.M. of four experiments. *p<0.05, **p<0.01, ***p<0.001 vs. cilostazol alone. Significant differences were shown between cilostazol alone and cilostazol plus probucol 0.03 µM or cilostazol plus 0.1 µM probucol groups by two-way repeated measures ANOVA.

Fig. 2

Inhibitory effect of cilostazol in combination with probucol on the intracellular ROS levels stimulated by in the HCAECs LPS (1 µg/ml). LPS-stimulated intracellular ROS was significantly decreased by cilostazol plus 0.1 µM probucol in combination. ***p<0.001 vs. cilostazol alone. Results are expressed as mean±S.E.M. of four experiments. Significant differences were shown between cilostazol alone and cilostazol plus 0.1 µM probucol groups by two-way repeated measures ANOVA.

Fig. 3

Effects of cilostazol and probucol alone and their combination on the TNF-α formation stimulated by in the HCAECs LPS (1 µg/ml). Increased TNF-α by LPS was significantly decreased by cilostazol and probucol in combination. Results are expressed as mean±S.E.M. of three experiments. **p<0.01, ***p<0.001 vs. vehicle, #p<0.05 vs. 0.3 µM probucol alone, †p<0.05 vs. 0.3 µM cilostazol alone, §§p<0.01 vs. 1 µM cilostazol alone.

Fig. 4

Inhibitory effect of cilostazol in combination with probucol on the VCAM-1expression in the HCAECs. TNF-α (50 ng/ml)-induced increased VCAM-1expression was significantly decreased by treatment with cilostazol and 0.1 µM probucol in combination. Results are expressed as mean±S.E.M. of four experiments. ***p<0.001 vs. cilostazol alone. Significant differences were shown between cilostazol plus 0.03 µM probucol and cilostazol plus 0.1 µM probucol groups by two-way repeated measures ANOVA.

Fig. 5

Effects of cilostazol and probucol in combination on the MCP-1 expression stimulated in the HCAECs by TNF-α (50 ng/ml). Concurrent treatment with 0.3 µM probucol plus 0.3 or 1 µM cilostazol significantly decreased MCP-1 expression in comparison to effect of probucol and cilostazol alone. Results are expressed as mean±S.E.M. of three experiments. *p<0.05 vs. vehicle.

Fig. 6

Effects of cilostazol and probucol alone and their combination on the monocyte adhesion to HCAECs. TNF-α (50 ng/ml)-stimulated adherent monocytes were significantly decreased by treatment with cilostazol and probucol in combination. Results are expressed as mean±S.E.M. of four experiments. *p<0.05, **p<0.01 vs. vehicle, #p<0.05 vs. probucol alone.