Inhibitory Effects and its Mechanism of Pinostilbene Hydrate on Melanogenesis and Adipogenesis in Mouse Cells

Abstract

Resveratrol is a phytoalexin with multiple bioactive properties, including antioxidative, neuroprotective, cardioprotective, and anticancer effects. However, resveratrol exhibits structural instability in response to UV, alkaline pH, and oxygen exposure. Thus, resveratrol derivatives have gained considerable research interest. In this study, we aimed to evaluate the anti-melanogenic and anti-adipogenic effects of pinostilbene hydrate (PH), a methylated resveratrol derivative, in B16F10 and 3T3-L1 cells. We also evaluated the mechanisms underlying the effects of PH on melanogenesis and adipogenesis in B16F10 cells and 3T3-L1 adipocytes. The results in B16F10 cells indicated that PH significantly inhibits melanin content and cellular tyrosinase activity in cells without causing cytotoxicity. In addition, Western blot analysis showed that PH downregulated the protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and other melanogenic enzymes, such as tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2). Although PH activated the phosphorylation of extracellular signal-regulated kinase (ERK), it inhibited p38 mitogen-activated protein kinases (MAPK). Furthermore, the inhibition of tyrosinase activity by PH was attenuated by treatment with PD98059 (a specific ERK inhibitor). Additionally, phosphorylated-protein kinase B (p-AKT) was upregulated by PH treatment. Finally, the inhibitory effects of PH on melanin content and tyrosinase activity were confirmed in normal human melanocytes. These results suggest PH downregulates melanogenesis via the inhibition of MITF expression, followed by the MAPK signaling pathways. The results in 3T3-L1 adipocytes showed that Oil Red O staining, lipid accumulation assay, and triglyceride (TG) content assay revealed that PH significantly inhibited lipid and TG accumulation on day 8 without cytotoxicity. In addition, we determined that PH decreased the expression of adipogenesis-related transcription factors such as CCAAT/enhancer-binding protein alpha (C/EBPα), peroxisome proliferator activated receptor gamma (PPARγ), sterol response element binding protein-1c (SREBP-1c), and fatty acid binding protein 4 (FABP4). In the mechanistic study, PH decreased the phosphorylation of mitogen-activated protein kinases (MAPK) and protein kinase B (AKT). Moreover, PH attenuated the expression of cAMP response element binding protein (CREB) and C/EBPβ. Furthermore, PH increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and decreased the expression of fatty acid synthase (FAS) and FABP4. Based on these results, we suggest that PH suppresses adipogenesis in 3T3-L1 cells via inhibition of MAPK and AKT-dependent insulin signaling, and AMPK signaling pathways. Thus, PH may be used as potential therapeutic agent to treat or prevent hyperpigmentation disorders, obesity and obesity-related metabolic disorders.