Human hepatoma HepG2 cells were obtained from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank/CAS Shanghai Institute for Biologic Science Cell Resource Center (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained in an incubator at 37°C in a 5% CO₂ atmosphere.

HepG2 cells were plated in 6-well plates at a density of ~4×10⁵ cells/well. The following day, cells were stimulated with 100 mM ethanol for 48 h, and simultaneously treated with 50 µM zopolrestat (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A total of 48 h post-treatment, cells were washed with ice-cold PBS, fixed with 10% formalin, and stained with Oil Red O to detect lipid droplets in cells.

Total RNA was extracted from cells using TriPure Isolation Reagent (Roche Applied Science, Mannheim, Germany), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The primer sequences for hepatic tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, interleukin (IL)-6, sterol regulatory element binding protein (SREBP)-1c, fatty acid synthase (FAS), peroxisome proliferator-activated receptor (PPAR)α, acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase (CPT)-1 are presented in Table I. qPCR was conducted using the FastStart Universal SYBR-Green Master (Rox; Roche Applied Science). The reaction was run at 95°C for 10 min, followed by 35 cycles at 95°C for 15 sec, 53–60°C for 30 sec, 72°C for 30 sec and a final extension at 72°C for 10 min. The relative expression of each gene was normalized to GAPDH and was calculated using the comparative Cq method (ΔΔCq) (13).

Whole cell extracts were prepared by dissolution of cell pellets in ice-cold radioimmunoprecipitation assay lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerin, 10 mM Na₄P₂O₇, 20 mM glycerophosphate, 10 mM NaF, 10 mM sodium orthovanadate and proteinase inhibitor mixture) until the cells were completely lysed. Following centrifugation at 12,000 × g, 4°C for 5 min, supernatants were collected and stored at −80°C before use. The protein concentrations of the extracts were measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of extracted protein samples (40 µg) were separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in 0.1% Tween-20 TBS for 1 h at room temperature and incubated with primary antibodies in TBS-0.1% Tween-20 with 5% non-fat milk at 4°C overnight. Primary antibodies used were: 5′ adenosine monophosphate-activated protein kinase α (AMPKα; cat. no. #2532; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-AMPKα (cat. no. #2535; 1:1,000 dilution; Cell Signaling Technology, Inc.), AR (cat. no. sc-17735; 1:500 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. A2228; 1:2,000 dilution; Sigma-Aldrich; Merck KGaA). After several washes with TBS-0.1% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin G secondary antibodies (dilution 1:2,000; cat. nos. AP307P and AB324P, respectively; Merck KGaA) in TBS-0.1% Tween-20 with 5% non-fat milk. The bands were visualized using the SuperSignal Chemiluminescent Substrate kit (Beyotime Institute of Biotechnology). Densitometric analysis of protein bands was performed using ImageJ v. 1.40 software (National Institutes of Health, Bethesda, MD, USA).

The statistical significance of the difference between groups was assessed by Student's t-test for pair-wise comparisons or one-way analysis of variance, followed by a post hoc Bonferroni's test for multiple comparisons. Data are expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference. The analysis was performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Previous studies have demonstrated that stimulating cells with ethanol may induce hepatic steatosis (14,15). In the present study, protein expression levels of AR were assessed in HepG2 cells stimulated with 100 mM ethanol. AR protein expression in cells stimulated with ethanol for 36 h was ~2.1x higher (P<0.05) compared with in control cells without ethanol stimulation (Fig. 1). To further investigate the role of AR in the development of ethanol-induced steatosis, AR activity was inhibited via treatment with the AR inhibitor zopolrestat (50 µM). Oil Red O staining revealed a marked lipid accumulation in ethanol-stimulated HepG2 cells, whereas control cells without ethanol stimulation did not exhibit steatosis (Fig. 2). When AR activity was inhibited, lipid accumulation in ethanol-stimulated cells was markedly attenuated. These findings suggested that AR is involved in the development of ethanol-induced steatosis in hepatocytes.

AMPK inactivation has been reported during the development of ethanol-induced hepatic steatosis (16,17). SREBP-1c is a transcription factor regulated by AMPK, which modulates the expression of several lipogenic enzymes, including FAS (18,19). To investigate the role AR serves in the development of ethanol-induced steatosis, the effect of AR inhibition on AMPK activity and on SREBP-1c and FAS mRNA expression was assessed in ethanol-stimulated HepG2 cells. Treatment with 100 mM ethanol resulted in a significant reduction in AMPK phosphorylation in HepG2 cells; the reduction in p-AMPK levels was markedly attenuated when ethanol-stimulated cells were treated withzopolrestat (Fig. 3A). In addition, treatment with ethanol caused a significant increase in SREBP-1c mRNA expression levels compared with in untreated cells; the increase was significantly attenuated by zopolrestat treatment (P<0.05). FAS mRNA expression levels were significantly reduced in ethanol-stimulated cells treated with zopolrestat compared with in cells that didn't receive the AR inhibitor (Fig. 3B). Furthermore, the effect of AR inhibition on PPARα, a transcription factor that regulates several enzymes involved in fatty acid oxidation, such as ACO and CPT-1, was investigated (20,21). AR inhibition did not alter the mRNA expression levels of PPARα, ACO or CPT-1 (Fig. 3C). Taken together, the present results suggested that the inhibition of AR may ameliorate ethanol-induced steatosis in HepG2 cells through activating AMPK and inhibiting SREBP-1c-regulated lipogenesis.

TNF-α has been reported to serve a pivotal role in the development of ethanol-induced hepatic steatosis (22,23). In the present study, mRNA expression levels of proinflammatory cytokines, including TNF-α, IL-6 and TGF-β1, were investigated. Cells stimulated with ethanol exhibited significantly elevated TNF-α mRNA expression levels compared with in unstimulated cells (Fig. 4), whereas treatment with zopolrestat significantly attenuated the ethanol-induced increase in TNF-α mRNA expression (P<0.05). Conversely, AR inhibition had no effect on IL-6 and TGF-β1 expression. These results suggested that AR inhibition may affect the production of proinflammatory cytokines, such as TNF-α, to prevent ethanol-induced steatosis in HepG2 cells.