Additional data for flavonoids inhibit 3T3-L1 adipocyte differentiation through reducing ROS generation
The data are mainly to explore the dosage effect and intervention stage of flavonoids including chrysin (CH), apigenin (AP), luteolin (LU), kaempferol (KA), quercetin (QU), and myricetin (MY) on 3T3-L1 preadipocyte viability, differentiation, reactive oxygen species (ROS) regulated adipogenesis. MTT assay was used to investigate the cytotoxicity of the six flavonoids on 3T3-L1 preadipocytes. 5, 10, 20, 30, 40, or 50 μM LU, QU, MY, AP, KA, or CH were added to 3T3-L1 for 4 h, respectively. 40 μM LU and QU, 50 μM LU, QU, MY, AP, and CH significantly inhibited cell viability in 3T3-L1 preadipocytes. All the flavonoids did not damage cell viability until 30 μM. To measure the anti-adipogenic activity of six dietary flavonoids, we added non-toxic 10 or 30 μM flavonoids to 3T3-L1 adipocytes culture media during the full stage of 8 days differentiation. Oil red O staining showed that 10 μM LU, QU, MY, AP, KA, or CH did not affect lipid storage in 3T3-L1 adipocytes. However, 30 μM LU, QU, MY, AP, KA, or CH prominently decreased lipid droplet content in 3T3-L1 adipocytes. Adipogenesis during adipocyte differentiation can be divided into growth arrest, post-confluent mitosis and clonal expansion, and lipid accumulation. 3T3-L1 preadipocytes were induced to mature adipocytes for 8 days. To explore which stage of 3T3-L1 adipocyte differentiation was inhibited by the dietary flavonoids, 20 μM flavonoids were added to 3T3-L1 differentiated media at intervals of 2 days, as shown in. Oil red staining showed that, during the different stages of 3T3-L1 differentiation, 20 µM QU, MY, AP, KA, or CH effectively inhibited the lipid droplet content of 3T3-L1 when the flavonoids were added during 0-2, 0-4, 0-6 or 0-8 days, but not during 2-8, 4-8 or 6-8 days. The level of intracellular ROS increases during adipocyte differentiation, and exogenous hydrogen peroxide causes lipid storage increasing in adipocytes. N-acetylcysteine is a cell-permeable cysteine precursor and removes intracellular ROS. To test whether LU inhibits 3T3-L1 adipocyte adipogenesis through lowering ROS generation, we used 20 μM LU and 10 mM NAC to treat 3T3-L1 cells with or without 100 μM exogenous hydrogen peroxide, respectively. Exogenous hydrogen peroxide significantly increased adipogenic gene expression including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/ enhancer binding protein α (C/EBPα), fatty acid binding protein 4 (AP2), CD36, fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) in 3T3-L1 cells. LU and NAC treatments not only directly resisted insulin cocktail-induced adipogenic gene expression, but also reversed the action of exogenous hydrogen peroxide on adipocyte differentiation.
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3T3-L1 preadipocytes were purchased from Cell Culture Center of Peking Union Medical College. For differentiation, the post-confluent 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% FBS (Gibco, Auckland, New Zealand) with 10 μg/mL insulin (Sigma, MO, USA), 1 μM dexamethasone (Sigma, MO, USA) and 0.5 mM isobutylmethylxanthine (Sigma, MO, USA) for 2 days, and then the cells were cultured in DMEM with 10 μg/mL insulin for another 2 days. Since day 4, the cells were cultured in normal DMEM for 4 days and the medium was freshly replaced every two days. MTT assay was used to investigate the cytotoxicity of the flavonoids. 3T3-L1 preadipocytes were incubated in 96-well plates. After confluent, 5mg/mL MTT was added to the medium and incubated at 37℃. The supernatant was removed and 150 μL dimethyl sulfoxide (DMSO) was added to each hole. The absorbance was detected by SpectraMax Absorbance Reader (Molecular Devices, SV, USA) at 490nM. Oil red staining specifically stain triglyceride in adipocytes, which be used to analyze the storage lipids of 3T3-L1 preadipocytes. 3T3-L1 were differentiated to mature adipocyte, washed twice with PBS, and fixed with 4% formaldehyde. After rinsing with 100% propylene glycol, the cells were stained with 5% filtered oil red dye. Then, the cells were rinsed with 98% and 85% propylene glycol, and PBS in turn. The stained cells were photographed using the light microscope (Nikon, Tokyo, Japan), and then, dissolved in 200 μL ethanol. The dissolved oil red O staining in ethanal was transferred to 96 wells plate and detected in ELx800 microplate reader (Bio-Tek Industries, GA, USA) at 510 nM. In order to analyze the effect of flavonoid in 3T3-L1 differentiation more clearly, the gene expression level in adipose cells was detected by real-time fluorescence quantitative PCR. Cells were lysed with Trizol reagent (Takara, Osaka, Japan). After static placement of 5 min, trichloromethane was added. The supernatant was collected after centrifuged at 12000g for 15 min at 4 ℃. After static placement of 10 min, equivalent isopropanol was added. Next, the supernatant was discarded after centrifuged, and 75% ethanol was added. After centrifuged for another 5 min, the RNA was dried, dissolved in double distilled water, and reverse transcripted to cDNA by using reverse transcription reagent (Takara, Osaka, Japan) at 37 ℃ for 45 min and terminated at 85 ℃. Quantitative real-time PCR was performed in CFX Connect Real-Time PCR Detection System (CFX ConnectTM Optics Module, Bio-Rad, CA, USA).