Figure 5.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) siRNA suppresses adipogenesis and oxidative stress in Graves’ orbitopathy (GO) fibroblasts. Orbital fibroblasts from GO (n=3) patients were cultured in adipogenic medium to induce differentiation into adipocytes for 7 days. (A) Oil red O staining showed treatment with PCSK9 siRNA (50 nM, 24 h) attenuated adipogenesis. (B) Quantification by measurements of optical density of cell lysates at 490nm echoed the histochemical results. The results are presented as the mean optical ratio ± SD (*p < 0.05 between sicon and siPCSK9). (C) Western blot analyses showed that throughout the 7-day period of adipogenesis, the cell lysates of fibroblasts collected at different time points showed a gradual increase in production of PCSK9, which was markedly decreased when PCSK9 siRNA (50 nM, 24 h) was treated. The levels of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein β (C/EBPβ), were substantially curtailed in fibroblasts transfected with PCSK9 siRNA. The levels of mature adipocyte marker, leptin, were also significantly reduced in the PCSK9 siRNA-treated group. The levels of antioxidants, manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1), and oxidative stress-related protein, hypoxia-inducible factor 1α (HIF-1α) were significantly decreased in the PCSK9 siRNA-transfected group. Data in the columns indicate the mean density ratio ± SD, normalized to the level of β-actin in the same sample, and representative gel images are shown (*p < 0.05 between sicon and siPCSK9 on days 0, 3, 5, and 7 of adipogenesis).