Both Ldlr−/− mice and NLRP3−/− mice were purchased from Cyagen Laboratories. Ldlr−/− mice on a C57BL/6 J background were crossed with NLRP3−/− mice to generate NLRP3−/−Ldlr−/− mice. Diet used in the study included a standard chow diet and a high-fat diet (20% fat and 0.5% cholesterol) purchased from Jiangsu Xietong Pharmaceutical Bioengineering Co. Ltd. Male Ldlr−/− mice (8 weeks old, n = 8) were randomly divided into four groups, including chow diet (CD) group, 50 mg/kg/day apigenin (APN 50) group, high-fat diet (HFD) group, HFD + 5 mg/kg/day apigenin (HFD + APN 5) group, and HFD + 50 mg/kg/day apigenin (HFD + APN 50) group. These mice were kept on CD or HFD for 8 weeks. Male NLRP3−/−Ldlr−/− mice (8 weeks old, n = 16) were randomly divided into two groups and treated with HFD with or without APN (50 mg/kg/day). APN was administrated by gavage every day. All of the mice were housed in pathogen-free and standard conditions (12:12-h light-dark cycle, a relative humidity of 60%, and room temperature of 22 °C) and had free access to water and food. All animal work was approved by the Animal Care Ethics Committee of the Henan Provincial People’s Hospital and was carried out in accordance with the Animals Act 1986, the National Institutes of Health Laboratory Animal Application Guidelines and the Regulations for the Administration of Affairs Concerning Experimental Animals published by the State Science and Technology Commission of China, and the ARRIVE guidelines.
Blood metabolic analysis
Blood was obtained by retro-orbital bleeding. Plasma total cholesterol (TC) and triglyceride (TG) concentrations were determined by enzymatic methods (Sigma Kits, USA).
Atherosclerotic lesion analysis
The aortas from the origin at the hearts were removed from the mice and fixed with 4% paraformaldehyde. The proximal aortas were embedded in OCT medium, and 4 μm-thick sections were prepared. Then, aortic sinus sections were stained using Oil Red O. The whole aortas were cut longitudinally and stained with Oil Red O. The lesion areas of the aorta and the aortic sinus were analyzed by using ImageJ software.
The livers were fixed in 10% formalin, embedded in paraffin, and then cut into 5 μm serial sections. The tissue sections were subjected to standard hematoxylin-eosin (H&E) staining for the determination of hepatic fat accumulation. OCT-embedded frozen livers were sectioned at 7 μm for Oil Red O staining.
ELISA. IL-1β and IL-18 levels in aortas were measured using commercial ELISA kits (cat# PMLB00C & DY122-05, Minneapolis, USA) in accordance with the manufacturer’s instructions.
Macrophage contents in atherosclerotic lesions were measured using immunohistochemistry staining. Briefly, frozen aortic sinus sections were incubated with 3% H2O2 for 10 min, blocked with 3% BSA (Siama) for 1 h, and incubated with anti-F4/80 antibody (1:200, Abcam, Inc., CA, USA; Cat.No. ab-300421) overnight at 4 °C. After incubation with anti-rabbit lgG for 1 h at room temperature, the slides were developed with 3,3-diaminobenzidine (DAB Quanto Kit, TA-060-QHDX, ThermoFisher) and stained with hematoxylin. Images were recorded uding a light microscope.
Human liver cancer lines, HepG2 (JCRB1054, lot no. 04202017) were purchased from JCRB Cell Bank. HepG2 were cultured in DMEM supplemented with 15% FBS and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The HepG2 cells used in the present study were regularly authenticated via morphologic observation and tested for the absence of mycoplasma contamination. Mycoplasma testing was performed using a MycoProbe® Mycoplasma Detection kit (R & D System, Inc) in accordance with the manufacturer’s protocol. The cells were starved in serum-free DMEM for 12 h followed by the incubation with OA for additional 24 h in the absence or presence of APN (25 and 50 μM).
Cell viability assays
For cell viability, the cells were cultured at a density of 4–5 × 104 cells per well in 96-well plates for 24 h. The cells were treated with different concentrations of APN for 24 h. Then cell viability was determined by the MTT reduction assay. The cells were incubated with MTT solution (5 mg/ml) for 4 h at 37 °C. The dark blue formazan crystals 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 was 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 (20 μg) were separated using 10% or 12% SDS-PAGE and were transferred onto polyvinylidene difluoride membranes (PVDF). Next, the 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), and GAPDH (Abcam, Inc., CA, USA; Cat. No.ab-181602) overnight at 4 °C. 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 mRNA expression using reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from the frozen tissues or treated cells using Trizol reagent (cat. No. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.), as per the manufacturer’s protocol. First strand cDNA was synthesized using an RT kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was then performed using TB Green Premix Ex Taq II (Tli RNaseH Plus; cat. No.RR820A, Takara Bio, Inc.). The thermocycling conditions comprised an initial denaturation at 94 °C for 5 min, 40 cycles of 10 s at 94 °C and 20 s at 60 °C, and a final extension of 30 s at 72 °C. A single melting curve peak confirmed the presence of a single product. GAPDH was used as the reference control gene. Results were expressed as fold differences relative to GAPDH using the 2-ΔΔCq method. All the primers were synthesized by Sangon Biotech (Shanghai, China) and the sequences are listed in Table 1.
All data are presented as means ± SEM. SPSS 21.0 was used to perform a statistical analysis of the data. Statistical differences were assessed with a two-tailed Student’s t test (for comparison of two conditions) and ANOVA(for comparison of more than two conditions). A P value lower than0.05 was considered statistically significant.
All of the animal work was approved by the Animal Care Ethics Committee of the Henan Provincial People’s Hospital and was carried out in accordance with the Animals Act 1986, the National Institutes of Health Laboratory Animal Application Guidelines and the Regulations for the Administration of Affairs Concerning Experimental Animals published by the State Science and Technology Commission of China, and the ARRIVE guidelines.
Consent for publication
All the authors approved the publication.