Endothelial cells have a critical physiological role in maintaining normal vessel and organ function. Damage to endothelial cells may cause the alteration of endothelial permeability barrier and vascular tone. Endothelial cell injury induced by oxidative stress is very common. Therefore, the protection of endothelial cells against damage caused by oxidative stress is an important therapeutic strategy.
Ginkgo biloba extract (EGb761) is known to act on cardiac, cerebral, and pulmonary disorders, which is a standardized product of the amount of 24% ginkgo-flavone glycosides and 6% terpenoid (1). One of the mechanisms of the beneficial pharmacologic effects of EGb761 is its antioxidant action. EGb761 has the ability to scavenge free radicals such as superoxide anion, hydroxyl and peroxyl radicals, and nitric oxide (1).
Recent studies have shown that EGb761 had protective effects on myocyte, neuronal cells, and vascular endothelial cells (2–5). EGb761 inhibited monocyte and neutrophil adhesion to bovine cerebral microvascular endothelial cell (6), relaxed porcine basilar artery, and enhanced the TNS-induced relaxation via a NO pathway (7).
To study the effects of EGb761 on endothelial cells induced by oxidative stress, the protective effects of EGb761 on bovine aortic endothelial cells (BAECs) against damage induced by H2O2 were investigated.
MATERIALS AND METHODS
The fura 2-AM, propidium iodide (PI), and sulforhodamine B (SRB) were obtained from Sigma Chemical (St. Louis, MO, U.S.A.). An in situ cell apoptosis detection kit was purchased from Sino-America Biotechnology Company (Shanghai, China). Hydrogen peroxide (H2O2) and other agents were purchased from Beijing Chemical Reagents Company (Beijing, China) at AR grade. EGb761 was provided by the IPSEN Institute (Paris, France) and dissolved in redistilled water.
The BAECs were isolated from bovine artery and cultured in Dulbecco modified Eagle medium containing 15% bovine serum, benzylpenicillin 100 kU/l and streptomycin sulfate 100 mg/l at 37°C in humidified 5% CO2 of air. After confluence, BAECs were passaged every 2–3 days. Cells from passage 6–10 were used for the experiments.
Endothelial cell survival test
Cultured confluent BAECs were washed twice with PBS and incubated with 10 and 100 mg/l EGb761 for 1 h at 37 C, then 0.1 m M H2O2 was added and incubation was continued for 8 h. The survival of BAECs was measured by the method of SRB described by Liu and Jan (8) with slight modification. Briefly, after treatment with EGb761 for 8 h, cells were washed twice with phosphate-buffer saline (PBS) and fixed with 10% trichloroacetic acid in Hanks solution at 4°C for 1. Following fixation, cells were washed with tap water and stained with 0.4% SBR for 30 min. After removal of the SRB solution, the cells were washed three times with 1% acetic acid and air dried. The SRB dye was extracted with 200 μl of 10 m M tris(hydroxymethyl)-aminomethane buffer (pH 10.5) for measuring the absorbance at 565 nm with a spectrophotometer from FLUOstar Galaxy (BMG LabTechnologies GmbH, Offenburg, Germany).
Determination of apoptosis
After the BAECs had been incubated with 100 mg/l EGb761 at 37°C for 1 h, H2O2 was added at the final concentration of 0.1 m M and incubation was continued for 18 h. Apoptosis of BAECs was evaluated by the DNA propidium iodide staining method described by Ding et al. (9) and TUNEL assay according to the apoptosis kit (Sino-American Biotechnology).
Caspase-3-enzyme activity was performed according to Hermann et al. (10). Briefly, BAECs (1 × 106 cells) were lysed in buffer (1% Triton X-100, 0.32 mol/l sucrose, 5 m M ethylenediaminetetraaceticacid (EDTA), 1 m M phenylmethylsulfonyl fluoride, 1 mg/l aprotinin, 1 mg/l leupeptin, 2 m M dithiothreitol, and 10 m M Tris-HCl, pH 8) for 15 min at 4°C followed by centrifugation (20,000 g, 10 min). Capase-3 activity was detected in the resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC). The H2O2 increased caspase-3 activity by 175.5% using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
Measurement of Ca2+i
The Ca2+i was measured as follows (11). After confluence, BAECs were collected and loaded by fura 2-AM in Hanks solution at 37°C in the dark for 30 min. Hanks solution contains NaCL 137 m M, KCL 5 m M, CaCL2 1.3 m M, MgSO4 · 7H2O 0.8 m M, Na2HPO4 0.6 m M, KH2PO4 0.4 m M, NaHCO3 3 m M, glucose 5.6 m M, pH 7.4. The final concentration of fura 2-AM was 5 μM. After loading with fura 2-AM, the cells were centrifuged at 200 g for 5 min twice and resuspended in Hanks solution containing 0.2% bovine serum albumin at 109 cell/l. The fura 2-AM loaded cells were incubated with EGb761 at 37°C for 10 min. Then 0.1 m M H2O2 was added and mixed for 6 min. The intracellular concentration of Ca2+i was measured with the Fluostar Galaxy (Germany) at λex 340 nm and 380 nm, λem 520 nm. The Ca2+i was calculated with Kd of 224 nmol/l by the following formula (12):EQUATION
where R indicates the rate of F340/F380, and Fmin and Fmax indicate the density of fluorescence with EDTA and triton X-100.
Data are expressed as mean ± SD. The statistical analysis was evaluated by Student t test. A value of p < 0.05 indicated statistical significance.
Effect of EGb761 on survival of BAECs
The survival of BAECs was significantly decreased after treatment with 0.1 m M H2O2 for 8 h, and the absorption value of SRB in the control group was much lower than that of the black group (p < 0.01). Findings showed that 10 mg/l and 100 mg/l EGb761 could improve the survival of BAECs and increase the absorption value by 2.9% and 20.6% (p < 0.01), respectively (Table 1). The EGb761 treatment alone did not affect cell viability (not shown).
Effect of EGb761 on apoptosis of BAECs induced by H2O2
In normal cultured BAECs, the apoptotic rate was 7.1 ± 0.7% in current experiment. After treatment with 0.1 m M H2O2 for 18 h, the apoptotic rate increased to 38.1 ± 2%, as tested by the PI staining. The 100 mg/l EGb761 significantly reduced the apoptotic rate induced by H2O2 to 27 ± 1% (Fig. 1).
There were about 5% to 7% of cells stained positive in the normal BAEC group (black group) when tested by the method of TUNEL. BAECs cultured with 0.1 m M H2O2 for 18 h showed an increase in the number of apoptotic cells to 37%–44%. After pretreatment with 10 mg/l and 100 mg/l EGb761, the number of apoptotic cells markedly decreased to 26.5 ± 3.1% and 17.5 ± 1.7%, respectively (Fig. 2).
Influence of EGb761 on caspase-3
The H2O2 increased caspase-3 activity by 175.5 ± 13.8%. Findings showed that 10 mg/l and 100 mg/l EGb761 reduced the H2O2 -induced increase of caspase-3 activity by 7 ± 1.6% and 33.2 ± 5.9%, respectively (Fig. 3).
Effect of EGb761 on the intracellular Ca2+i increase induced by H2O2 in BAECs
The resting Ca2+i in BAECs was 111.14 ± 12.19 nM in Hanks solution (Fig. 4). It was found that 0.1 m M H2O2 increased the Ca2+i by 52.13% (p < 0.001). Findings also showed that 10 mg/l and 100 mg/l EGb761 inhibited the H2O2-induced Ca2+i elevation by 4.5% and 20.6% (p < 0.01), respectively, after BAECs were preincubated with EGb761 for 10 min.
Some studies have already demonstrated the neuroprotective potency of EGb761 (13). But the protective effect of EGb761 on endothelial cells is less known. In the current experiments, the results showed that EGb761 was able to protect endothelial cells against H2O2-induced endothelial cell injury.
Our results showed that H2O2 decreased the survival rate of BAECs and increased the apoptotic rate of BAECs. In light of the lack of specificity of the SRB assay to differentiate between necrosis and apoptosis, we reasoned that not only necrosis but also apoptosis was involved in the H2O2-induced endothelial cell death. Several reports have shown that endothelial permeability increase, lipid peroxidation of cellular membrane, and denaturation of protein and nucleic acids induced by oxygen free radicals were involved in the pathways leading to irreversible injury (14–17). Moreover, lipid peroxidation may alter plasma membrane fluidity, leading to altered transport and enzyme properties. In particular, it has been hypothesized that the alternation in cytosolic free Ca2+i plays an important role in oxidative stress–induced endothelial cell injury (18–21). In addition, oxygen free radicals could induce cell apoptosis in various models (22). It has been shown that the overexpression of the endogenous antioxidant system or the protooncogene bcl-2 prevented apoptosis (23,24). Taken together, oxygen free radicals could result in necrosis and apoptosis through different pathways.
In our results, EGb761 increased the survival of endothelial cells and decreased the apoptotic rate of endothelial cells. Although the mechanisms by which EGb761 prevents endothelial cell damage induced by H2O2 were not fully clarified, the antioxidant property of EGb761 might be one of the protective mechanisms. EGb761 contains 24% ginkgo-flavone glycosides and 6% terpenoid. The flavonoid glycosides have been shown to scavenge hydroxyl radicals (25), superoxide anions (26), and lipid peroxides. The terpene lactones scavenge superoxide anions (27). Heron et al. (28) indicated that suppression of the production of reactive oxygen species reduced lipid peroxidation and membrane viscosity and enhanced membrane fluidity. Therefore, the radical scavenging capacity of EGb761 might play a critical role in inhibiting H2O2-induced endothelial cell injury.
Ca2+ is a widely used second messenger that regulates a variety of biologic processes including gene expression, neurotransmission, cell motility, and cell growth. The elevation of the intracellular level of Ca2+ could activate a lot of cellular proteases such as PKC and Ca2+-calmodulin kinases (29). Moreover, Ca2+ influx was a signal that initiated platelet-activating factor (PAF) synthesis (30), stimulated NOS activity (31), and appeared to serve as a common early signal for the initiation of apoptosis (32,33). Some investigations have indicated that H2O2 caused an increase in Ca2+I due to the influx of Ca2+ from the extracellular medium (34). Therefore, the intracellular Ca2+ elevation might in part be responsible for the necrosis and apoptosis of BAECs induced by H2O2. In the current study, we also found that EGb761 depressed the intracellular free Ca2+i elevation induced by H2O2. Thus, EGb761 might inhibit Ca2+-activated pathways through suppressing the elevation of intracellular Ca2+, which was beneficial in the protection of endothelial cells against oxidative injury.
In addition, our data also showed that EGb761 inhibited caspase-3 activity, which could in part explain the antiapoptotic action of EGb761. It is well known that the cysteine protease family of caspases plays an important role in apoptotic signal transduction. Caspase-3 is one of the best-characterized caspases and has been termed the “central executioner” of apoptosis. Once activated, caspase-3 can cleave numerous proteins involved in cell structure, signaling, and repair, and is essential for DNA fragmentation (35). Our studies confirmed the observation of other investigators that caspase-3 is activated during endothelial cell apoptosis induced by H2O2 (36). Besides inhibiting caspase-3 activity, the effect of EGb761 on other components of the apoptotic pathway, including up- and downstream caspases, remains undetermined. This awaits further investigation.
Mitochondria participate in the initiation of apoptotic programs either by releasing cytochrome c or by opening mitochondrial membrane transitions. These two events are upstream of caspase-3 activation. Mitochondrial dysfunction due to oxidative stress has been reported to be suppressed by EGb (13). Our previous studies also showed that EGb761 protected mitochondria against damage induced by anoxia–reoxygenation (1). These results suggest that protecting mitochondria against reactive oxygen species–induced dysfunction also might be involved in the protective actions of EGb761.
The PAF was a potent phospholipid autocoid implicated in a number of pathophysiologic conditions including inflammation, ischemia–reperfusion, and shock (37). Recent reports have indicated that PAF is a critical factor contributing to oxidant-mediated endothelial dysfunction. H2O2 has been shown to stimulate the production and release of PAF in endothelial cells (31). EGb761 was able to suppress the PAF-induced generation of reactive oxygen species and other actions of PAF due to its PAF antagonistic properties (38,39).
In summary, the results of the current study suggest that EGb761 is able to protect cultured endothelial cells against damage induced by H2O2. These effects of EGb761 are, at least in part, attributable to its antioxidant property and suppression of the intracellular Ca2+i elevation and caspases-3 activity. Certainly, further studies are needed to clarify the mechanisms of action of EGb761.
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