Herbal plant extract
AR was purchased from Daqing Fu Rui Bang pharmacy, China. The raw herbs were soaked in distilled water overnight followed by decocting twice in boiling water (60 min each time). The combined aqueous extract was filtered through gauze and then heated until evaporation [14]. Insoluble particles were removed by low-speed centrifugation, the supernatant sterilized by filtration through a 0.22 µm Millipore filter (MILLEX, GP) and stored at 4 °C for use. Main components of AR were analyzed by high-performance liquid chromatography-electrospray ionization/mass spectrometry (LC/MS).
Animals and treatment
Male Kun Ming mice (20–22 g; 8 weeks) were obtained from Harbin Medical University (Daqing, China). Mice were housed in cages with a 12 h light/dark cycle in a temperature-controlled environment. The mice were acclimatized to laboratory conditions for 1 week before the study and then randomly divided into five groups of six: control group fed a standard diet, NAFLD group fed a high fat diet (HFD) (60% kcal fat), low dose group fed HFD + 1.8 g/kg AR given orally (0.1 mL per 10 g body weight), medium dose group (HFD + 4.5 g/kg AR) and high dose group (HFD + 9.0 g/kg AR). HFD feeding was initiated at 8 weeks of age and continued for an additional 8 weeks at which point mice were fasted for 12 h prior to sacrifice with ether. Blood was collected just before sacrifice for serum biochemical analysis. The liver was quickly excised, cleaned completely with ice-cold phosphate-buffered saline (PBS), weighed and preserved in liquid nitrogen until use. All animal studies were approved by the Ethics Committee of Heilongjiang Bayi Agricultural University in accordance with the Chinese guidelines for the care and use of laboratory animals.
Histological Examination
A portion of liver tissue was fixed with 4% paraformaldehyde and embedded in paraffin. For hematoxylin and eosin (H&E) staining [15], rehydration was done in a decreasing ethanol series, and then stained with H&E. Frozen sections were prepared and stained with Oil red O to determine hepatic lipid accumulation. The most severe areas with hepatic inflammation in the representative histology sections were photographed using a microscope. Cells were fixed with 4% paraformaldehyde and stained with freshly diluted Oil Red O solution. Representative photomicrographs were captured using a system incorporated in the microscope.
ELISA assays
To detect liver biochemical indicators, tissue was first placed in pre-cooled PBS and ground into a homogenate, followed by centrifugation to recover the supernatant for analyses. Determination of alanine aminotransferase (ALT) (Catalog No. BPE20168), aspartate aminotransferase (AST) (Catalog No. BPE20184), tumor necrosis factor-α (TNF-α) (Catalog No. BPE20220), interleukin-6 (IL-6) (Catalog No. BPE20012), malondialdehyde (MDA) (Catalog No. BPE20347), glutathione (GSH) (Catalog No. BPE20879), superoxide dismutase (SOD) (Catalog No. BPE20348), triglyceride (TG) (Catalog No. BPE20754) and total cholesterol (TC) (Catalog No. BPE20095) were quantified via ELISA (Shanghai Lengton Bioscience Co.,LTD) (ShangHai, China). All assays were performed according to the manufacturer’s instructions.
Cell culture
Alpha mouse liver 12 (AML12) cells, a hepatocyte cell line from a mouse transgenic for human transforming growth factor α, were kindly provided by Stem Cell Bank, Chinese Academy of Sciences and cultured in the manufacturer's recommended medium composed of DMEM-F12 (gibco, 12400-024) medium containing 10% fetal bovine serum (CLARK, FB25015), 1% streptomycin (100 µg/mL) and penicillin–streptomycin (100 U/mL) (Solarbio, P1400), 1% transferrin (gibco, 41400-045), and 40 ng/mL dexamethasone (SIGMA, D4902-25MG). Cells were incubated with fresh medium at 37 °C in 95% air, 5% CO2, and used in each experiment after 3 days.
Cell viability analysis
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Solarbio, M8180) was used to analyze cell viability. Cells were treated with different concentrations of AR for 20 h, and then 10 µL MTT was added for another 4 h. Culture medium was then totally removed and DMSO added, followed by measurement of absorbance using a microplate reader. All MTT assays were performed at least 3 times for each group. Subsequently, results of the MTT assay were used to select 5 different concentrations of AR to add to cell culture fluid. Real-time cell growth curves were measured through the Real-time label-free cell analysis (RTCA) system (ACEA Biosciences).
Cell treatment
AML12 cells were seeded in 6-well plates. Hepatic steatosis in vitro was induced according to previously established methods [16] in which AML12 cells were treated for 24 h with a mixture of FFA containing a 2:1 ratio of oleate (SIGMA, O1383-5G) and palmitate (SIGMA, P5585-10G), the final concentration of FFA being 1 mM. For the AR supplementation experiment, the herbal extracts were added to the above medium containing 1 mM FFA for 24 h at a high (25 mg/mL), medium (12.5 mg/mL), or low (6.25 mg/mL) concentration.
Protein extraction and Western blotting
Liver or AML12 cell samples were prepared to lysates containing protease inhibitors by adding frozen RIPA buffer before determining protein concentration with the BCA kit (Beyotime, P0010). Protein samples (25 µg) were separated on 10% Bis–Tris SDS-PAGE gel and then transferred onto PVDF membranes. After blocking for 1 hour in a TBST (0.1% Tween 20, pH 7.4) with 5% nonfat milk, the membranes were incubated overnight with the indicated primary antibodies. After dilution with TBST (1:1000 dilution), ACC1 (abcam, ab45174), FAS (CST, 3180S), SREBP1 (NOVUSBIO, NB100-2215), NF-κB (CST, 6956S), p-NF-κB (CST, 3033S), IκBα (CST, 4814S), p-IκBα (CST, 2859S), IKKα (CST, 2682S), ATF6 (abcam, ab203119), PERK (CST, 3192S), IRE1 (abcam,ab37073), monoclonal antibody was used to detect protein expression levels in the samples. Membranes were washed 3x with TBST, followed by a 30-min room temperature incubation with HRP labeled goat antimouse or goat anti rabbit (3:5000; Beyotime, A0208, A0216) in TBST plus 5% milk. Membranes were washed as before and then developed using HaiGene (M2301) detection kit and imaged with AI600. ImageJ software was used to detect protein abundance.
Confocal laser fluorescence imaging
For immunofluorescence [17], cells were plated on coverslips at a density of 0.5 × 105 cells per well followed by treatment, and fixed with 4% paraformaldehyde for 30 min. After incubating in blocking solution (3% Bovine Serum Albumin, 5% Goat serum, 0.5% Triton 100 in PBS, pH 7.4) for 30 min, cells were incubated overnight in 4 ℃ with NF-κB (1:800) antibody. Cells were then washed with PBS 5 times and incubated with FITC conjugated goat anti-mouse IgG (E1216, Santa Cruz Blotechnology) for 1 h. Hochest 33342 (C1026, Beyotime) was used for nuclear staining. Fluorescence images were observed and photographed by using an immunofluorescence microscope (Leica microsystems).
Flow cytometry
To measure the production of ROS, we employed the Reactive Oxygen Species Assay Kit (APPLYGEN, C1300) according to the manufacturer’s instructions. Results were then analyzed by fluorescent microscopy and a flow cytometer (BD Biosciences, USA) [18].
Statistical analysis
All results are expressed as the mean ± SD. Statistical analyses were performed using the Student t-test. For multiple comparisons, one-way analysis of variance (ANOVA) was used. P < 0.05 was considered statistically significant.