MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triglyceride (TG), Total Cholesterol (TC), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT), and Alkaline Phosphatase (ALP) kits were purchased from Asan Pharmaceutical company (Seoul, Republic of Korea). Fatty Acid Synthase (FAS), Carnitine Palmitoyl Transferase-1 (CPT-1), and Peroxisome Proliferator-Activated Receptor-α (PPAR-α) antibodies were purchased from santacruz Biotechnology Inc. (Santa Cruz, CA, USA), 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase (HMGCR) was purchased from Cell Signaling (Beverly, MA, USA). β-Actin was purchased from Bethyl Laboratories (Montgomery, TX, USA).
Preparation of Yuja extracts
Yuja was provided by Goheung Country Office (Goheung, Republic of Korea), where a voucher specimen was deposited. Yuja peel extracts (YE) were prepared as described previously . Briefly, yuja peels were dissolved in a 10-fold volume of 70% ethanol by shaking for 24 h at 25 °C, and precipitates were removed by centrifugation at 8000xg for 30 min. Supernatants were dried using freeze dryer. Yuja extract was dissolved in DMSO and used to treat HepG2 cells and mice fed high fat diet. The cells were incubated with 1% BSA-supplemented low-glucose DMEM (None), 0.5 mM oleic acid (OA) in 1% BSA-supplemented low-glucose DMEM, and 0.5 mM OA in DMEM with 50, 100, and 200 μg/mL YE as a treatment group for 24 h.
Cell culture and sample treatment
HepG2 cells were purchased from the American Type Culture Collection (Mannassas, VA, USA). These cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, which were purchased from Welgene Inc. (Daegu, Republic of Korea). The cells were maintained at 37 °C in a humidified atmosphere under 5% CO2. When processing the sample on the cells, cells were treated with 1% BSA in low-glucose DMEM (None), 0.5 mM oleic acid (OA) in 1% BSA-supplemented low-glucose DMEM, and 0.5 mM OA in DMEM with 50, 100, and 200 μg/mL YE as a treatment group for 24 h.
Cytotoxicity (MTT assay)
HepG2 cells were cultured in 24-well plates and treated with YE at the indicated concentrations. Then, 10 μL of MTT solution (5 mg/mL in PBS) was added and incubated for 3 h. After removing the medium, the cells were dissolved in dimethylsulfoxide (DMSO), and 100 μL of supernatant was transferred to 96-well plates. Absorbance was measured at 540 nm using microplate reader (Molecular Device Co., Sunnyvale, CA, USA).
Oil Red O staining
HepG2 cells were cultured in 24-well plates and then treated with YE for 24 h. After treatment, they were stained with Oil Red O to measure lipid droplet accumulation, washed with 200 μL of PBS, and fixed with 200 μL of 4% formaldehyde for 15 min, at room temperature (RT). The cells were then washed three times with PBS, incubated with 200 μL of 60% isopropanol for 5 min, and then stained with 200 μL of 0.1% Oil Red O staining solution for 60 min at RT. The cells were further washed 3 times with 1 mL of water. The images of these cells were captured by microscopy (Olympus, Tokyo, Japan). To measure lipid accumulation, the cells were dissolved in isopropanol for 10 min. The dissolved dye was transferred to 96-well plates and the absorbance was measured at 510 nm.
Western blot assay
The proteins were harvested in RIPA buffer (Elpis, Daejeon, Republic of Korea) containing a protease inhibitor and a phosphatase inhibitor (Roche, Basel, Switzerland). After this treatment, the protein was quantified using Bicinchoninic Acid (BCA) methods. The protein samples (20 μg) were loaded onto 10% Bis-Tris gel and transferred to nitrocellulose membranes. The membrane was blocked using 5% skim milk solution for 1 h and probed with each antibody at 4 °C overnight. After triplicate washing with PBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. It was washed again with PBS and the protein expression was detected by chemiluminescence methods.
Animal fed a high-cholesterol diet
Male C57BL/6 J mice (3 weeks old) were housed at the Korea Food Research Institute (KFRI) in a climate-controlled environment (24 °C at 50% relative humidity) with a 12 h light/dark cycle. After 1 week of adaptation, the mice were randomly divided into four groups (n = 8): N (Normal diet), HC (high-cholesterol diet), YL (HC + 1% YE), and YH (HC + 5% YE). D12336 diet (high-cholesterol diet, Purified diet to match Paigen’s atherogenic rodent diet, Research Diets, Inc.) was used as HC. The composition of the high cholesterol diet was as follow: 46% carbohydrate, 16% fat, 21% protein, 12.5% cholesterol, 0.5% cholic acid, mineral mixture and vitamin mixture (source of carbohydrate = maltodextrin, sucrose and corn starch; source of fat = soybean oil, cocoa butter and coconut oil; source of protein = casein, soy protein and DL-methionine, source of mineral mixture = calcium carbonate, sodium chloride, potassium citrate; vitamin mixture = choline bitartrate). The mice were fed these diets for 10 weeks with free access to autoclaved tap water in cages. The total daily intake and weight of mice were recorded every week for up to 10 weeks. At the end of the experiment, animals were sacrificed to collect their blood and liver tissue samples, which were then stored at −70 °C. Our experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea Food Research Institute.
Hematoxylin and eosin (H&E) staining
Liver tissues were fixed using 4% formaldehyde solution and cut into 4-μm-thick portions, which were stained with hematoxylin and eosin, and images were captured using a microscope (Olympus, Tokyo, Japan).
Measurement of enzyme and lipid change in blood
The collected blood samples were centrifuged at 12000 rpm for 15 min at 4 °C. After centrifugation, the serum was transferred to a new centrifuge tube and then stored at −70 °C. Blood serum levels of TG, TC, HDL-cholesterol, GOT, GPT, and ALT were analysed according to the manufacturer’s protocol.
Results obtained from at least three independent in vitro studies were expressed as mean ± standard deviation (SD). The results were determined by nonparametric methods using the SPSS computer-based statistics programs (Ver. 20; SPSS Inc., Chicago, IL, USA). In case of in vivo studies using mice, the results are expressed as mean ± standard error of mean (SE). Statistical differences between mean values were evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni test. A P value of <0.05 was considered statistically significant.