Apigenin (C15H10O5, MW: 270.24) was purchased from Sigma-Aldrich (St. Louis, MO). Total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Oil Red O kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Trizol reagent and SYBR Green Master Mix were purchased from Vazyme (Nanjing, China). Total protein Extraction kit were purchased from TRANSGEN BIOTECH (Beijing, China). BCA protein kit were purchased from Thermo (USA). Antibiotic-antimycotic solution (10,000 units/ml of penicillin, 10,000 ug/ml of streptomycin) was obtained from Sigma Chemical Co. (St Louis, MO, USA). OA were purchased from Energy Chemical (Shanghai, China). MTT was purchased from Solarbio (Beijng, China). Rabbit anti-NLRP3 (ab-270449), Rabbit anti-NF-κB/p-65 (ab-76302), rabbit anti-GAPDH (ab-181602) were purchased from Abcam (Cambrige, UK).
Ldlr-/- mice were purchased from Jackson Laboratories (Bar Harbor, ME). All experimental procedures were approved by the Animal Care Ethics Committee of the Henan Provincial People’s Hospital, and were conducted in accordance with the American Physiology Institutes of Health. All mice were maintained on a 12:12-h light-dark cycle and have free access to water and food. Mice were randomly divided into three groups (8 mice/group), including control group, HFD group, HFD plus APN (50 mg/kg) (HFD+APN) group. Control group mice were fed with a normal chow diet. HFD group mice were fed with the HFD diet, containing 20% fat and 0.5% cholesterol. HFD+APN group (50mg/kg/day, dispersed in 0.5% sodium carboxymethyl cellulose) was administrated by gavage every day. Control group and HFD group mice were treated with the same volume of 0.5% sodium carboxymethyl cellulose as vehicle group.
Blood metabolite analysis
Blood was obtained by retro-orbital bleeding. Plasma total cholesterol (TC), and triglyceride (TG) were enzymatic methods (Sigma Kits, USA).
HepG2 cells were cultured in DMEM supplemented with 15% FBS and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37℃. Cells were starved in serum-free DMEM for 12 hour followed by incubation with OA for additional 24 hour in the absence or presence of APN (50 μM).
The livers were fixed in 10% formalin and embedded in paraffin, and then cut into 5μm serial sections. Tissue sections were subjected to standard haematoxylin-eosin (H&E) staining for determination of hepatic fat accumulation. OCT-embedded_frozen livers were sectioned at 7μm for Oil Red O staining.
Methylthiazolyl tetrazolium assay for cell viability
Cells were cultured at a density of 4-5 x 104 cells per well in 96-well plates for 24 h. The cells were treated with different concentrations of APN for 24 h. And then cell viability was determined by the MTT reduction assay. Cells were incubated with MTT solution (5 mg/mL) for 4 h at 37 °C. The dark blue formazan crystals that formed in intact cells were solubilized with 150 μL of DMSO, and the absorbance at 490 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA).
Total protein were extracted from cells and liver tissues using RIPA lysis buffer and phenylmethylsulfonyl fluoride (Beyotime, China). The protein concentration was detected by using a BCA protein assay kit. Equal amounts of protein (20μg) were separated using 10 or 12% SDS-PAGE and were transferred onto a polyvinylidene difluoride membrane (PVDF). Next, PVDF membranes were blocked with 5% fat-free milk and incubated with primary antibodies to NLRP3 (Abcam, Inc., CA, USA, Cat. No. ab-270449), NF-κB/p-65 (Abcam, Inc., CA, USA, Cat. No. ab-76302), GAPDH (Abcam, Inc., CA, USA, Cat. No.ab-181602) overnight at 4℃. Subsequently, the membranes were washed and incubated with secondary antibodies at room temperature. The optical density of the bands was visualized by an ECL system (Pierce). GAPDH was used as an endogenous control. Data was normalized to GAPDH levels.
RNA isolation and qPCR analysis
Total RNA was extracted from the frozen tissues or treated HepG2 cells using Trizol reagent. First strand cDNA was synthesized using an RT kit. Amplifications were performed using an opticon continuous fluorescence detection system with SYBR green fluorescence. A single melting curve peak confirmed the presence of a single product. Results were expressed as fold differences relative to GAPDH using the 2-ΔΔCT method. All the primers were synthesized by Sangon Biotech (Shanghai, China) and the sequence are listed in Table 1.
All data are presented as means ± SEM. SPSS 21.0 was used to perform statistical analysis of the data. Statistical differences were calculated with the 2-tailed Student t test when comparing 2 conditions, and ANOVA was used when comparing ＞2 conditions. A value of P <0.05 was considered statistically significant.