Reagent, cell culture, and cell intervention

Ox-LDL was obtained from Yeasen Biotechnology Co., Ltd. (Shanghai, China). PCB2 was purchased from Selleck Co., Ltd. (Shanghai, China). HUVECs were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Ham’s F-12 K medium supplemented with 0.1 mg/mL Heparin, 0.03 mg/mL Endothelial Cell Growth Supplement, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (cat. no. CM-0122, Procell Life Science&Technology Co., Ltd., Wuhan, China). For the ox-LDL-treated cell model, HUVECs were treated with 100 µg/ml of ox-LDL for 24 h. After 24 h of ox-LDL (100 µg/ml) treatment, HUVECs were cultured in the complete medium containing low (L, 0.5 µg/ml), medium (M, 1 µg/ml), or high (H, 2 µg/ml) concentration of PCB2 for another 12 h.

THP-1 human leukemia monocytic cell line, isolated from peripheral blood from an acute monocytic leukemia patient, is often used as a cell model to investigate the functions and activities of monocytes and macrophages along with related regulatory mechanisms [21]. THP-1 cells (Procell Life Science&Technology) were cultured in RPMI-1640 medium containing 10% FBS, 0.05 mM β-mercaptoethanol, and 1% penicillin/streptomycin (cat. no. CM-0233, Procell Life Science&Technology).

Cell counting kit-8 (CCK-8) assay

Cell viability was analyzed by CCK-8 assay using the CCK-8 kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the protocols of the manufacturer. Briefly, cells were seeded into 96-well plates and treated with ox-LDL alone (100 µg/ml) for 24 h or along with different concentrations of PCB2 (0.5, 1, or 2 µg/ml) for an additional 12 h. At the indicated time points after treatment, CCK-8 solution (10 µl per well) was inoculated into 96-well plates and incubated for 1 h. Next, the absorbance was measured at 450 nm.

Cell apoptotic rate detection

Cell apoptotic rate was examined using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology) referring to the manufacturer’s protocols. Briefly, HUVECs were stimulated with ox-LDL (100 µg/ml) for 24 h and then treated with different concentrations of PCB2 (0, 0.5, 1, or 2 µg/ml) for an additional 12 h. HUVECs were collected and resuspended in Annexin V-FITC binding solution. Next, HUVECs were incubated with Annexin V-FITC and propidium iodide staining solution for 15 min at 20–25˚C in a dark environment. Finally, the cell apoptotic rate was measured by flow cytometry. The cell percentage in the Q2 and Q3 regions denoted the apoptotic rate.

Reverse transcription-quantitative PCR (RT-qPCR) assay

HUVECs were stimulated with DMSO or ox-LDL (100 µg/ml) for 24 h. After 24 h of ox-LDL (100 µg/ml) stimulation, HUVECs were treated with or without PCB2 (1 µg/ml) for an additional 12 h. Next, total RNA was isolated from HUVECs using the TRI Reagent Solution (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The synthesis of the cDNA first strand was performed using the above RNA template and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Quantitative PCR reactions were conducted using the SYBR Select Master Mix (Thermo Scientific) and specific primers for LOX-1, MCP-1, VCAM-1, ICAM-1, CXCL1, and CXCL8 on ABI QuantStudio6 Felx Real-Time System (Thermo Scientific). The relative expression levels of genes were measured using the 2−ΔΔCt method. The primer sequences were shown in Table 1.

Table 1 The primer sequences for RT-qPCRWestern blot assay

HUVECs were collected after 24 h of ox-LDL (100 µg/ml) stimulation alone or along with 12 h of PCB2 (1 µg/ml) treatment and then lysed using the RIPA lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Beyotime Biotechnology). After the quantitative analysis using the BCA Protein Assay Kit (Beyotime Biotechnology), protein (35 µg per lane) was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Next, the membranes were sequentially incubated with 5% skimmed milk (1 h, room temperature), the primary antibody against LOX-1 (cat. no. 17958-1-AP, Proteintech, Wuhan, China), nuclear factor kappa-B (NF-κB) p65 (cat. no. 80979-1-RR, Proteintech, Rosemont, IL, USA), IKBα (cat. no. 10268-1-AP, Proteintech), IKBα (phospho S36) (cat. no. ab133462, Abcam, Cambridge, UK), Lamin B (cat. no. 12987-1-AP, Proteintech), GAPDH (cat. no. GB11002, Servicebio, Wuhan, China), or β-actin (cat. no. GB11001, Servicebio) and a secondary antibody conjugated with horseradish peroxidase (1 h, room temperature) (cat. no. G1213, Servicebio, Wuhan, China) were used. Finally, Pierce ECL Western Blotting Substrate (Thermo Scientific) was used to detect the protein signals.

Enzyme linked immunosorbent assay (ELISA)

HUVECs were treated with ox-LDL (100 µg/ml) for 24 h. HUVECs after ox-LDL stimulation were incubated with or without PCB2 (1 µg/ml) for an additional 12 h. Next, cell supernatants were collected. MCP-1 (MultiSciences, Huangzhou, China), VCAM-1 (MultiSciences), ICAM-1 (MultiSciences), CXCL1 (Elabsciences, Wuhan, China) and CXCL8 (MultiSciences) secretion levels in the cell supernatants were measured using corresponding ELISA kits following the instructions of the manufacturer.

Monocyte recruitment assay

HUVECs were treated with ox-LDL (100 µg/ml) for 24 h and stimulated with or without PCB2 (1 µg/ml) for another 12 h. Next, cell supernatants were collected and placed in the lower chambers of Transwell chambers (8-µm pore size, Costar Corning Inc., Corning, NY, USA). THP-1 cells were added to the upper chambers. Five hours later, THP-1 cells in the low chambers were stained with calcein, imaged, and counted.

Bioinformatics analysis

Procyanidin oligomers belong to the procyanidins. To elucidate the downstream molecular targets underlying action of PCB2, we utilized the GSE9647 dataset that analyzed gene expression alterations in response to the stimulation of apple procyanidin oligomers in HUVECs. The GSE9647 dataset was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9647). Gene differential expression analysis in HUVECs treated with apple procyanidin oligomers and DMSO-treated controls based on the GSE9647 dataset was performed using the GEO2R software with the criterion of |log2fold-change| > 0.58 and adjusted p-value < 0.05. GO and KEGG enrichment analysis of these differentially expressed genes was performed using the KOBAS software [22].

Oxidative stress index determination

HUVECs were stimulated with ox-LDL (100 µg/ml) for 24 h and then treated with different concentrations of PCB2 (0, 0.5, 1, or 2 µg/ml) for an additional 12 h.

Malondialdehyde (MDA) level was measured using a Lipid Peroxidation MDA Assay Kit (cat. no. S0131S, Beyotime Biotechnology) following the protocols of the manufacturer. MDA is an indicator of oxidative stress and a natural product of lipid oxidation. The lipid peroxidation MDA assay kit uses a color reaction of MDA and thiobarbituric acid (TBA) to yield the red MDA-TBA adduct.

The reactive oxygen species (ROS) level was examined by ROS Assay Kit (cat. no. S0033S, Beyotime Biotechnology) according to the instructions of the manufacturer. Oxidative stress can induce the accumulation of ROS. The ROS assay kit uses the fluorescent probe DCFH-DA to determine the ROS level. Briefly, DCFH-DA is a fluorescent probe that doesn’t produce fluorescence itself and can penetrate the cell membrane freely. Next, DCFH-DA is hydrolyzed into DCFH by the esterase in the cells. DCFH cannot pass through the cell membrane and is loaded into the cells. Non-fluorescent DCFH is oxidized into fluorescent DCF by ROS in the cells. Thus, the ROS level in the cells can be assessed by the fluorescent intensity of DCF.

Mitochondrial membrane potential (MMP) was determined through a mitochondrial membrane potential assay kit with tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (cat. no. C2006, Beyotime Biotechnology) according to the manufacturer’s protocols. JC-1 is predominantly a monomer that produces green fluorescence when the MMP is low. At high MMP, JC-1 is gathered in the mitochondrial matrix to form JC-1 aggregates, which can yield red fluorescence. Thus, the relative percentage of red/green fluorescence is often used to examine the MMP.

Statistical analysis

Data were analyzed using the GraphPad Prism software (Version 7, La Jolla, CA, USA) and the results were displayed as mean ± standard deviation. The differences between groups were examined by student’s t-test. The differences among groups were analyzed using one-way or two-way Analysis of Variance (ANOVA) along with the turkey test. The differences were defined to be statistically significant at p-value < 0.05.