AML12 mouse liver cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium/nutrient mixture F12 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 100 units/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) at 37°C in an incubator with 5% CO₂.

Cell viability was assessed by an MTT assay. Briefly, AML12 cells were seeded in 96-well plates at the density of 1×10⁴ cells/well. The following day, cells were treated with 800 mM ethanol and co-cultured with 5, 10, 20 or 40 µM myricitrin for 16 h. Subsequently MTT solution (5 mg/ml) was added to each well and the cells were cultured for another 4 h. MTT formazan precipitate was dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at a wavelength of 570 nm using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The vehicle control cells were treated with ultrapure water rather than ethanol, and DMSO rather than myricitrin. Cell viability values are expressed as percentage of the vehicle control (100%). All experiments were performed in triplicate.

AML12 cells were seeded into 24-well plates at a density of 1×10⁵/well. The following day, cells were stimulated with 100 mM ethanol and co-treated with 20 µM myricitrin. Following 36 h of treatment, cells were washed with ice-cold PBS buffer, fixed with 10% formalin at room temperature for 10 min, and stained with oil red O at room temperature for 30 min to detect lipid droplets in cells. Following staining, the cultures were washed and the dye was extracted by isopropanol. Quantification was performed by measuring optical density at a wavelength of 510 nm.

AML12 cells were seeded in 24-well plates at a density of 1×10⁵ cells/well. After 24 h, cells were stimulated with 100 mM ethanol and co-cultured with 20 µM myricitrin. A total of 48 h after ethanol and myricitrin treatment, cells were washed and resuspended in PBS and incubated with 10 µM 2′,7′-dichlorofluorescin diacetate (DCF-DA) for 1 h at 37°C. Subsequently, 1×10⁶ cells/200 µl/ well were seeded in a 96-well microplate and DCF fluorescence was measured using a fluorescence microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

AML12 cells were plated and treated with ethanol and myricitrin as described above. At 48 h after ethanol and myricitrin treatment, total lipoperoxides were measured as thiobarbituric acid reactive substances (TBARS) in culture medium using a Lipid Peroxidation MDA Assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. TBARS were quantified using malondialdehyde (MDA) present in each sample.

AML12 cells were plated and treated with ethanol and myricitrin as described above. A total of 48 h after ethanol and myricitrin treatment, the cells were centrifuged at 400 × g at 4°C for 5 min, the supernatant was collected and samples were stored at −20°C until cytokine levels were measured. The concentrations of TNF-α, interleukin (IL)-6 and TGF-β1 in cell supernatants were tested using the following ELISA kits: TNF-α (cat. no. ml002095), IL-6 (cat. no. ml002293) and TGF-β1 (cat. no. ml002115). All kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China), and performed according to the manufacturer's protocol.

Total RNA was isolated from AML12 cells using the TRIpure reagent (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. cDNA was synthesized from hepatic mRNA using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The following temperature protocol was used for cDNA synthesis: Incubation for 5 min at 25°C, followed by 60 min at 42°C and then termination of the reaction via incubation for 5 min at 70°C. Hepatic sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), TNF-α, IL-6, and TGF-β1 were analyzed using specific primers listed in Table I. qPCR was performed using FastStart Universal SYBR Green Master (Rox; Roche Applied Science). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 10 min; 35 cycles of 95°C for 15 sec, 53–60°C for 30 sec and 72°C for 30 sec; and a final extension at 72°C for 10 min. Relative expression of each gene was normalized to β-actin and was calculated using the comparative 2^−∆∆Cq method (19).

Cells were homogenized in ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Total protein concentration in extracts was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Each protein sample (40 µg/lane) was loaded and separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in TBS containing 0.1% Tween-20 for 1 h at room temperature and subsequently incubated with anti-phosphorylated (p)-adenosine 5′-phosphate-activated protein kinase (AMPKα; cat. no. 2535; 1:1,000 dilution), anti-AMPKα (cat. no. 2532; 1:1,000 dilution; both Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. G9545; 1:2,000 dilution; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) antibodies at 4°C overnight. Subsequently, horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (cat. no. A6154; 1:2,000 dilution; Sigma-Aldrich; Merck KGaA) was used for an incubation at room temperature for 2 h. Detection was performed using a Supersignal Chemiluminescent Substrate kit (Beyotime Institute of Biotechnology).

All data were processed and analyzed using GraphPad software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) and are presented as the mean ± standard error of the mean. One-way analysis of variance with Bonferroni's multiple comparison test were used for analyses. P<0.05 was considered to indicate a statistically significant difference.

The present study aimed to determine the effect of various concentrations of myricitrin (5, 10, 20 and 40 µM) on ethanol-induced hepatotoxicity in AML12 cells. Myricitrin alone at 5, 10, 20, or 40 µM did not exert a cytotoxic effect on AML12 cells (Fig. 1A). Incubation of AML12 cells with 800 mM ethanol for 16 h induced ~45.5% growth inhibition compared with the untreated control (P<0.001; Fig. 1B). Administration of 5, 10, 20, and 40 µM myricitrin to ethanol-stimulated cells exerted a protective effect and reduced cell viability inhibition to ~41.6 (P<0.01), 28.5 (P<0.001), 24.2 (P<0.001) and 25.9% (P<0.001), respectively. The above data suggest that myricitrin exerts a significant protective effect against ethanol-induced cytotoxicity. Myricitrin at a dose of 20 µM exerted the most beneficial effect on ethanol-induced cytotoxicity and therefore, this dose was used in the subsequent experiments.

Previous studies demonstrated that ethanol may induce steatosis (20,21). To determine the effect of myricitrin on ethanol-induced steatosis, cells were treated with 100 mM ethanol or 20 µM myricitrin, or both. Oil red O staining demonstrated lipid accumulation in ethanol-stimulated cells while the control cells did not exhibit steatosis (P<0.001; Fig. 2). Following co-culture with myricitrin, lipid accumulation in ethanol-stimulated cells was significantly attenuated (P<0.001). The results indicate that myricitrin can suppress the development of ethanol-induced steatosis in hepatocytes.

Oxidative stress is reported to be involved in the development of ethanol-induced steatosis (2). The present study investigated the effect of myricitrin treatment on MDA and ROS production in AML12 cells. Treatment with myricitrin resulted in a 32.1% decrease in ROS levels (P<0.001) and 22.4% decrease in MDA levels (P<0.001) in ethanol-stimulated AML12 cells (Fig. 3). The above data indicate that myricitrin alleviates ethanol-induced oxidative stress in liver cells. Certain pro-inflammatory cytokines, including TNF-α, IL-6 and TGF-β1 have been reported to be associated with the development of ethanol-induced hepatic steatosis (8,22). Expression levels of TNF-α, IL-6 and TGF-β1 were also been determined in the present study. Cells stimulated with ethanol exhibited markedly elevated mRNA expression levels of TNF-α, compared with control cells (Fig. 4A). However, ethanol-induced increase in TNF-α mRNA levels was significantly attenuated in cells treated with myricitrin (47.9%; P<0.001). Similarly, cells stimulated with ethanol exhibited markedly increased expression of IL-6 and TGF-β1 mRNA compared with control cells. This ethanol-induced elevation of mRNA levels of IL-6 and TGF-β1 was significantly attenuated, by 27.1% (P<0.01) and 51.8% (P<0.001) respectively, in cells treated with myricitrin. Furthermore, ethanol-induced secretion of TNF-α, IL-6 and TGF-β1 proteins was also suppressed by 28.3% (P<0.01), 44.6% (P<0.05) and 43.8% (P<0.01), respectively, in cells treated with myricitrin (Fig. 4B). The results suggest that myricitrin may reduce oxidative stress and affect the production of pro-inflammatory cytokines to prevent ethanol-induced steatosis in AML12 liver cells.

Previous studies indicated that AMPK inactivation may be involved in the development of ethanol-induced hepatic steatosis (23,24). To determine the mechanism underlying myricitrin-induced amelioration of ethanol-induced steatosis, the present study investigated the effect of treatment with myricitrin on AMPK activity. pAMPK protein expression levels in ethanol-stimulated AML12 cells treated with myricitrin increased compared with myricitrin untreated ethanol-stimulated cells, indicating that myricitrin treatment activated AMPK in ethanol-stimulated AML12 cells (Fig. 5A). Furthermore, the effect of treatment with myricitrin on mRNA expression of SREBP-1c and FAS was determined. SREBP-1c and FAS genes are regulated by AMPK and control the synthesis of fatty acids in ethanol-stimulated liver cells (21,25,26). Treatment with 100 mM ethanol resulted in a significant increase in SREBP-1c mRNA expression levels compared with the control group and this increase was significantly attenuated following treatment of ethanol-stimulated cells with myricitrin (P<0.05; Fig. 5B). mRNA expression of FAS significantly decreased in myricitrin and ethanol-stimulated cells compared with the ethanol treatment group (P<0.01; Fig. 5C). The above results indicate that treatment with myricitrin may ameliorate ethanol-induced steatosis in AML12 cells through activation of AMPK and subsequent inhibition of SREBP-1c-regulated lipogenesis.