The experimental protocol was approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), and performed according to the principles of the Institutional Animal Care and Ethics Committee guidelines (IACUC no. S619). A total of 60 male C57BL/6J mice weighing 22 g (6–7 weeks of age) were purchased from Beijing Huafukang Bioscience Co., Ltd., and maintained in a temperature-controlled room (23±2°C) with a 12-h light-dark lighting cycle.

NASH mice models were established via an MCD for 4 weeks and an HFD for 20 weeks (19). These models demonstrated increased adipose tissue inflammation, enlarged fat cells and less oxidized fat in their muscles. DADS (purity >75%; Sigma-Aldrich; Merck KGaA) dissolved in corn oil was prepared immediately prior to treatment. Following a 2-week acclimation period with standard pellet chow and water ad libitum given prior to the experiments, the mice were randomly divided into one of the following 12 groups (n=5 mice per group): i) Mice fed a normal diet with daily oral gavage of corn oil [Normal + negative control (NC) group]; ii) mice fed a normal diet with daily oral gavage of 20 mg/kg DADS (Normal + DADS-L group); iii) mice fed a normal diet with daily oral gavage of 50 mg/kg DADS (Normal + DADS-M group); iv) mice fed a normal diet with daily oral gavage of 100 mg/kg DADS (Normal + DADS-H group); v) mice fed an MCD diet with daily oral gavage of corn oil (MCD + NC group); vi) mice fed an MCD diet with daily oral gavage of 20 mg/kg DADS (MCD + DADS-L group); vii) mice fed an MCD diet with daily oral gavage of 50 mg/kg DADS (MCD + DADS-M group); viii) mice fed an MCD diet with daily oral gavage of 100 mg/kg DADS (MCD + DADS-H group); ix) mice fed an HFD diet with daily oral gavage of corn oil (HFD + NC group); x) mice fed an HFD diet with daily oral gavage of 20 mg/kg DADS (HFD + DADS-L group); xi) mice fed an HFD diet with daily oral gavage of 50 mg/kg DADS (HFD + DADS-M group); and xii) mice fed an HFD diet with daily oral gavage of 100 mg/kg DADS (HFD + DADS-H group). The DADS concentrations were selected based on previous studies with minor modifications (5,17).

Following treatment with MCD for 4 weeks and HFD for 20 weeks, the mice were sacrificed following a 12-h fast via pentobarbital sodium injection. Blood samples were collected from the heart following euthanasia and serum was isolated via centrifugation at 4°C for 15 min at 2,000 × g. Liver tissues were collected and weighed following washing with physiological saline, and then a portion of the tissue was fixed with 4% paraformaldehyde for 24 h at room temperature to prepare for histological staining. The remaining hepatic tissues were stored at −80°C for molecular biological assays.

Paraformaldehyde-fixed liver tissues were used for hematoxylin and eosin (H&E) staining and oil red O staining, as described in a previous study (20). The NASH activity score, a histological feature scoring system, was utilized to assess the liver lesions: Steatosis (score, 0–3), lobular inflammation (score, 0–3), and hepatocellular ballooning (score, 0–2). Samples with scores >4 were diagnosed as ‘NASH’, samples with scores <3 were designated as ‘not NASH’, and samples with scores between 3–4 were designated as ‘possible for NASH’ (21). The expression of F4/80, a macrophage-surface marker, was detected via immunohistochemistry using an immunohistochemistry kit (Wuhan Boster Biological Technology, Ltd.) with the rat anti-F4/80 monoclonal antibody (1:3,000; cat. no. MCA497; Bio-Rad Laboratories, Inc.) at 37°C for 2 h, and subsequent incubation at 37°C for 2 h with the secondary antibodies (horseradish peroxidase-conjugated anti-mouse IgG; 1:2,000; cat. no. SV0004; Wuhan Boster Biological Technology, Ltd.). Detection of positive staining was performed using 3,3′-diaminobenzidine reagent (Wuhan Boster Biological Technology, Ltd.) for 3 min at room temperature. Finally, all sections were counterstained with 10% hematoxylin for 30 sec at room temperature, and were then left to dry and covered with a coverslip. For every section, 5 fields were randomly selected to evaluate the quantification of F4/80 using Image-Pro Plus 6.0 (Media Cybernetics, Inc.) under a light microscope (magnification, ×40).

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using biochemical methods according to protocols by the Nanjing Jiancheng Bioengineering Institute. Serum triglyceride (TG) and total cholesterol (TC) levels were measured using GPO-PAP and COD-PAP enzymatic colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute), respectively, according to the manufacturer's protocol.

The plasma concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using specific ELISA kits (BioLegend, Inc.) according to the manufacturer's protocol. The absorbance of the samples and standards was detected at 450 and 570 nm for the respective kits using a microplate reader (Bio-Rad Laboratories, Inc.).

Paraffin-embedded liver sections (4-µm thickness) were dewaxed with dimethyl-benzene, permeabilized with 0.3% Triton X-100, and blocked with normal donkey serum (GeneTex, Inc.) for 60 min at room temperature. The sections were then co-incubated with cyclic AMP-responsive element-binding protein H (CREBH) antibody (1:100; cat. no. sc-377332; Santa Cruz Biotechnology, Inc.), and peroxisome proliferator-activated receptor α (PPARα) antibody (1:100; cat. no. ab8934; Abcam) or stearoyl-coenzyme A desaturase 1 (SCD-1) antibody (1:100; cat. no. 2794; Cell Signaling Technology, Inc.) overnight at 4°C. After being washed with phosphate buffer, the samples were incubated with secondary antibodies, including Alexa Fluor^® 488-linked (green) anti-mouse and Alexa Fluor^® 647-linked (red) anti-rabbit antibodies (1:200; cat. nos. R37114 and A-31573; both from Thermo Fisher Scientific, Inc.). Nuclei were stained using DAPI at room temperature for 10 min. All slides were examined under a fluorescent confocal microscope (magnification, ×40; FV-500; Olympus Corporation). Images were acquired using simultaneous dual-channel scanning.

Total RNA from the liver tissues was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). Next, 5 µg of RNA was used for cDNA synthesis using an M-MLV RTase cDNA Synthesis kit (Takara Bio Inc.). Then, RT-qPCR was performed using a SYBR Green PCR kit (Takara Bio Inc.) with the LightCycler480 sequence detector system (Roche Applied Science), using the following reaction conditions: 95°C for 5 min, 45 cycles of 95°C for 10 sec, 60°C for 20 sec and 70°C for 30 sec. The primer sequences for the RT-qPCR are listed in Table I. The 2^−ΔΔCq method was used to calculate the relative mRNA expression (22), which was normalized against the internal control gene, β-actin. Gene expression levels for each sample were analyzed in duplicate.

Tissue lysates were prepared using RIPA lysis buffer containing 0.1 mmol/l phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich; Merck KGaA). After being quantified using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.), protein extracts (50 µg/lane) were separated via 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore). Membranes were blocked with 5% fat-free milk at room temperature for 60 min, followed by incubation with primary antibodies overnight at 4°C. The membranes were washed and subsequently incubated at room temperature for 2 h in the next day with the secondary antibodies [horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000; cat. no. GTX213110-01) and horseradish peroxidase-conjugated anti-mouse IgG (1:2,000; cat. no. GTX213111-01); both GeneTex, Inc.]. Immunoreactive proteins were visualized using chemiluminescence kits (Thermo Fisher Scientific, Inc.). The protein expression of each sample was analyzed three times and normalized against the internal reference antibody, GAPDH or tubulin.

The following primary antibodies were used: Anti-sterol regulatory element-binding transcription factor 1 (SREBP1c; 1:1,000; cat. no. ab28481; Abcam); anti-CREBH (1:1,000; cat. no. sc-377332; Santa Cruz Biotechnology, Inc); anti-PPARα (1:2,000; cat. no. ab8934; Abcam); anti-SCD-1 (1:1,000; cat. no. 2794; Cell Signaling Technology, Inc.); anti-phospho (p)-nuclear factor (NF)-κB p65 (1:2,000; cat. no. 3033; Cell Signaling Technology, Inc.); anti-tubulin (1:2,000; cat. no. GTX76511; GeneTex, Inc.); and anti-GAPDH (1:2,000; cat. no. GTX100118; GeneTex, Inc.).

Values are expressed as the mean ± standard error of the mean (SEM). All statistical analyses were performed using SPSS software (version 16.0; SPSS Inc.), and statistical significance was calculated using one-way analysis of variance. Post hoc multiple comparison testing among groups was performed using the least significance difference test. All experiments were repeated independently a minimum of three times. P<0.05 was considered to indicate a statistically significant difference.

Following 4 weeks of feeding with MCD or 20 weeks of feeding with HFD, the liver morphology and liver sections in the mice exhibited significant vacuolated hepatocytes, marked inflammatory cell infiltration and severe micro- and macro-vesicular steatosis, which indicated the successful establishment of two NASH mouse models (Fig. 1A and B). The inclusion of DADS (50 and 100 mg) in the MCD or HFD models, respectively, greatly ameliorated the hepatic dysfunction, as determined via H&E staining (Fig. 1B). It is worth noting that supplementation with DADS had no effect on liver histology in mice fed a normal diet, demonstrating its non-hepatotoxic properties (P>0.05; Fig. 1B, E and F). Mice fed with MCD had the lowest liver weight and mice fed with HFD had the heaviest liver weight compared with those mice fed with a normal diet (P<0.01; Fig. 1C). DADS (50 and 100 mg) significantly increased the liver weight in MCD-fed mice and decreased it in HFD-fed mice, respectively (P<0.01; Fig. 1C). The trend of changes in body weight was similar to the liver weight in mice (P<0.05; Fig. 1D). Furthermore, the serum ALT and AST levels were significantly increased in these two NASH models, and these levels were significantly decreased by DADS treatment in a dose-dependent manner (P<0.05; Fig. 1E and F).

Oil Red O staining was performed in liver sections in order to investigate the effect of DADS on lipid deposition and lipid droplet accumulation. As presented in Fig. 2A, the hepatic lipid accumulation was greater in mouse models of NASH than in the mice fed with a normal diet (P<0.05). DADS treatment caused a marked decrease in the number and size of lipid droplets (P<0.01; Fig. 2A). The liver TG and TC contents in MCD-fed mice were elevated by 2.51- and 1.81-fold in MCD-fed mice, respectively, compared with the mice fed a normal diet (P<0.05; Fig. 2B and C). These contents were also increased by 2.42- and 1.72-fold in HFD-fed mice, respectively, when compared with those mice fed a normal diet (P<0.05; Fig. 2B and C). Interestingly, DADS treatment significantly decreased MCD-induced or HFD-induced elevations in the liver TG and TC contents in a dose-dependent manner (P<0.05; Fig. 2B and C). These results clearly indicated the anti-steatotic role of DADS on MCD- or HFD-induced NASH. Since the high-dose DADS group displayed the best hepatoprotective effects, 100 mg/kg DADS was chosen for the following experiments.

To investigate the molecular mechanisms behind DADS in ameliorating hepatic steatosis, the mRNA levels of key lipid metabolism-associated genes were examined. MCD or HFD caused a significant increase in the mRNA levels of SREBP-1c and apolipoprotein A1 (ApoA-I), while they led to a significant decrease in the mRNA levels of CREBH and fibroblast growth factor (FGF)21 (P<0.05; Fig. 3A and B). In addition, DADS treatment reversed the effect of MCD or HFD on the expression of these genes (P<0.05; Fig. 3A and B). As the major regulators of lipid biosynthesis, SREBP-1c and CREBH regulate the expression of several genes associated with TG and fatty acid (FA) synthesis. Thus, the protein levels of SREBP-1c and CREBH were detected and the results revealed that DADS decreased the protein expression levels of SREBP-1c and increased the protein expression levels of CREBH (P<0.05; Fig. 3C and D).

To investigate the role of DADS in lipotoxicity and lipid peroxidation in HFD-fed mice, the expression levels of PPARα and SCD-1 were detected in the present study. As presented in Fig. 4A and B, the expression levels of PPARα or SCD-1 were significantly decreased or increased, respectively, in HFD-fed mice when compared with the normal control group (P<0.05). DADS treatment led to an increase in PPARα expression and a decrease in SCD-1 expression in HFD-fed mice (P<0.05; Fig. 4A and B). These results were further verified via an immunofluorescence assay, demonstrating the expression levels of PPARα and SCD-1 (Fig. 4C). The abnormalities in MDA and SOD levels in the plasma could also be alleviated by DADS treatment in HFD-fed mice (P<0.05; Fig. 4D).

The results of immunohistochemical staining of F4/80-positive cells revealed that the number of macrophage cells had increased in the liver in MCD-fed group, while it had decreased following DADS treatment (P<0.05; Fig. 5A). TNF-α and IL-6 are the two most common inflammatory cytokines. TNF-α is the earliest and most important inflammatory mediator in the inflammatory process; it activates neutrophils and lymphocytes and allows them to pass through the vascular endothelial cells. In addition, TNF-α regulates other metabolic activities in the tissues and promotes the synthesis and release of other cytokines. IL-6 induces B-cell differentiation and produces antibodies. It also induces the activation, proliferation and differentiation of sputum cells, and participates in the immune response of the body. IL-6 is a trigger for inflammatory reactions. Therefore, these two most representative inflammatory factors were chosen to assess the therapeutic effects of DADS in the present study. The plasma levels of IL-6 and TNF-α were higher in the MCD-fed mice than in the mice that were fed a normal diet (P<0.05; Fig. 5B). In addition, DADS treatment in MCD-fed mice effectively reversed these elevations (P<0.05; Fig. 5B). We then detected the expression of the NF-κB pathway, which was revealed to play a pivotal role in the pathogenesis of NASH. As presented in Fig. 5C and D, MCD led to an increase in the protein expression levels of p-NF-κB p65 in the liver of MCD-fed mice, and DADS treatment blocked this increase (P<0.05).