Chemicals, Reagents, and Antibodies
EMPA and 3-methyladenine (3-MA) (purity > 98%) were purchased from MedChemExpress (MCE, Shanghai, China). The anti-GAPDH antibody (10494-1-AP), anti- SQSTM1/P62 antibody (18420-1-AP), anti-LC3B antibody (18725-1-AP), anti-Beclin1 antibody (11306-1-AP), anti-IL-6 antibody (66146-1-lg) and anti-TNF-α antibody (60291-1-lg) were purchased from Proteintech Group, Inc. Human oxidized low density lipoprotein (oxLDL) and Oil Red O staining solutions were purchased from Yiyuan Biotechnologies (Guangzhou, China). The CCK-8 kit, cell cycle apoptosis kit and antibody diluent were obtained from Beyotime Biotechnology (Shanghai, China). The KeyFlour 488 Click-iT EdU imaging detection kit was obtained from KEYGEN Biotech (Nanjing, China). The HRP-conjugated secondary antibodies, goat-anti-mouse secondary antibody and goat-anti-rabbit secondary antibody were purchased from Beyotime Biotechnology (Shanghai, China).
Male C57BL/6J and ApoE-/- mice (8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China), and ApoE-/- mice were used to establish animal models of AS. All in vivo experiments followed the ARRIVE guidelines . ApoE -/- mice were fed a high-fat diet (containing 21% fat and 0.15% cholesterol) for 8 weeks to induce AS. After 8 weeks of modeling, different doses of EMPA (1.5 and 3.5 mg/kg/d) were injected intraperitoneally for 8 weeks. After 8 weeks, samples were collected for the follow-up test, and mice were randomly divided into the following four groups (n = 6 mice per group): blank control group (C57BL/6J), model control group (DMSO), low-dose group (1.5 mg/kg/d; EMPA) and high-dose group (3.5 mg/kg/d; EMPA). To avoid the potential confounding effects of dietary differences between different batches, we retained and used a single batch of diets.
The three parts of the aortic arch, thorax and abdomen of C57BL/6J- mice and ApoE -/- mice were collected, fixed for 24 hours, embedded in paraffin and sectioned. The paraffin sections were dewaxed in water, and nuclei were stained with hematoxylin. The sections were then stained with eosin staining solution for 1–3 minutes, dehydrated, mounted with neutral gum and observed under a microscope.
The mouse macrophage (RAW 264.7) and RAW 264.7 cell complete culture medium were purchased from Procell Life Science & Technology Co. Ltd. (Shanghai, China). Human vein endothelial cells (HUVECs) were obtained from Icell Bioscience Inc. (Shanghai, China) and cultured in an endothelial cell culture system containing 93% endothelial cell culture medium, 5% fetal bovine serum (FBS), 1% P/S and 1% endothelial cell culture additive. Human aortic smooth muscle cells (HASMCs)(CL-0517) were obtained from Procell Life Science & Technology Co. Ltd. (Shanghai, China) and cultured in DMEM supplemented with 10% FBS (HyClone) and 1% P/S. All cells were cultured in a humid air incubator containing 5% CO2 at 37°C, and after culturing to 80–90% confluence, they were passaged, frozen or used for experiments.
A GFP-LC3 plasmid (2.5 µg/ml, Genomeditech (Shanghai) Co., Ltd., Shanghai, China) was transfected into RAW 264.7 cells, HASMCs and HUVECs with Lipofetamine®8000 (Invitrogen; Thermo Fisher Science, Inc.) for 24–48 h and then incubated with oxLDL (80 µg/mL) and EMPA (50 µM) at 37°C for 24 h. Finally, autophagosomes were observed under a laser confocal microscope.
Western Blot Analysis
After cell treatment, cells were lysed, and total protein was extracted. The protein concentration was measured by the BCA method. Protein samples were separated by SDS–PAGE, transferred to PVDF membranes and blocked with 5% skim milk for 2 h. Then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature and detected with an enhanced chemiluminescence system. Relative target protein expression levels were calculated using Image Lab software (Bio–Rad Laboratories, Inc.).
Oil Red O Staining
Cells were seeded into a 6-well plate, treated with oxLDL (80 µg/ml) for 24 h, pretreated with different doses of EMPA (30 and 50 µM) in DMEM containing 10% fetal calf serum for 24 h and washed three times with PBS. Subsequently, cells were fixed with paraformaldehyde at 37°C for 30 minutes, stained with Oil Red 0 staining solution at 37°C for 20 minutes and rinsed with 60% isopropanol for 10 seconds. After quickly washing twice with PBS, the accumulation of lipids in macrophages was observed under an inverted microscope (Nikon Ti-S, Japan).
Cell Viability Assay
Cell viability assays were performed using the Cell Counting Kit-8 (CCK8). HASMCs were cultured in 96-well plates (1×104 cells/well) for 24 h. Cells were then treated with different concentrations of EMPA (10,30,50 and 100 µM) for 24 h. After treatment, 10 µl of CCK8 solution was added to each well at 37°C for 1 hour, and the absorbance values at 450 nm were then measured with spectroscopy (µQuant spectrophotometer, Bio-Tek Instruments, Winooski, VT).
Cell Cycle Analysis
HASMCs were cultured with different concentrations of EMPA for 24 h, and cells were then collected and washed with PBS precooled at 4 ℃. The supernatant was removed by centrifugation at 1000 g and incubated overnight in 70% ethanol at 4 ℃. The supernatant was discarded and then resuspended in PBS precooled at 4 ℃ on the second day. After centrifugation, the supernatant was discarded and treated with propidium iodide (50 µg/ml) and ribonuclease A (100 µg/ml) for 30 min. DNA fluorescence was detected by a Beckman Coulter CyAn ADP cell analyzer (Brea, CA). Flow cytometry data were evaluated with ModFit software (ModFit wintrial).
HASMCs were counted and seeding into 24-well plates (2×104 cells/well). When the cell density reached 70%-80%, different concentrations of EMPA (50 and 100 µM) were added followed by incubation for 24 hours. EdU reagent was then added into the medium for a final concentration of 100 µM, and cells were incubated for an additional 24 h. Cells were then fixed, processed according to the instructions of the Fluor 488 EdU cell proliferation detection kit and stained with DAPI. Finally, cells were observed under a fluorescence microscope.
HASMC migration was evaluated by a scratch assay. HASMCs were plated into 6-well plates. A 20 µl Axygen pipet tip was used to make a scratch in the cell monolayer after cells had grown to 90% confluence, and floating cells in each well were washed away twice using PBS. After adding culture medium, cells were treated with different concentrations of EMPA (0, 50 and 100 µM) for 24 hours in a 5% CO2 incubator at 37 ℃. Images of scratches were acquired at the beginning of the experiment and 24 hours later.
All data represent at least three independent experiments and are expressed as mean ± standard deviation. The paired-sample t test was used to compare the differences between the two groups, All statistical analyses were performed using Prism 8 (GraphPad Software, San Diego, USA). *P < 0.05 is considered a statistically significant difference.