Regents and materials. FLD is a classical Chinese herbal formulae and their ingredients are listed in Table 1. FLD is composed of Radix Codonopsis (15 g), Rhizoma Atractylodis Macrocephalae (9 g), Radix Glycyrrhizae (6 g), Rhizoma Zingiberis (9 g), and Radix Aconiti Lateralis Preparata (9 g). Herbs were purchased from Hubei Tianji Chinese Herbal Sliced Medicine Co., Ltd.. Decoction according to the method of Yang Xin (18). Polyene phosphatidylcholine was purchased from Sanofi Beijing pharmaceuticals Co., Ltd (cat No. 5JD065B). IL-2 (RA20132), IL-6 (RA20607) and TNFα (RA20035) ELISA kit were purchased from Bioswamp (Wuhan, China). Penicillin, streptomycin and antimycotic were obtained from Sigma-Aldrich (USA). TLR4 (ab13867, 1:1000 dilution), NFκB p65 (ab16502, 1:2000 dilution), p-NFκB (ab86299, 1:2000 dilution), MyD88 (ab2064, 1:1000 dilution) and TRAF6 (ab33915, 1:5000 dilution) antibodies were purchased from Abcam (USA). Anti-GAPDH antibody (2118, 1:10000 dilution) was purchased from CST (USA).
Animals and experimental groups. Forty-eight male Wistar rats were obtained from Hubei Provincial Academy of Preventive Medicine (certification No. 42000600013948). Rats were housed in a specific pathogen-free facility (SPF) at Wuhan First Hospital. Following an acclimatization period, rats were randomly divided into the control group (CON), NAFLD model group (MOD), positive control group (PC) and FLD with high dose group (HIG), middle dose group (MID), and low dose group (LOW), n = 8 animals per group. The rats in control group received standard laboratory diet, the model group received high-fat diet (standard laboratory diet + 2% cholesterol + 10% lard + 2.5% vegetable oil), the rats in various FLD group received 5, 10, and 20 g/kg/d FLD treatment, and the rats in positive group received 30 mg/kg/d polyene phosphatidylcholine. Rats were maintained under a 12 h light-dark cycle at 23 ± 2oC. After the experiment, the mice were euthanized by intraperitoneal injection of 200 mg/kg pentobarbital, packaged and eventually burned. All experimental procedures were approved by the Animal Ethics Committee of Wuhan Integrated TCM and Western Medicine Hospital (certificate no. 42000600013948).
Cell culture and treatment. Human liver HL-7702 cell line was obtained from Procell (CL-0111). HL-7702 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (ThermoFisher, Waltham, USA), 100 U/mL penicillin, 100 mg/ml streptomycin, and 100 mg/ml antimycotic. The cells were grown in an incubator with a humidified atmosphere (95% air/5% CO2 v/v) at 37oC for 48 h until 80% confluence, then washed, and exposed for 48 h to FLD at different concentrations (0.5, 1.0, and 1.5 g/ml in serum-free DMEM) or polyene phosphatidylcholine (5 µmol/l). In vitro steatosis was induced by incubating the hepatocytes with 6 mmol/l of a 1:1 v/v mixture of oleic (18:1) and linoleic acid (18:2).
Histological and serological examination. Liver tissue were fixed in 4% paraformaldehyde for 30 min and stained by 0.5% Oil Red O. The stained sections were observed under a microscope (Olympus CX31-LV320, Tokyo, Japan). Serum levels of total cholesterol (TC), triglyceride (TG) and blood glucose (Glu) were detected using an automatic biochemical analyser (model 7180, Tokyo, Japan). The free fatty acid (FFA) level was detected by Nonesterified Free Fatty Acids Assay Kit (A042-1, Nanjing Jiancheng, Nanjing, China).
ELISA assay. IL-2, IL-6 and TNFα levels in the serum or cell culture supernate were evaluated by ELISA kits and the assay was performed in accordance with the manufacturer’s protocols.
Total RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from liver tissue or HL-7702 cells by using TRIzol reagent (Takara Bio Inc., Dalian, China) and assessed using an ultraviolet spectrophotometer and 1% agarose electrophoresis. For each sample, 1 µg RNA was reverse transcribed to obtain first-strand cDNA using the PrimeScript® RT reagent kit with gDNA Eraser (Takara Bio, Inc.) following the manufacturer’s instructions. The reaction mixture (20 µl total volume) contained 10 µl 2 X SYBR Premix Ex Taq™ (Takara Bio, Inc.), 0.5 µl each primer and 0.2 ± 0.02 µg cDNA template. The following three-step qPCR reaction was performed: Pre-denaturation at 95oC for 30 sec, followed by 40 cycles, including denaturation at 95oC for 3 min and annealing at 60oC for 20 sec and elongation at 72oC for 20 sec. The primes used were shown in Table 1. Levels of gene expression were then calculated using the 2−ΔΔCq method. For each group, three samples were measured and three technical replicates of each measurement were obtained.
Western blot. Protein expression levels were analyzed by Western blot analysis and conducted using standard methods with modification. Liver tissue samples were homogenizing in RIPA lysis buffer containing protease inhibitor at 4oC. For in vitro study, cells washed twice with phosphate-buffered saline (PBS) and lysed with RIPA buffer (Beyotime, China) containing protease inhibitor at 4oC. Both cell lysate and tissue lysate were centrifuged at 12000×g for 15 min and supernatants were collected. Protein concentration was detected using a BCA kit (Bio-Swamp Life Science). Equal amounts of protein (30 µg) were separated by 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline (20 mmol/l Tris, 500 mmol/l NaCl and 0.05% Tween 20). Subsequently, the membrane was incubated with primary antibodies. GAPDH was used as an internal reference. Membranes were subsequently washed with Tris-buffered saline and incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (cat. no. W4011; dilution, 1:3,000; Promega Corporation, Madison, WI, USA) for 2 h at room temperature. Immunoreactivity was visualized via a colorimetric reaction using enhanced chemiluminescent substrate buffer (EMD Millipore). Membranes were analyzed using a Gel Doc EZ imager and bans were quantified using Quantity One 5.0 (Bio-Rad Laboratories, Hercules, CA, USA).
Experimental outcomes. We took the effect of FLD on TLR4/MyD88/TRAF6 signaling pathway in rat liver and the HL-7702 cells as the primary experimental outcomes. The inflammation factors and hepatic lipid contents were the secondary experimental outcomes.
Statistical analysis. The statistical differences of the experimental data were evaluated by analysis of variance (ANOVA) using SPSS 19.0 software package. Differences were considered as statistically significant at P < 0.05. All results were expressed as mean ± SD.