Ginkgo biloba extract and Aspirin synergistically attenuate activated platelet-induced ROS production and LOX-1 expression in human coronary artery endothelial cells

Abstract

Aim

In the present study, we investigated whether the therapeutic dosages of Ginkgo biloba extract (EGb) and Aspirin (ASP) might synergistically suppress oxidative stress through regulating the expressions of LOX-1 and phosphorylated p38MAPK (p-p38MAPK) in human coronary artery endothelial cells (HCAECs) ex vivo.

Methods

HCAECs were stressed with activated platelets (2 × 10⁸/ml) and followed by ASP (1, 2 or 5 mmol/l), EGb (4, 40 or 400 μg/ml) and combinational (1 mmol/l ASP and 40 μg/ml EGb) treatments in three groups for 12 h. Superoxide anion in HCAECs was measured with H2DCF-DA probe. The expressions of LOX-1 and p-p38MAPK were examined by Western blot.

Results

After stimulation of activated platelets, intracellular superoxide anion was increased about 3-folds in HCAECs. Both ASP and EGb reduced superoxide anion in HCAECs in a dosage dependent manner. Combinational administration of ASP and EGb showed synergistic effect. By Western blot analysis, we were able to show that activated platelets markedly enhanced the expressions of LOX-1 and p-p38MAPK. Both ASP and EGb only inhibited LOX-1 expression in a concentration-dependent manner, but not p-p38MAPK. As expected, the combination of ASP and EGb markedly reduced not only the expression of LOX-1 but also the phosphorylation of p38MAPK.

Conclusions

Both EGb and ASP attenuate the oxidative stress of HCAECs stimulated by activated platelets ex vivo. It appears that the synergistic effect of EGb and ASP may correlate with the inhibition of ROS production, LOX-1 expression and p38MAPK phosphorylation.

Introduction

Cardiovascular diseases (CVD) have the highest level of morbidity and mortality worldwide, the basic pathology of which is atherosclerosis. Decelerating the progression of atherosclerosis can decrease the mortality of cardiovascular diseases.

In Ross's response-to-injury hypothesis (Ross and Glomset, 1976), repetitive stimulation of various pathogenic mechanical, chemical and immune factors first induced the damage of the integrality of intima. Plasma lipid then penetrated the damaged intima and precipitated between subintima and smooth muscle cells, which resulted in atherosclerosis in the end. Other reports suggested that oxidative stress created reactive oxygen species (ROS) that induced the generation of oxidized low-density lipoprotein (ox-LDL), and then ox-LDL binding to lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) resulted in the activation of NADPH oxidase and p38 mitogen-activated protein kinase (p38MAPK)-mediated signaling pathway. Release of inflammatory factors caused endothelial dysfunction and atherosclerosis (Steinberg, 1983).

Blood platelet cells of patients with atherosclerosis are at a high degree of activation. Some reports revealed that platelet–endothelium interaction mediated by LOX-1 resulted in the generation of ROS (Kakutani et al., 2000) and decreased the expression of nitric oxide (NO) (Cominacini et al., 2003) in endothelial cells. In the present study, we hypothesized that activated platelet–endothelium interaction mediated by LOX-1 may trigger the activation of p38MAPK signaling pathway.

Statins, Aspirin (ASP), traditional Chinese medicines and their extract, vitamin E and C and so on have been reported retarding the atherosclerosis progression. Ginkgo biloba leave extract is an herbal dietary ingredient supplied widely in the United States, which contains flavone glycosides, terpene lactones (ginkgolides A, B, and C, bilobalide) and ginkgolic acids (Chan et al., 2007, Singh et al., 2008, Yang et al., 2011). Ginkgo biloba extract (EGb) may have multiple pharmacological effects, such as scavenging free radicals (Chen et al., 2003, Kumar et al., 2007, Mansour et al., 2011, Ou et al., 2009) and inhibiting the generation of platelet activating factor (PAF) (Koch, 2005, Kudolo et al., 2002). We asked whether EGb and ASP might synergistically regulate the generation of ROS through p38MAPK signaling pathway.