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

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Keywords: Ginkgo biloba Atherosclerosis Activated platelets ROS LOX-1 p38MAPK Isobole

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 phosphorylatal p38MAPK (p-p38MAPK) in human coronary artery endothelial cells (HCAECs) ex vivo.

Methods: HCAECs were stressed with activated platelets (2 x [10sup.8]/m1) and followed by ASP (1, 2 or 5 mmol/1), EGb (4,40 or 400 [micro]g/m1) and combinational (1 mmol/1 ASP and 40 [micro]g/m1 EGb) treatments in three groups for 12 h. Superoxide anion in HCAECs was measured with 1-12DCF-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.

[c] 2012 Elsevier GmbH. All rights reserved.

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 (ginkgolicles 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.

Materials and methods

Cell culture

The methodology for culture of HCAECs was described previously (Chen et al., 2010; Mehta et al., 2004). Human coronary artery endothelial cells (HCAECs) for initial batch were purchased from ScienCellTm Research Laboratories (Carlsbad, CA, USA). HCAECs were cultured in endothelial cell medium (ECM) supplemented with endothelial cell growth supplement (ECGS) (Carlsbad, CA, USA) at 37 C in a 5% CO2 and 95% ambient air environment. The purity of endothelial cells was verified based on morphology and immunostaining for factor VIII-related antigen and acetylated LDL, and were 100% negative for smooth muscle cell a-actin expression. Passages 5-7 were used for next experiments in the study.

Preparation and activation of platelets

Platelets were isolated using the method described by Baenziger and Majerus (1974). Briefly, human blood from healthy volunteers was mixed with 3.8% (w/v) sodium citrate solution, which was then centrifuged at 200 x g for 15 min. The upper phase was used as platelet-rich plasma. To obtain washed platelets, one part of acid-citrate-dextrose (2.5% (w/v) trisodium citrate, 1.5% (w/v) citric acid and 2% (w/v) glucose) was mixed with nine parts of platelet-rich plasma. The suspended platelets were centrifuged at 1000 x g for 15 min. The pellet was resuspended with Hepes-Tyrode's buffer (10 mmo1/1 Hepes, 137 mmol/1 NaCl, 2.68 mmol/1 KCI, 0.42 mmol/1NaH2PO4, 1.7 mmol/IMgC12, 11.9 mmol/INaHCO3, 5 mmol/1 glucose). After two washes, the pellet was resuspended in Hepes-Tyrocle's buffer and used as washed platelets. To obtain activated platelets, the washed platelets were treated with 5 pmol/1 ADP (Sigma-Aldrich, St. Louis, MO, USA) for 20 min.

Study design and treatment

In all experiments, HCAECs, not stimulated by activated platelets but directly administered with vehicle (phosphate buffer solution, PBS), were used as endothelial cell group. In other groups, stimulated by activated platelets (2 x [10.sup.8]/m1) for 12 h, HCAECs were administered with vehicle (vehicle control group), ASP (1, 2 or 5 mmo1/1, Sigma-Aldrich, St. Louis), EGb (4, 40 or 4001.t.g/ml, National Institutes for Food and Drug Control, Beijing) and their combination (ASP 1 mmol/1 and EGb 40 [micro]g/m1) for another 12 h to be used for next experiments.

ROS assay of HCAECs

ROS generated in activated platelet stimulated-HCAECs was measured with a fluorescence probe H2DCF-DA (I nvitrogen, Carlsbad, CA, USA), which was converted to the highly fluorescent 2',7'-dichlorofluorescein by ROS (Chen et al., 2010). In brief, treated by drugs for 12h, activated platelet stimulated-HCAECs were incubated with 2[micro]mo1/1 H2DCF-DA for 30 min at 37[degrees]C. The ROS-mediated fluorescence was immediately micrographed under the fluorescent microscope, Olympus Bx60 (Japan), with excitation set at 485 nm and emission at 530 nm. The integrated fluorescence intensity corresponds to the level of ROS. Image-Pro Plus 6.0 software was used to quantify the integrated fluorescence intensity.

Western blot analysis for LOX-l and p-p38 MAPK of HCAECs

The expressions of LOX-1 and p-p38 were examined by Western blot. Western blot analysis was performed using whole cell extracts from HCAECs as previously described (Choi et at., 2005). HCAECs lysates containing equal amounts of total protein were fractionated by electrophoresis on 10% SDS-PAGE gels and transferred onto a nitrocellulose membrane. Nonspecific binding was blocked by soaking the membrane in TBS-T buffer Tris-HCI (pH 7.5), 1.5 mo1/1 NaC1, and 1%Tween 201 containing 5% nonfat dry milk for 3 h. The membrane was incubated with primary antibodies (anti-LOX-1 diluted in 1:250. R&D systems, Minneapolis, MN, USA; anti-p-p38MAPM diluted in 1:300, Santa Cruz, CA, USA; anti-I3-actin 1:3000, Zhongshan Golden-bridge Biotechnology, Beijing) overnight at 4[sup.0]C. After 3 washes with TBS-T, the membrane was then incubated for 1 h at room temperature with HRP-labeled secondary antibodies (Zhongshan Goklenbridge Biotechnology, Beijing). Chemiluminescence intensity of the target protein bands was analyzed by Scan-gel-it software.

Data analysis

The synergism of drug combination was evaluated as follows: interaction index (I)=da/Da +db/Db, where Da and Db are the doses of a and b separately that are isoeffective with the combination of da and d[sub.b]. I <1,= 1 or >1, respectively represent synergism, zero-interaction or antagonism (Berenbaum 1989: Wagner and Ulrich-Merzenich 2009).

All data represent three separately performed experiments. Data are displayed as mean [+ or -] SD. Statistical significance was determined by ANOVA and the SNK-q test. A p value <0.05 or 0.01 is considered as significant.

Results

EGb and ASP inhibited activated platelet-induced oxidative stress

EGb has been reported playing multiple roles to benefit CVD patients, but the underlying mechanism is still elusive. We initially examined the effect of EGb on superoxide generation. After activated platelets stimulation for 12 h, intracellular superoxide anion production was elevated about 3-folds in HCAECs compared to endothelial cell group. But then HCAECs treated with EGb (4, 40 or 400[micro],g/m1) for another 12 h, activated platelet-induced superoxide anion generation in HCAECs was significantly inhibited as compared to the cells treated with vehicle solution, furthermore, whose inhibition was in a dosage-dependent manner (Fig. 1A). ASP possessed antioxidant properties (Chen et al., 2010; El Miclaoui et al., 2002; Mehta et al., 2004). Consistent with the reported, ASP (I, 2 or 5 mmo1/1) markedly reduced activated platelet-induced ROS production after treating HCAECs for another 12 h, and also had a dosage-dependent inhibitory effect on superoxide generation (Fig. 1B).

The inhibition of activated platelet-induced oxidative stress by combinational treatment with EGb and ASP

We then asked whether EGb and ASP could inhibit oxidative stress in a synergistic way. Lower dosage of ASP (1 mmol/l) and EGb (40 [micro],g/m1) were applied on stressed HCAECs. The significantly inhibitory effect was observed on superoxide generation in HCAECs (Fig. 2). lsobole showed synergy between EGb and ASP in inhibiting ROS production for /(10s) was less than I (Fig. 3). So our data argue that ASP and EGb can synergistically regulate superoxide generation.

The inhibition of activated platelet-induced expressions of LOX-1 and p-p38MAPK by EGb or ASP

ox-LDL is a marker of oxidative stress in the plasma and in the atherosclerotic arteries of patients (Mehta 2004). We then examined the expressions of LOX-1, the ox-LDL receptor, and p-p38MAPK in response to activated platelets. Treating HCAECs for 12 h, activated platelets greatly elicited the expressions of LOX-1 and p-p38MAPK in HCAECs compared to their endothelial cell groups. But then treating HCAECs for another 12 h, EGb (4, 40 or 400 [micro],g/m1) as well as ASP (1, 2 or 5 mmol/l) had all inhibitory effect on the expressions of LOX-1 in a concentration-dependent manner (Fig. 4B and E), but not p-p38MAPK (Fig. 4C and F).

Combinational application of EGb and ASP down-regulated the expressions of LOX-1 and p-p38MAPK

We then investigated whether combinational application of EGb and ASP down-regulated the expressions of LOX-I and p-p38MAPK after activated platelet stimulation. ASP, EGb and their combination were applied on activated platelet-induced HCAECs for 12h. In comparison with the individual application, combinational administration markedly reduced LOX-1 expression (Fig. 5B). Surprisingly, the expression of p-p38MAPK was clown-regulated significantly only by the combination of EGb and ASP (Fig. 5C). Isobole showed synergy between EGb and ASP in inhibiting the expressions of LOX-1 (Fig. 6A) and p-p38MAPK (Fig. 6B) in HCAECs induced by activated platelets respectively for /(Lox_) and /(p_p38mApK) were all less than 1.

Discussion

Activated platelets binding to LOX-1 induces the generation of ROS in endothelial cells, which may decrease endothelial function and involve atherogenesis (Cominacini et al., 2003; Kakutani et al., 2000). Exposing HCAECs to activated platelets induced the generation of ROS ex vivo. ASP, an antioxidant, was able to inhibit ROS production (Mehta 2004; Mehta et al., 2004). In this study, our data showed that EGb as well as ASP were able to inhibit ROS production in HCAECs. Furthermore, we observed a synergistic effect when EGb and ASP were applied in combination.

LOX-1 was identified from endothelial cells as the receptor of ox-LDL that might induce endothelial dysfunction triggered by its ligand, ox-LDL (Sawamura et al., 1997). Recent studies has demonstrated that LOX-1 contributes to all stages of atherogenesis by binding to ox-LDL and other stimuli, such as homocysteine, TNF-a and activated platelets. When binding to those ligands. LOX-1 activates downstream signaling pathways including p38MAPK, p44/42-MAPK, PKC, PKB and PTK, which consequently enhance NADPH oxidase activity and NF-03 expression (Navarra et al., 2010; Ogura et al., 2009). In the present study, we demonstrated that both ASP and EGb significantly inhibited LOX-1 expression at functional protein level in HCAECs and showed a dosage-dependent inhibitory effect. Note that the combination of ASP and EGb at lower dosage (1 mmol/l and 40 [micro],g/ml, respectively) exerted a markedly inhibitory effect, which suggested a synergistic effect between EGb and ASP. We speculate that they target different molecules if this combination did not generate a new compound in the experiments (Wagner and Ulrich-Merzenich 2009). Our data also suggest that inhibiting phosphorylation of p38MAPK might correlate with the synergistic effect of EGb and ASP. The underlying mechanism is still under investigation. We consider using pharmacological inhibitors of p38MAPK pathway to decipher this question in our future study.

Mitogen-activated protein kinases, including extracellular signal-regulated protein kinase1/2 (EKR1/2), c-jun N-terminal kinases/stress-activated protein kinases UNK/SAPK) and p38MAPK, play multiple roles in cells growth, proliferation, differentiation and apoptosis. p38MAPK has been shown to contribute to endothelial dysfunction (Aramaki et al., 2008; Chen et al., 2011). When the ligands bind to LOX-1, it induces the activation of p38MAPK and the increase of AP-1 that enhance the generation of inflammatory factors, which may accelerate the progression of atherosclerosis (Wang etal., 2010; Werle et al., 2002). Here we detected the effect of ASP and EGb on the phosphorylation of p38MAPK in HCAECs stimulated with activated platelets. Our data clearly showed that the combination of ASP and EGb remarkably decreased the expression of p-p38 MAPK. However, separate administration of either EGb or ASP showed insignificantly inhibitory effect on the expression of p-p38MAPK. In conclusion, the combination of ASP and EGb may attenuate the development and progression of atherosclerosis by down-regulating the generation of ROS, the expressions of LOX-1 and p-p38 (Fig. 7). These findings indicate that the combination could be administered in preventive and clinical application against atherogenesis.

Abbreviations: EGb, Ginkgo biloba extract; ASP, Aspirin; p-p38MAPK, phospho-rylated p38 mitogen-activated protein kinase; HCAECs, human coronary artery endothelial cells; CVD, cardiovascular diseases; ROS, reactive oxygen species; ox-LDL, oxidized low-density lipoprotein; LOX-1, lectin-like oxidized low-density lipoprotein receptor-I; ECM, endothelial cell medium; ECGS, endothelial cell growth supplement; EKR 1/2, extracellular signal-regulated protein kinase1/2; JNK/SAPK, c-Jun N-terminal kinases/stress-activated protein kinases.

Acknowledgment

This work was supported by Capital Medical Development Foundation, China (No. SF-2007-11-06).

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* Corresponding author. Tel.: +86 010 66927605; fax: +86 010 66928505. E-mail address: kzdw66@sohu.com (1. Wang).

0944-7113/S-see front matter 2012 Elsevier GmbH. All rights reserved. http://clx.cloi.org/10.10I6/j.phymed.2012.10.005

Xianguan Zhu (a), Zhongdong Li (b), Cunxi Li (c), Jing Zhang (a), Zhikang Zou (a), Jianchang Wang (a), *

(a.) Geriatrics Research Center. General Hospital of Air Force, PM, Beijing 100142, China

(b.) Department of Pharmacology, General Hospital of Air Force. PM, Beijing 100142, China

(c.) Department of Medicine, Vanderbilt University Medical Center. Nashville, TN 37232, USA
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Title Annotation:Reactive oxygen species; lectin-type oxidized LDL receptor 1
Author:Zhu, Xianguan; Li, Zhongdong; Li, Cunxi; Jing, Zhang; Zou, Zhikang; Wanga, Jianchang
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jan 15, 2013
Words:2890
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