Pinoresinol Diglucoside Alleviates oxLDL-Induced Dysfunction in Human Umbilical Vein Endothelial Cells

Abstract

Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. Deposition of oxidized low-density lipoprotein (oxLDL) is one of the initiators and promoters of atherosclerosis. Eucommia lignans were shown to possess antihypertensive effects. This study aimed to investigate the effects of pinoresinol diglucoside (PD), a Eucommia lignan, on oxLDL-induced endothelial dysfunction. HUVECs were treated with oxLDL and/or PD followed by assessing radical oxygen species (ROS), apoptosis, nitrogen oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) activity with specific assays kits, mRNA levels with quantitative real-time polymerase chain reaction (PCR), and protein levels with western blot. PD abolished oxLDL-induced ROS and MDA production, apoptosis, upregulation of lectin-like oxidized LDL recptor-1 (LOX-1), intercellular Adhesion Molecule 1 (ICAM-1), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), and activation of p38MAPK (mitogen-activated protein kinases)/NF-κB signaling. Meanwhile, PD alleviated oxLDL-caused inhibition of SOD activity, eNOS expression, and NO production. These data demonstrated that PD was effective in protecting endothelial cells from oxLDL-caused injuries, which guarantees further investigation on the clinical benefits of PD on cardiovascular diseases.

Figure 1

Pinoresinol diglucoside inhibits oxLDL-induced apoptosis. (a) The apoptosis of HUVECs was analyzed by Annexin V/PI staining followed by flow cytometry. HUVECs were cultured for 24 hrs without treatment, with 100 μg/mL of oxLDL, 1 μmol/L of PD, 100 μg/mL of oxLDL plus 0.1 μmol/L of PD, and 100 μg/mL of oxLDL plus 1 μmol/L of PD. (b) Quantitative analysis of percentage of apoptotic cells of HUVECs with the different treatment listed in (a). Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 2

Pinoresinol diglucoside alleviates oxidative stress caused by oxLDL. (a) Representative pictures of HUVECs stained with DCFH-DA after being cultured without treatment, with 100 μg/mL of oxLDL, 1 μmol/L of PD, and 100 μg/mL of oxLDL plus 1 μmol/L of PD for 24 hrs. (b) The relative ROS levels of HUVECs were analyzed by flow cytometry following DCFH-DA staining after being cultured without treatment, with 40 μg/mL of oxLDL, 1 μmol/L of PD, and 40 μg/mL of oxLDL plus 1 μmol/L of PD for 24 hrs. (c) The lipid peroxidation was assessed by measuring the contents of malondialdehyde in HUVECs treated with oxLDL and/or PD. Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 3

Pinoresinol diglucoside relieves the inhibition of SOD activity by oxLDL. The total SOD enzymatic activity of HUVECs treated with 40 μg/mL oxLDL and/or 1 μmol/L PD was analyzed using a commercially available assay kit. Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 4

Pinoresinol diglucoside alleviates oxLDL-induced inhibition of NO production. (a) The total NO metabolites levels in HUVECs treated with 40 μg/mL oxLDL and/or 1 μmol/L PD were measured using a commercial assay kit. (b) The mRNA levels of eNOS in HUVECs treated with 40 μg/mL oxLDL and/or 1 μmol/L PD were assessed by quantitative real-time PCR. Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 5

Pinoresinol diglucoside inhibits oxLDL-induced increase of LOX-1, ICAM, and NF-κB expression. (a) The mRNA levels of LOX-1, ICAM1, and NF-κB were analyzed using quantitative real-time PCR. (b) The protein levels of LOX-1 and ICAM were evaluated by western blot. The results of quantitative analysis of ICAM (c) and LOX-1 (d) western blot were shown. Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 6

The oxLDL promoted activation of p38MAPK/NF-κB signaling is inhibited by pinoresinol diglucoside. (a) HUVECs were treated with or without 1 μmol/L of PD for 60 min before being treated with 40 mg/mL of oxLDL for 45 min. The levels of p-p38MAPK, p38MAPK, p-NF-κB p65, and NF-κB p65 were analyzed by western blot. The ratio of p-p38MAPK/p38MAPK (b) and p-NF-κB p65/NF-κB p65 (c) was shown. Experiments were performed 3 independent times with 3 replications. ^∗ p < 0.05 compared to control cells; ^# p < 0.05 compared to oxLDL-treated cells.

Figure 7

The proposed working model for pinoresinol diglucoside protecting HUVECs against oxLDL-induced injuries. Refer to test for details.