Journal List > Korean J Physiol Pharmacol > v.13(2) > 1025660

Yun, Park, Seo, Kim, Yoon, and Kim: Cilostazol Attenuates 4-hydroxynonenal-enhanced CD36 Expression on Murine Macrophages via Inhibition of NADPH Oxidase-derived Reactive Oxygen Species Production

Abstract

Although anti-atherogenic effects of cilostazol have been suggested, its effects on the expression of SR in macrophages are unclear. This study investigated the role of cilostazol on CD36 expression of murine macrophages enhanced by HNE, a byproduct of lipid peroxidation. The stimulation of macrophages with HNE led to an increased expression of CD36, which was significantly attenuated by NAC, an antioxidant. Moreover, the increased production of ROS by HNE was completely abolished by NADPH oxidase inhibitors, DPI and apocynin, as well as by the 5-LO inhibitor, MK886, but not by inhibitors for other oxidases. This suggested that NADPH-oxidase and 5-LO were major sources of ROS induced by HNE. In addition, HNE-enhanced expression of CD36 was reduced by these inhibitors, which indicated a role for NADPH oxidase and 5-LO on CD36 expression. In our present study, cilostazol was a significant inhibitor of ROS production, as well as CD36 expression induced by HNE. An increase in NADPH oxidase activity by HNE was significantly attenuated by cilostazol, however cilostazol had no effect on HNE-enhanced 5-LO activity. Together, these results suggest that cilostazol attenuates HNE-enhanced CD36 expression on murine macrophages thorough inhibition of NADPH oxidase-derived ROS generation.

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Fig. 1.
Effect of HNE on the expression of CD36 in J774A.1 macrophages. (A) Total RNA was extracted from macrophages treated with 10 μM HNE for the indicated time (0 ~ 18 hrs). 2 μg RNA was subjected to RT-PCR. Band intensity for CD36 mRNA was quantified by densitometric scanning and normalized to that of GAPDH. (B) Macrophages were incubated with 10 μM HNE, and cellular lysates were analyzed by Western blotting using anti-CD36 antibody. β-actin as used to normalize density. Each photograph is the representative of 5 independent experiments. Data is presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. value at time 0.
kjpp-13-99f1.tif
Fig. 2.
Role of ROS on HNE-induced CD36 expression in J774A.1 macrophages. Macrophages were treated with 10 μM HNE for the indicated time in the absence or presence of N-acetylcysteine (Nac, 5 mM). The levels of CD36 mRNA and protein were determined by RT-PCR (A) and Western blot analysis (B), respectively. Each fig is the representative of 4 independent experiments. Data was presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. control; ##p<0.01 vs. veh (vehicle).
kjpp-13-99f2.tif
Fig. 3.
Effects of inhibitors of NADPH oxidase or lipoxygenase on HNE-induced ROS production in J774A.1. macrophages. (A) Cells were treated with different concentrations of HNE for 45 min, and then ROS generation was measured by flow cytometry (FACS) using DCF fluorescence. Fluorescence intensity was analyzed by CellQuest Software. Data was presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. value at concentration 0. (B) Cells were incubated with 10 μM HNE for 45 min after pre-treatment with various inhibitors for ROS including N-acetylcysteine (Nac, 5 mM), diphenylamine iodonium (DPI, 10 μM), apocynine (Apo, 300 μM), nordihydroguaiaretic acid (NDGA, 10 μM), allopurinol (Allo, 100 μM), or rotenone (Ro, 1 μM) plus stigmatellin (Stig, 1 μM) for 30 min. (C) Cells were pre-treated with eicosanoid inhibitors including MK886 (10 μM), baicalein (10 μM), or indomethacin (Indo, 100 μM) for 30 min, and then stimulated with 10 μM HNE for 45 min. ROS generation was analyzed by FACS. Data was presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. control; ##p<0.01 vs. veh (vehicle).
kjpp-13-99f3.tif
Fig. 4.
Effect of inhibitors for NADPH oxidase or 5-lipoxygenase on HNE-induced CD36 expression in J774A.1. macrophages. Macrophages were treated with diphenylamine iodonium (DPI, 10 μM), apocynin (Apo, 300 μM), or MK886 (10 μM) for 30 min prior to HNE application. Cells were stimulated with 10 μM HNE for the indicated time. The levels of CD36 mRNA and protein were determined by RT-PCR (A) and Western blot analysis (B), respectively. Each fig is the representative of 5 independent experiments. Data was presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. control; #p<0.05; ##p<0.01 vs. veh (vehicle).
kjpp-13-99f4.tif
Fig. 5.
Effects of cilostazol on the expression of CD36 in J774A.1. macrophages. Effects of cilostazol on the expression of CD36 mRNA (A) and protein (B) were analyzed using RT-PCR and Western blot, respectively. Each fig is the representative of 4 independent experiments. Data was presented as mean±SE from 4 independent experiments. ∗∗p<0.01 vs. control; ##p<0.01 vs. HNE alone.
kjpp-13-99f5.tif
Fig. 6.
Effects of cilostazol on the generation of ROS in J774A.1. macrophages. After pre-treatment of various concentrations of cilostazol, macrophages were stimulated with 10 μM HNE for 45 min. ROS generation was measured by flow cytometry (FACS) using DCF fluorescence. Data was presented as mean±SE from six independent experiments. ∗∗p<0.01 vs control; #p<0.05; ##p<0.01 vs. HNE alone.
kjpp-13-99f6.tif
Fig. 7.
Effect of cilostazol on the activities of NADPH oxidase and 5-lipoxygenase. After pre-treatment with various concentrations of cilostazol, macrophages were stimulated with 10 μM HNE for the indicated time. The activities of NADPH oxidase (A) and 5-LO (B) were quantified by chemiluminescence assay and ELISA, respectively. Data was presented as mean±SE from 5 independent experiments. ∗∗p<0.01 vs. control; ##p<0.01 vs. HNE alone.
kjpp-13-99f7.tif
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