Male C57BL/6J mice (8-week-old; 20–25 g) were obtained from the Experimental Animals Center of the Chinese Academy of Medical Sciences (Beijing, China), and were bred in a temperature-controlled animal facility with a 12 h light-dark cycle. The mice had free access to water, and were fed with a standard rodent diet ad libitum for 1 week prior to the experiment to enable adaptation to the food and environment. The mice were randomly divided into four groups (n=6 per group): i) Control group, fed an MCD diet supplemented with choline bitartrate (2 g/kg) and DL-methionine (3 g/kg; both ICN, Aurora, OH, USA); ii) MCD group, fed an MCD diet (ICN); iii) MCD+pioglitazone (PIO) group, fed an MCD diet with pioglitazone (50 mg/kg/day; chow; GlaxoSmithKline Co., Ltd., Tianjin, China); iv) MCD+GW9662 group, fed an MCD diet and administered intraperitoneally with 2-chloro-5-nitrobenzani-liden (GW9662; 1 mg/kg; three times per week; Alexis Biochemicals, Lausen, Switzerland). The duration of the experiment lasted up to 8 weeks. Following overnight fasting at the end of the experiments, all animals were sacrificed via exposure to 0.08 ml/l aether anesthesia (1.9%), which was presented on a cotton ball inside a conical tube for 5–10 min. Blood samples were collected from the femoral arteries for biochemical analysis. The livers were weighed and either fixed in 10% formalin for histological analysis, or snap-frozen in lipid nitrogen and stored at −80°C until required. All protocols and procedures were performed in accordance with the the guidelines of the Hebei Committee for Care and Use of Laboratory Animals (Hebei, China) and were approved by the Animal Experimentation Ethics Committee of Hebei Medical University (Shijiazhuang, China).

Prior to euthanasia, 0.5 ml blood was harvested from each mouse via an orbital sinus puncture and the serum was separated by centrifugation at 1,500 × g for 15 min at 4°C. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an the enzymatic kinetic method with an automatic biochemical analyzer (UA2700; Olympus Corporation, Tokyo, Japan), according to the manufacturer's protocol.

Liver tissues were cut into 5 µm-thick sections, paraffin-embedded and fixed for 16 h in 4% phosphate-buffered formalin at 4°C prior to hematoxylin and eosin and Masson's trichromatism staining. The liver sections were subsequently scored for hepatic steatosis, inflammation and fibrosis using a Leica DM 2000 microscope (Leica Microsystems, Inc., Buffalo Grove, IL), as described previously, in accordance with Brunt's criteria (12) and the histological scoring system for NAFLD issued by the Pathology Committee of the Nonalcoholic Steatohepatitis Clinical Research Network (13). The histological scoring system for NAFLD was as follows: Steatosis (0–3), lobular inflammation (0–2), hepatocellular ballooning (0–2), and fibrosis (0–4).

Liver tissue samples were homogenized using 1 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) per 50–100 mg tissue using a glass homogenizer. A total of 0.2 ml chloroform was subsequently added per 1 ml TRIzol reagent and the samples were vigorously vortexed for 15 sec prior to incubation at room temperature for 2 min. Following this, samples were centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase of the sample was transferred to a new tube and supplemented 0.5 ml 100% isopropanol prior to incubation at room temperature for 10 min and centrifugation at 12,000 × g for 10 min at 4°C. Total RNA was isolated from the frozen liver tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Subsequently, 1 µg total RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Thermo Fisher Scientific Inc.), according to the manufacturer's instructions The hepatic mRNA levels of PPARγ, TLR4, myeloid differentiation primary response gene 88 (MyD88), inhibitor of κB kinase-β (IKK-β), nuclear factor-κB (NF-κB); c-Jun N-terminal protein kinase 1 (JNK1), activator protein-1 (AP-1), bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), collagen type I (Col-1), tumor-necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), were determined by RT-qPCR using an ABI PRISM 7500 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was performed at a final reaction volume of 50 µl containing 2 µl cDNA, 5 pmol specific primers, 25 µl SYBR Green reagent (CWBio, Beijing, China) and 23 µl water. Thermal cycling was completed as follows: 95°C for 5 min, followed by 20 cycles of 95°C for 30 sec and 60°C for 30 sec. Expression levels of the target genes were normalized against an endogenous reference gene, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), and were calculated using the 2^−ΔΔCq method (14) using ABI PRISM 7500 Sequence Detection software. The specific primers for PPARγ, TLR4, MyD88, IKK-β, NF-κB, JNK1, AP-1, TNF-α, IL-1β, MCP-1, MIP-1α, BAMBI, TGF-β1, α-SMA, Col-1 and GAPDH were designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA), and the sequences are listed in Table I. All data were obtained using Sequence Detector software.

Total hepatic proteins were extracted using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.), and the protein concentration was measured using the Bradford method (DC protein assay; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Loading buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was added to each lysate, which contained equal quantities of protein (100 µg/well). The lysate was boiled for 5 min and then electrophoresed on 10% SDS-polyacrylamide gels. The proteins were then transferred onto equilibrated polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) by electroblotting. The membranes were incubated, respectively, with the following primary antibodies overnight at 4°C: Mouse polyclonal anti-PPARγ (1:400; sc-122729; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit polyclonal anti-TLR4 (1:1,000; ab13556; Abcam, Cambridge, MA, USA); rabbit monoclonal anti-MyD88 (1:,1000; 4283; Cell Signaling Technology, Inc., Danvers, MA, USA); rabbit polyclonal anti-IKK-β (ab55404; Abcam); and mouse monoclonal anti-NF-κB (1:400; sc-8414; Santa Cruz Biotechnology, Inc.). And the following rabbit polyclonal primary antibodies against: JNK1 (bs-10562R); AP-1 (bs-0670R); TNF-α (bs-2081R); IL-1β (bs-0812R); MCP-1 (bs-0562R); MIP-1α (bs-1045R); BAMBI (bs-12418R); TGF-β1 (bs-0086R); α-SMA (bs-0819R); Col-1 (bs-7158R); and β-actin (bs-0061R; all 1:400; all Biosynthesis Biotechnology, Co., Ltd., Beijing, China). The membranes were then incubated with appropriate peroxidase-conjugated secondary antibodies at room temperature for 1 h. Bands were visualized using enhanced chemiluminescence (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), followed by exposure to an X-ray film (Kodak, Rochester, NY, USA). The protein expression levels were normalized to that of β-actin in the same sample, and the expression levels were quantified by scanning densitometry using Quantity One V4.62 software (Bio-Rad Laboratories, Inc.).

All data are expressed as the mean ± standard deviation. Statistical analyses of the data were performed using one-way analysis of variance (15) or a Kruskal-Wallis H-test, with a least significant difference t-test or Mann-Whitney U test for post-hoc comparison using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The MCD diet induced steatohepatitis and liver fibrosis in the mice, as shown in Fig. 1, which was in accordance with the effects observed in our previous study (16). Compared with the control group, the MCD group showed disordered lobule structure, macrosteatosis in Zone 3, spot or focal hepatocyte necrosis, inflammatory infiltration (Fig. 1A), and portal and persinusoidal fibrosis (Fig. 1B) in the liver sections, which were accompanied with significantly downregulated mRNA and protein expression levels of hepatic PPARγ, which were decreased further by GW9662 administration (P<0.01). Pioglitazone administration markedly ameliorated hepatic steatosis, inflammation (Fig. 1A) and progressive fibrosis (Fig. 1B), which were associated with marked decreases in serum ALT (1) and AST (Fig. 1C and D; P<0.01). However, the opposite effect was observed in the MCD+GW9662 group, which exhibited aggravated steatosis, inflammation and fibrosis (Fig. 1A and B), and further increase in the serum aminotransferase levels (Fig. 1C and D).

The hepatic expression levels of TLR4 and MyD88 were significantly higher in the MCD-fed mice, compared with those in the control mice (P<0.01). Pioglitazone administration significantly upregulated the mRNA expression of PPARγ (Fig. 2). Following the induction of PPARγ by pioglitazone, the mRNA expression levels of TLR4 and MyD88 were downregulated, compared with those in the MCD group. However, the administration of GW9662 led to further upregulation in the mRNA levels of TLR4 and MyD88 (Fig. 2B and C; P<0.01). The same results were observed on examination of the protein expression levels of PPARγ (Fig. 2D), TLR4 (Fig. 2E) and MyD88 (Fig. 2F).

To further investigate the TLR4-mediated inflammatory response and determine the anti-inflammatory effects of pioglitazone, the present study investigated hepatic mRNA and protein expression levels of the MyD88-dependent signaling pathway and downstream genes, IKK-β, NF-κB, JNK1 and AP-1. Compared with the controls, the hepatic mRNA and protein expression levels of IKK-β, NF-κB, JNK1 and AP-1 were markedly increased in the MCD-fed mice (P<0.01), and these levels were significantly reduced by pioglitazone (P<0.01), as shown in Fig. 3. By contrast, the hepatic mRNA and protein expression levels of IKK-β, NF-κB, JNK1 and AP-1 were further upregulated by GW9662 treatment, compared with the MCD group (Fig. 3; P<0.01).

To further examine the mechanism underlying PPARγ-induced amelioration of liver histology, the present study examined the hepatic expression levels of inflammatory cytokines and chemokines. Compared with the control group, the hepatic expression levels of TNF-α, IL-1β, MCP-1 and MIP-1α were upregulated in the MCD diet-fed mice (P<0.01; Fig. 4). These induced expression levels were significantly decreased by pioglitazone treatment. Treatment with GW9662 led to further increases in the expression levels of TNF-α, IL-1β, MCP-1 and MIP-1α, compared with the levels in the mice fed an MCD diet (Fig. 4; P<0.01).

To evaluate the effect and mechanisms of PPARγ-induced fibrotic steatohepatitis, the present study assessed the hepatic expression levels of fibrosis-associated genes. The mice fed an MCD diet showed enhanced hepatic mRNA and protein expression levels of TGF-β1, α-SMA and Col-1 (P<0.01), which were depressed by pioglitazone administration. By contrast, the mRNA and protein expression levels of BAMBI were significantly lower in the MCD-fed mice, compared with the control mice, and were markedly increased following treatment of pioglitazone (both P<0.01). The opposite changes were observed in the GW9662 group, compared with the MCD group (Fig. 5; P<0.01).