Materials

The following biochemical kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China) and used to estimate the corresponding biomarkers: TG (cat. no. A110–1-1); total cholesterol (TC) (cat. no. A111–1-1); low-density lipoprotein cholesterol (LDL-C) (cat. no. A113–1-1); high-density lipoprotein cholesterol (HDL-C) (cat. no. A112–1-1); AST (cat. no. C010–2-1); ALT (cat. no. C009–2-1); glucose (GLU) (cat. no. A154–1-1); insulin (cat. no. H203–1-1); malondialdehyde (MDA) (cat. no. A003–1-2); glutathione (GSH) (cat. no. A006–2-1); superoxide dismutase (SOD) activity (cat. no. A001–1-2). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-alpha (TNF-α) (cat. no. ml002859), interleukin-1 beta (IL-1β) (cat. no. ml037361), and interleukin-6 (IL-6) (cat. no. ml064292) were purchased from Enzyme-Linked Biotechnology (Shanghai, China). Reagents and kits used for RNA extraction (cat. no. 9767), reverse transcription (cat. no. RR037A), and real-time polymerase chain reaction (PCR) (cat. no. RR430A) were purchased from Takara Biotechnology (Dalian, China). Primers were purchased from BioSune Biotechnology (Shanghai, China). Akt (cat. no. sc-5298) and p-Akt (S473) (cat. no. sc-293,125) antibodies were purchased from Santa Cruz Biotechnology (Shanghai, China). All other chemicals used in this study were of analytical grade.

Preparation of WEA

WEA was supplied by Huimei Agricultural Science and Technology Co. Ltd. (Hunan, China). Briefly, the extraction process was as follows: fresh artichoke was milled, followed by extraction with hot water (1:5, w/w) at 90 °C for 1.5 hours. The filtered extract was then concentrated by rotary evaporation. Next, a spray-drying method was used to obtain a water extract of the artichoke product. The artichoke extract product was quantified using a high-performance liquid chromatography (HPLC) system (Santa Clara, CA, USA). Briefly, the mobile phase was composed of 0.2% acetonitrile and phosphomolybdic acid in an aqueous solution. The concentration of acetonitrile was gradually increased from 5 to 20% in 10 min and then increased from 20 to 30% in 15 min. Next, the concentration of acetonitrile was gradually decreased from 30 to 5% in 25 min and then maintained at 5% for 30 min. The flow rate was set at 1.0 mL/min and the column temperature was 30 °C. The detection wavelength was set at 330 nm.

Animals

Male Sprague Dawley (SD) rats (8-weeks-old) (? = 40) (certificate No. SCXK (Hu)2012–0002) weighing 220 ± 5 g were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and used for experimental procedures after 7 days of acclimation. All animals were maintained under controlled conditions (22 ± 2 °C, 60 ± 5% humidity, and 12–12 h light–dark cycle), and were given free access to standard laboratory animal feed and water. A normal chow diet (fat contributed 10% calories) and HFD (fat contributed 45% calories) were purchased from Jiangsu Medicine Biological & Pharmaceutical Company (Yangzhou, China). All animal procedures were approved by the Institutional Animal Care and Use Committee at Huaqiao University (Approval No. HQ-ECLA-20200316).

Experimental method

The male SD rats were randomly assigned to five groups (n = 8 per group): chow diet control, HFD, HFD + WEA 0.4 (WEA 0.4 g/kg body weight, BW), HFD + WEA 0.8 (WEA 0.8 g/kg BW), and HFD + WEA 1.6 (WEA 1.6 g/kg BW). Rats were fed a HFD for 8 weeks to induce NAFLD and then treated with WEA for 8 weeks. Rats in the WEA treatment groups received WEA at doses of 0.4, 0.8, and 1.6 g per kg BW daily via oral administration. The chow diet and HFD groups were gavaged with an equal volume of saline solution (0.9% w/v). After intervention, the body weights of the rats were measured, and the animals were fasted overnight. Following this, blood samples were obtained from the hearts by cardiac puncture, and serum samples were preserved at − 80 °C for biochemical analyses. Liver weights were measured, and liver samples isolated after perfusion with PBS were processed for biochemical and histopathological studies, RNA extraction, and western blotting analysis.

Liver index calculation

Liver index, an indicator of hepatic steatosis, was calculated using the following formula: liver index = [liver weight (g)/body weight (g)] × 100 [16].

Determination of biochemical parameters

Serum TG, TC, LDL-C, HDL-C, AST, ALT, GLU, and insulin levels were determined using commercially available kits following the manufacturer’s instructions. Lysis of rat liver tissue was performed as previously described [15], with minor modifications. Briefly, liver tissue (~ 100 mg) was cut into slices and then homogenized with saline solution (0.9% w/v) in an ice bath. The samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected and stored at − 80 °C until analysis. Liver MDA and GSH levels, SOD activity, and TG content were determined using commercial kits according to the manufacturer’s instructions.

Histopathological analysis

Liver histopathological changes were assessed as previously described [17]. Briefly, 200 mg tissue sections were removed from the same region of the right lobe of the liver from animals in each group, fixed in 10% neutral formalin, embedded in paraffin, and then 5 μm sections were cut and stained with hematoxylin and eosin (H&E). Histopathological analysis was performed, and quantitative scoring of the morphological data was evaluated by an expert blinded to the analysis of hepatic steatosis. Hepatic fat accumulation was observed by three examiners independently using a light microscope and scored as follows: steatosis (0, < 5%; 1, 6–33%; 2, 34–66%; 3, > 66%), ballooning (0, 0 foci; 1, < 2 foci; 2, 2–4 foci; 3, > 4 foci, per 200× field), and inflammation (0, 0 foci; 1, < 2 foci; 2, 2–4 foci; 3, > 4 foci, per 200× field).

RNA extraction and real-time PCR analysis

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Real-time PCR was performed as previously described [18]. Briefly, cDNA was synthesized from 1 μg of RNA using a primer mix (oligo-dT/random primers) and Superscript reverse transcriptase (Takara, Dalian, China), according to the manufacturer’s instructions. Real-time PCR was performed in triplicate for each sample, using a 20 μL reaction volume and a SYBR Green universal PCR mix (Takara). The thermal cycling conditions were as follows: 95 °C for 3 min (initial denaturation), 95 °C for 10 s repeated in 40 cycles (denaturation), and 60 °C for 30 s (annealing/extension). The relative expression of the target genes was normalized to the expression of GAPDH, and the fold change was calculated using the 2−ΔΔCt method. The primer sequences used for the real-time PCR are listed in Table 1.

Table 1 Primer Sequences for real-time PCRWestern blotting

Western blotting was performed as previously described [18]. Briefly, liver tissue proteins were extracted using a lysis buffer containing protease or phosphatase inhibitors. Protein concentrations were determined using a BCA protein assay kit (cat. no. 23225, Thermo Scientific, USA). Protein samples were separated using 10% SDS-PAGE and transferred to a PVDF membrane (cat. no. IPVH00010, Millipore, USA). Western blotting was performed using antibodies against Akt (cat. no. sc-5298; Santa Cruz Biotechnology, USA), phospho-Akt (Ser473) (cat. no. sc-514,032; Santa Cruz Biotechnology, USA), and β-actin (cat. no. 4967; Cell Signaling Technology, USA). Specific secondary antibodies labeled with horseradish peroxidase (HRP; 1:5000 dilution; cat. no. ab205719, Abcam, USA) and an enhanced chemiluminescence (ECL) detection kit (cat. no. 32106, Thermo Scientific, USA) were used to detect protein signals. Protein band intensities were quantified using ImageJ software (ver. 1.44p; National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s test using GraphPad Prism software (version 6.0; GraphPad Software, Inc., California, USA). Statistical significance was set at P < 0.05.