The J774A.1 murine macrophage cell line was purchased from the China Center for Type Culture Collection (Wuhan, China). The cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Co., Waltham, MA, USA), supplemented with 10% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd., Hangzhou, China), at 37°C in an atmosphere containing 5% CO₂. Ox-LDL was purchased from Yiyuan Biotechnology Co., Ltd (Guangzhou, China). Simvastatin was purchased from Tokyo Chemical Industry Co., Ltd (Shanghai, China). Oil Red O dye and Total Cholesterol Detection kit were purchased from Nanjing Jiancheng Biotechnology Institute Co., Ltd (Nanjing, China). Lipofectamine^® 2000 was obtained from Life Technologies (Grand Island, NY, USA). Rabbit anti-microtubule-associated protein 1 light chain 3(LC3) and rabbit anti-Beclin1 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit Immunoglobulin G was purchased from Beijing ZSGB-Biotechnology, Co. (Beijing, China). The Bicinchoninic Acid (BCL) Protein Assay kit was obtained from Beijing YPH-Bio Company (Beijing, China), and the Enhanced Chemiluminescence (ECL) Detection system was purchased from Pierce Biotechnology, Inc. (Rockville, MD, USA).

The J774A.1 cells were seeded into 6-well plates (5×10⁵ cells per well) and grown to 50–60% confluency. A total of 0, 25, 50 or 100 μg/ml ox-LDL was added to the cell supernatants, and the cells were incubated for 24 h. The cells were washed with phosphate-buffered saline and fixed with formaldehyde. The fixed cells were stained with oil Red O dye, for 30 min at 37°C. The status of intracellular lipid accumulation was detected by microscopy (Olympus CKX41; Olympus, Tokyo, Japan), in order to assess the formation of macrophage foam cells.

The J774A.1 cells were plated in 6-well plates and grown to 50–60% confluency. Following stimulation with ox-LDL (or/and simvastatin at 0.5 and 1.0 μM), the cells were harvested and lysed with RIPA lysis buffer on ice. The cell lysates were centrifuged at 13,000 g for 30 min at 4°C. The protein concentrations of the supernatants were determined using the BCA kit. Equal quantities of protein (30 μg) from the homogenized samples were separated by 15% SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). The membranes were blocked with tris-buffered saline-Tween^® (TBST), containing 5% non-fat milk powder, for 2 h at room temperature. The membranes were then incubated with polyclonal rabbit anti-microtubule-associated protein 1 light chain 3(LC3; 1:1,000) and polyclonal rabbit anti-Beclin1 (1:1,000) primary antibodies at 4°C overnight, washed with TBST, and incubated for 1 h with HRP-conjugated goat anti-rabbit antibody (1:10,000 dilution), at room temperature. β-actin was used as a control. Immunoreactive bands were visualized using the ECL detection system and images were captured using an automatic digital gel image analysis system (4500SF; Tanon Science & Technology Co., Ltd., Shanghai, China).

Green fluorescent protein (GFP)-LC3 plasmids were previously prepared in our laboratory (15) and were transfected into the J774A.1 cells, using Lipofectamine^® 2000 according to the manufacturer’s instructions. The cells were treated with ox-LDL (and/or simvastatin) 24 h post-transfection, and incubated for a further 24 h. All GFP-LC3 plasmid-transfected cells with or without ox-LDL (and/or simvastatin) treatment were then fixed using Immunol Staining Fix Solution (Beyotime Institute of Biotechnology, Haimen, China) and GFP-LC3 fluorescence was detected by confocal laser scanning microscopy (Leica TCS SP5; Leica, Mannheim, Germany).

Following stimulation with ox-LDL (or/and simvastatin), the cells were harvested and sonicated on ice for a total of 5 sec, with 15 sec pauses between bursts using a sonicator (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). Following sonication for 10 min, the cells were centrifuged at 8,000 g for 10 min at 4°C. The supernatants were then used for measurement of intracellular cholesterol. Total cholesterol levels in the cells were determined by the CHOD-PAP method, using the Total Cholesterol Detection kit (Nanjing Jiancheng Biotechnology Institute Co., Ltd.), according to the manufacturer’s instructions.

The data are represented as the mean ± standard deviation, and were statistically analyzed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using Student’s t-test to compare the differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the possible roles of simvastatin on ox-LDL-induced macrophage autophagy and lipid accumulation, a macrophage foam cell model was generated. Groups A, B, C and D of the J774A.1 cells were treated with 0, 25, 50 and 100 μg/ml ox-LDL respectively, for 24 h, and the status of lipid accumulation was determined by oil red O staining. No obvious lipid accumulation was observed in the control group (Fig. 1A). Conversely, lipid accumulation was observed in all of the groups treated with ox-LDL (Fig. 1B–D). Lipid accumulation was more visible in groups C and D, as compared with group B, implying the existence of a dose-dependent effect. All of the cells treated with ox-LDL had similar morphological characteristics to foam cells, suggesting the successful establishment of a macrophage foam cell model. Thereafter, 50 μg/ml ox-LDL was selected to treat cells in further experiments.

Ox-LDLs have previously been shown to trigger autophagy in endothelial cells (8,9,16). The present study examined whether ox-LDL induced autophagy in macrophages. Ox-LDL (50 μg/ml) induced autophagy in the J774A.1 macrophage cell line by converting LC3 I to LC3 II, which is a well-known autophagy marker. The transformation of LC3 I to LC3 II increased gradually, in a time-dependent manner following treatment with 50 μg/ml ox-LDL, as compared with the control group (Fig. 2).

Simvastatin was previously shown to enhance autophagy in osteoblastic cells and coronary arterial myocytes (14,17). The present study determined whether simvastatin elevated ox-LDL-induced autophagy in macrophages. Concordant with the results presented in Fig. 2, the protein expression levels of the autophagy marker LC3 II were induced in group 2, following stimulation with 50 μg/ml ox-LDL for 24 h (Fig. 3). Notably, treatment with simvastatin markedly promoted the conversion of LC3 I to LC3 II (group 4 and 5, as compared with group 2). Treatment with 1.0 μM simvastatin enhanced ox-LDL-induced LC3 II expression more than treatment with 0.5 μM simvastatin, suggesting that simvastatin affects ox-LDL-induced autophagy in a dose-dependent manner (Fig. 3). Simvastatin (0.5 and 1.0 μM) also increased the expression levels of another ox-LDL-induced autophagy marker, Beclin1 (Fig. 3).

The GFP-LC3 plasmid was used to detect autophagosome formation. Stimulation with ox-LDL led to the redistribution of GFP-LC3, from diffusion distribution to puncta in the J774A.1 cells, as determined by confocal laser scanning microscopy (Fig. 4A and B). Treatment with simvastatin (0.5 and 1.0 μM) alone resulted in the formation of GFP-LC3 fluorescent puncta in the J774A.1 cells (Fig. 4C and D). Furthermore, treatment with simvastatin markedly promoted the ox-LDL-induced formation of GFP-LC3 puncta (Fig. 4E and F). Treatment with 1.0 μM simvastatin enhanced the formation of ox-LDL-induced GFP-LC3 puncta more so than 0.5 μM simvastatin (Fig. 4E and F). These results indicate that simvastatin may enhance ox-LDL-induced macrophage autophagy.

As a HMG-CoA reductase inhibitor, simvastatin is a well-known drug used to ameliorate atherosclerotic diseases, by lowering plasma cholesterol. The present study determined whether simvastatin may inhibit ox-LDL-induced cholesterol accumulation in J774A.1 cells. The number of lipid droplets, the existing form of cholesterol in cells, decreased following incubation with simvastatin (Fig. 5A–C). The CHOD-PAP assay indicated that treatment with 0.5 and 1.0 μM simvastatin led to a significant reduction in the intracellular total cholesterol levels, from 2.774±0.059 mmol/l, to 2.53±0.058 mmol/l and 2.27±0.085 mmol/l, respectively (Fig. 5D). These results suggest that simvastatin may inhibit ox-LDL-induced cholesterol accumulation in macrophages.