Novel derivatives of neohesperidin dihydrochalcone inhibit adipogenic differentiation of human adipose-derived stem cells through the Nrf2 pathway

Abstract

Background: Obesity is a global epidemic. The pathogenesis of obesity is complicated and interacted with multiple factors including distorted adipogenesis through oxidative stress and inflammation as well as excessive calorie intake and physical inactivity. Reactive oxygen species (ROS) are involved to adipogenic differentiation. Neohesperidin as one of strong antioxidants has a significant free radical scavenging activity and anti-inflammatory activity. There is lack of studies to elucidate that neohesperidin dihydrochalcone (NHDC) and its derivatives can suppress adipogenic differentiation and obesity development. Purpose: The main objective of this study is to determine the inhibitory effects of NHDC and its derivatives on in vitro adipogenic differentiation of human adipose-derived stem cells (hASCs). Materials and Methods: hASCs were cultured with supplementation of growth factor and L-glutamine. The culture medium was changed every 3 days and the cells were used in early passages (fourth to seventh). The cytotoxicity of NHDC and its derivatives on hASCs was estimated. hASCs were cultured in proliferation and differentiation medium in the presence or absence of NHDC or derivatives, and experimental groups are as follows: Control group, no NHDC and derivatives + proliferative media only; Differentiation group, no NHDC and derivatives + proliferation and differentiation media; Sample group, NHDC or derivatives + proliferation and differentiation media. The accumulation of intracellular lipid droplets was evaluated using Oil Red O staining. The expression of adipogenic marker at RNA level and protein level were assessed by RT-PCR and Western blotting, respectively. Results: The inhibition of adipogenic differentiation of hASC was tested against NHDC and its derivatives (compounds 9 and 12). In the NHDC treated group, slightly reduced Oil Red O staining was observed compared with the differentiated group. The amount of lipid accumulation decreased in the order of compounds 12 and 9. The expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in compound 9 and 12 treated groups was decreased by 46.7% and 70% respectively, compared to adipogenic differentiation group. Expression of fatty acid synthase (FAS) was decreased by 51.6% and 38.7% by compounds 9 and 12, respectively. Additionally, compounds 9 and 12 reduced the expression of sterol regulatory element-binding protein-1 (SREBP-1) by 65.7% and 60%, respectively. Although the fluorescent intensity of ROS in experimental groups including NHDC and compound 12 were not dramatically weaker than differentiation group, compound 9 showed strong reduction of ROS. Expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) protein did not show any statistically significant difference in the group treated with NHDC and compound 9, but significant increased expression was observed in the group treated with compound 12 compared to the differentiation group. Relatively, the expression of heme oxygenase 1 (HO-1) and NAD(P)H:quinine oxidorecductase-1 (NQO-1) was significantly increased in the NHDC-treated experimental group. Conclusions: NHDC derivatives inhibited adipogenic differentiation in hASC by regulation of ROS through the activation of Nrf2 pathway. NHDC derivatives, having strong effects of Nrf2 activation and ROS reduction in hASC could be a potential therapeutic tool to overcome obesity. Especially, compound 9 having stronger Nrf2 activation activity showed higher reduction of ROS production.