Materials

Gastrodin (purity > 98%) was purchased from Sigma (St. Louis, MO, USA). Antibodies against GAPDH (Cat. No. ab-9485), Histone H1 (Cat. No. ab-4270), TNF-α (Cat. No.ab-381238)), NF-κB p65 (Cat. No.ab-32536)), SR-B1 (Cat. No. ab-106572), ABCA1 (Cat. No.ab-18180) and CD36 (Cat. No. ab-252923) were purchased form Abcam CO (Cambridge, USA). IL-1β (Cat. No. 12242) were purchased form Affinity Biosciences (OH, USA). IL-18 (Cat. No. A16741) were purchased from ABclonal Technology (Wuhan, China). The secondary antibodies HRP-conjugated anti-mouse lgG and HRP-conjugated anti-rabbit lgG were purchased from Affinity Biosciences (OH, USA). Animals Eight-week-old male Ldlr−/−mice were purchased from Cyagen Laboratories. The mice were fed with a high fat diet (HFD, 20% fat and 0.5% cholesterol). Gastrodin-treated group was administered with Gastrodin daily via oral gavage at doses of 50 mg/kg, and normal saline for control group for 8 weeks. Mice were weighed once every week, and food intake was monitored throughout the experiments. All animals were maintained on a 12:12 h light-dark cycle and have free access to water and food.

All experiments involving mice were approved by the Institutional Animal Care Research Advisory Committee of the National Institute of Biological Science (NIBS) and Animal Care Committee of Zhengzhou University.

Plasma lipid measurements

Blood was obtained by retro-orbital bleeding after overnight fasting. Plasma triglyceride (TG) and total cholesterol (TC) were measured by enzymatic methods according to the manufacturer’s instructions (Sigma kits, USA).

Atherosclerosis lesion analysis

Hearts and proximal aortas were obtained and fixed in 4% paraformaldehyde. And aortas were removed from the iliac artery bifurcation to the origin at the heart. En face lipid accumulation were stained with Oil Red O and atherosclerotic lesions were analyzed. The upper sections of hearts were embedded in OCT medium. The aortic sinus Sects. (4 μm) were prepared and stained with Oil Red O. The lesion of aortas and aortic sinus were analyzed by using Image J software.

Immunohistochemistry

Macrophage contents in atherosclerotic lesion were measured using immunohistochemistry staining. Briefly, aortic sinus sections were incubated with 3% H2O2 for 10 min and blocked with 3% BSA (Sigma) 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 incubating with anti-rabbit lgG for 1 h at room temperature, slides were developed with 3,3′-diaminobenzidine (DAB Quanto Kit, TA-060-QHDX, ThermoFisher) and stained with heamatoxylin. Images were recorded using a light microscope.

Measurement of inflammatory cytokines by ELISA

Aorta was freshly isolated and homogenized in Tris buffer. And then aorta homogenate was centrifuged at 12,000 × g for 5 min. The supernatant was analyzed for protein concentration. The inflammatory cytokines in aorta homogenate were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Abingdon, UK) in accordance with the manufacturer’s instructions.

Cell culture

C57BL/6 mice were intraperitoneally injected with 4% solution of thioglycollate media. Three days after injection, peritoneal macrophages were isolated and cultured in RPIM 1640 with 10% fetal bovine serum with a humidified atmosphere of 5% CO2 at 37 °C.

MTT cell viability assay

Cell metabolic activity was analyzed by the MTT reduction assay as per manufacturer’s protocol after treated with different concentrations of gastrodin for 24 h. Briefly, cells at a density of 4 × 104 cells per well were cultured in 96-well plates for 24 h. Cells were incubated with MTT solution for 4 h at 37 °C. DMSO was used to dissolve the insoluble formazan product. The absorbance values at 570 nm were then read using a microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were repeated at least three times.

Foam cell formation

Macrophages were cultured on chamber slides in 12-well palates. Cells were incubated with 20 μg/ml ox-LDL (Yiyuan Biotech) for 24 h to induce the formation of foam cells.

Intracellular cholesterol measurement The peritoneal macrophage cultured in 12-well palates was incubated with 20 μg/ml ox-LDL for 24 h. Then cells were washed 3 times with PBS, and then cholesterol and triglyceride were determined using commercial kits from Applygen Technologies (Beijing, China).

Western blotting

Total protein and nuclear protein were extracted from cells and murine tissues. The protein concentration was detected by using a BCA protein assay kit. Equal amounts of protein (25 μg) were separated on SDS-PAGE gel and electro-transferred onto a polyvinylidene difluoride membrane (PVDF). Next, PVDF membranes were blocked with 5% fat-free milk for 1 h, and then incubated with primary antibodies overnight at 4 °C. After incubating with secondary antibodies at room temperature, the optical density of the bands was visualized by an ECL system (Pierce). Data was normalized to GAPDH or Histone H1 levels.

Nuclear protein isolation

Cells were washed with PBS and lysed by cell lysis buffer (10 mM Tris, 10 mM KCl, 1.5 mM MgCl2, and 1 mM DTT in 1 × complete protease inhibitor cocktail), and nuclei ware enriched by centrifugation at 4700 g for 10 min at 4 °C. Then, the nuclear proteins were extracted with chromatin digestion buffer (20 mM Tris (pH 7.5), 15 mM NaCl, 60 mM KCl, 1 mM CaCl2, 5 mM MgCls, 30 mM sucrose and 0.4% NP40) after incubating at 4 °C for 30 min. A BCA kit was used to quantify the unclear protein concentration.

RNA isolation and mRNA expression using quantitative reverse transcription PCR (RT-qPCR)

Total RNA from the cells was extracted using Trizol reagent (cat. No. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.), as per the manufacturer’s protocol. First strand cDNA was generated by using an RT kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was then performed using an opticon continuous fluorescence detection system with SYBR Green fluorescence (Molecular Probes, Eugene, USA). The RT-qPCR thermocycling parameters were as follows: initial denaturation at 94 °C for 5 min, followed by 40 cycles for 5 s at 94 °C, for 30 s at 60 °C, and for 30 s at 72 °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 sequence are listed in Table 1.

Table 1 Primer list for quantitative real-time PCRStatistical analysis

The data are presented as means ± standard error of mean (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.