Male C57B6/L mice (8 weeks old) were purchased from the Experimental Animal Center of Anhui Medical University (Hefei, China). Mice were randomly divided into four groups, the control group (CD-fed), model group (EtOH-fed), blank solvent group, and PT2399 treatment group (n = 10 per group). All experiments were performed according to the institutional ethical guidelines for laboratory animal care and use of Anhui Medical University. An AFLD model was established using the National Institute on Alcohol Abuse and Alcoholism (NIAAA)- recommended method of a Lieber-De Carli (LD) liquid diet and alcohol intragastric administration. Animal feed was obtained from TROPHIC Animal Feed High-Tech Co., Ltd. (Hai’ an, Jiangsu, China). The modeling process lasted a total of 16 days, including liquid diet adaptation (5 days), modeling (10 days), intragastric administration (1 time), and specimen acquisition stages (1 day). Mice in the EtOH-fed group were randomly fed a LD liquid diet containing 5% ethanol (vol/vol) for 10 days. Mice in the PT2399 treatment group received PT2399 (10 mg/kg) twice daily from day 10‒17 via intragastric administration. Mice were anesthetized 9 h after the final oral gavage of 33% ethanol (5 g/kg), and the blood and liver tissue samples were collected for subsequent analysis. At least six independent experiments were performed.
Liver tissue samples were fixed with 4% paraformaldehyde for 24 h, and then embedded in paraffin blocks and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed according to a standard procedure. Brown staining was considered as a positive result. Images were captured using a Panoramic 250 slide scanner (3DHistech Ltd).
The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the levels of triglycerides (TGs) and total cholesterol (T-CHO) were measured using commercial assay kits (Jiancheng, Nanjing, China) in a microplate reader (Biotek, USA) at the appropriate wavelength.
Transmission electron microscopy (TEM)
Grain size liver tissue samples were fixed in 2.5% glutaraldehyde at 4 °C and then in 1% osmium tetroxide for 4 h on ice. Ultrathin Sects. (60 nm thick) were cut on an Ultracut E Microtome (Reichert‐Jung; Leica Microsystems, Wetzlar Germany) with a diamond knife (Diatome, Hatfield, PA, USA), collected on copper grids, and stained with uranyl acetate and lead citrate. Sections were dehydrated sequentially in a graded series of ethanol, infiltrated with graded mixtures of propylene oxides, embedded in fresh resin, and incubated at 60 °C for 48 h. Electron micrographs were obtained using TEM (Hitachi, Japan).
Alpha mouse liver 12 (AML-12) cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Materials, China) and 1% penicillin–streptomycin (Biyuntian, China) at 37 °C under an atmosphere of 5% CO2. The medium was changed once a day. They were treated with 100 mM ethanol for 48 h. At least three independent replicates were performed for each experiment.
Oil red o staining
Cultured cells were fixed in 4% paraformaldehyde for 15 min. Sections were then stained with Oil red o for 30 min, and then washed with 60% dimethylcarbinol and double distilled water. The fat drops were observed using an inverted fluorescent microscope. The same procedure was performed for fixed liver tissue samples.
Hif-2α siRNA plasmid and BNIP3 shRNA plasmid (Gene Pharma, Shanghai, China) were designed to downregulate the expression of Hif-2α and BNIP3, respectively. pc-DNA3.1-BNIP3 plasmid was designed to upregulate the expression of BNIP3. Transfection was performed using LipofectamineTM2000 (Invitrogen, Carlsbad, MA), according to the manufacturer’s instructions. After 6 h of transfection, the medium was changed to DMEM and ethanol was added. The following primers were used: siRNA-Hif-2α (mouse): 5′-GCAACUACCUGUUCACCAATT-3′ (sense) and 5′-UUGG UGAA CAG GUAGUUG CT-3′ (antisense).
Cell viability assay
Cultured cells were seeded in a 96-well plate, and 20 µL of 3-(4,5-dimethylthiaz ol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, Sigma–Aldrich) was added to each well and then incubated for 4 h. Thereafter, 100 µL of dimethylsulfoxide (DMSO, Sigma-Aldrich) was added to each well and incubated for 10 min. Cell viability was determined by measuring the absorbance (A) at 490 nm using a spectrophotometer (BioTek Instruments, USA).
RNA extraction and quantitative real-time PCR (qRT–PCR)
Total cellular RNA was isolated using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using a kit. PCR was performed using the PIKO REAL 96 (Thermo Fisher Scientific) system. Real-time quantitative PCR (qRT-PCR) analysis was performed using SYBR® Prime Script™ RT–PCR Kit (TAKARA, Kusatsu, Japan). β-actin was used as the reference gene. The relative expression of each gene was calculated using the 2−ΔΔCt method. The specific sequences of each primer were as follows:
Upstream primer sequence (5ʹ–3ʹ)
Downstream primer sequence (5ʹ–3ʹ)
Liver tissue samples and cultured cells were lysed in RIPA buffer. The nuclear and cytoplasmic protein fractions were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Protein concentration was measured using a BCA protein assay kit (Boster, Wuhan, China). The protein samples were separated by SDS–PAGE (Bio–Rad Laboratories Inc., Berkeley, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Billerica, MA, USA). The PVDF membranes were incubated with primary antibodies against PGC-1α, BNIP3, Beclin-1, MCAD, CPT-1α (1:500; Proteintech, Wuhan, China), PPAR-α (1:500; Cell Signaling Technology, USA), Hif-2α and LC3B (1:1000; Abcam, Cambridge, MA, USA), and Lamin A/C (1:500; Zhongshan Jinqiao, China) for 24 h, and then incubated with secondary antibody (1:500; Zhongshan Jinqiao, China) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) assay (Thermo Scientific, USA), and the blots were analyzed using Image Lab 3.0 (Bio–Rad, Hercules, CA, USA). The gray value of protein bands was calculated using Image J 1.50i (National Institutes of Health, USA) and normalized to that of the internal control, β-actin.
Laser scanning confocal microscopy
The fluorescence of intracellular Fluo-3 was measured by confocal laser scanning fluorescence microscopy (Carl-Zeiss, Jena, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Grayscale images were collected at different time points from 0 to 5 min, and then archived as TIFF image files. Images were analyzed using Leica-sp5 software from the Leica Application Suite (LAS) AF software (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Cultured cells were fixed in 4% formaldehyde for 15 min, and then blocked with 10% bovine serum albumin (BSA, Sigma–Aldrich) to prevent nonspecific staining. Cells were incubated with primary antibodies against Hif-2α or BNIP3 (1:100) at 4 ℃ overnight, and then probed with Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG (Molecular Probes, Beijing, China) at 37 °C for 1 h. Nuclear staining was achieved by incubating cells with 4′,6-diamidino-2-phenylindole and dilactate (DAPI; Invitrogen, Carlsbad, CA, USA). Stained sections were examined using a Carl-Zeiss microscope.
Monitoring of mitophagy
To detect mitophagy, cultured cells were stained with 150 nM Mito Tracker Red (MTR, Invitrogen, L7528) in combination with LC3B. Stained cells were visualized using a Carl-Zeiss microscope within 24 h after mounting.
Data are presented as the mean ± standard deviation (SD). A Student’s t-test or one way ANOVA was used to assess statistically significant differences between groups in GraphPad Prism software. Significance was set at p < 0.05. Results are representative of at least three independent experiments.