Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 2

Research Article

Synergistic Effects of Cilostazol and Probucol on ER Stress-Induced Hepatic Steatosis via Heme Oxygenase-1-Dependent Activation of Mitochondrial Biogenesis

Figure 2

Effect of combinatorial treatment of cilostazol (CZ) and probucol (PB) on mitochondrial biogenesis-related genes expression. HepG2 cells were treated with cilostazol at various concentrations (0, 0.1, 0.3, 1, and 3 μM) with or without probucol (0.1 μM) for 4 h and PGC-1α, TFAM, and NRF-1 mRNA expression were detected by RT-PCR (a) and real-time RT-PCR (b, c, and d). Primary hepatocytes isolated from C57BL/6 mice were treated with probucol (0.1 μM) and cilostazol (3 μM) individually as well as with their combination for 4 h. Mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 were measured by RT-PCR (e), and mitochondrial-related proteins PGC-1α, COX III, and COX IV were measured by Western blotting (f). Primary hepatocytes from HO-1 WT mice were pretreated in the absence or presence of ZnPP (10 μM) and then were treated with probucol (0.1 μM) and cilostazol (3 μM) individually or in combination for 4 h and then the expression of mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 was detected by RT-PCR (g). Furthermore, primary hepatocytes from HO-1 KO mice were also analyzed by RT-PCR to detect mitochondrial biogenesis-related genes expression (h). Bar graphs, right panels of (a), (e), and (f), as well as lower panels of (g) and (h) are summary data of normalized densitometric ratios. Quantitative data are expressed as means ± SE; . , , and versus cells without treatment; versus cells treated with probucol; versus cells treated with cilostazol.