Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells
Introduction
Accumulation of β-amyloid (Aβ) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimer's disease (AD). Numerous in vitro and in vivo studies indicate that Aβ can directly induce neuronal cell death. The mechanism underlying Aβ-induced neurotoxicity is complex, involving several pathways as signaling events associated with apoptosis. For example, nuclear factor-kappaB (NF-kappaB), ERK/p38 MAPK pathways [1], [2], [3] and JNK pathways [4] are shown to be involved in Aβ-mediated neuronal apoptosis.
Aβ-induced neurotoxicity also implicates oxidative stress that is associated with an increase in lipid peroxidation, protein oxidation, alteration of antioxidants systems, and DNA damage as described in the AD brains [5], [6], [7], [8], [9], [10], [11]. Aβ has been substantiated to spontaneously generate free radicals [12], [13], [14] and the residue methionine 35 in the Aβ (1–42) peptides plays a critical role in oxidative stress and its neurotoxic properties [15]. In addition, mitochondrial dysfunction is also suggested to be involved in Aβ-induced oxidative cell death. Effects of the Aβ peptides (25–35 and 1) in NT2 cells with mitochondria (NT2–P+) and without mitochondria (NT2–P0) were investigated by Cardoso et al. [16]. It was found that, cell viability was decreased in NT2–P+ cells treated with Aβ (25–35) but not in NT2–P0 cells treated with Aβ (25–35), suggesting that Aβ peptides require functional mitochondria to induce cell toxicity [16]. Therefore, in addition to the oxidative damage directly induced by Aβ, mitochondrial dysfunction may also contribute to Aβ-induced oxidative cell death. A possible synthesis of these results is that ROS derived directly from Aβ may induce mitochondrial dysfunction that then amplifies the ROS signal.
The standardized Ginkgo biloba extract EGb761 is a well-known antioxidant [17]. It has also been shown to protect against Aβ-induced neurotoxicity. For instance, EGb761 was able to protect and rescue primary hippocampal neurons and PC12 cells against the Aβ toxicity by blockage of Aβ-induced events, such as ROS accumulation, glucose uptake and apoptosis in a concentration-dependent manner [18]. Another in vitro reconstitution study demonstrated that EGb761 inhibited, in a dose-dependent manner, the formation of Aβ-derived diffusible neurotoxic soluble ligands (ADDLs) [19]. Moreover, in an Aβ-expressing neuroblastoma cell line N2a, EGb761 could restore the impaired phosphorylation of cyclic AMP response element-binding protein (CREB) [20] and prevent the activation of NF-kappaB, ERK1/2, and JNK pathways [21]. In addition, EGb761 was also found to be able to attenuate the basal as well as the induced levels of hydrogen peroxide (H2O2)-related ROS in both Aβ-expressing neuroblastoma cell line N2a and Aβ-expressing transgenic Caenorhabditis elegans[22].
Two major groups of constituents of EGb761, flavonoids (24%) and terpenoids (6%), have also been actively investigated for their neuroprotective properties [20], [22]. The ginkgolides, known as potent antagonists of platelet-activating factor (PAF) [23] are unique to the G. biloba tree [24], [25], [26]. The flavonoids, such as quercetin, are involved in the antioxidant properties of EGb761. Despite the beneficial effects for AD dementia, the underlying mechanisms of the action of EGb761 and its single constituents are not clearly known.
Following the above-mentioned points, in this study, we used human neuroblastoma SH-SY5Y cells to test the effects of EGb761 and two of its constituents, quercetin and ginkgolide B, on Aβ-induced cell apoptosis, ROS accumulation, mitochondrial dysfunction, and activation of JNK, ERK1/2 and Akt signaling pathways. The results contribute to the better understanding of the neuroprotective effects of EGb761.
Section snippets
Reagents
Eagle's minimum essential medium (DMEM) was purchased from American Type Culture Collection (ATCC). Fetal bovine serum, penicillin and streptomycin, Donkey Anti-Goat IgG-HRP, Donkey Anti-Mouse IgG-HRP and Goat Anti-Rabbit IgG-HRP were from Invitrogen (Grand Island, NY). EGb761, a G. biloba extract standardized to contain 24% flavonol glycosides, 6% terpene trilactones (of these, 2.9% ginkgolides A, B, C and J and 3.1% bilobalide), and <5 ppm ginkgolic acidesis, was from Dr. Willmar Schwabe
Protective effect of EGb761 and its constituents quercetin and ginkgolide B against Aβ (1–42)-induced cell death in SH-SY5Y cells
As estimated by the MTT assay, cell viability dropped to about 50% after exposure to 100 μg/ml Aβ (1–42) for 24 h (Fig. 1A–C). By contrast, incubation with different concentrations of EGb761 (50–200 μg/ml), or quercetin (1.5–6 μg/ml), or ginkgolide B (5–20 μg/ml) alone for 24 h did not affect cell viability (Fig. 1A–C).
To investigate the effect of EGb761 and two constituents quercetin and ginkgolide B on Aβ (1–42)-induced cell death, we pre-incubated SH-SY5Y cells with various doses of EGb761, or
Discussion
Aβ is identified as the major protein component of amyloid, the defining feature of AD neuropathology. Aβ (1–42) constitutes the majority of the Aβ found in human brain [34]. Therefore, blockage of Aβ (1–42)-induced neurotoxicity may help to prevent the occurrence or progression of AD. In this study, protective effects of EGb761 and its constituents quercetin and ginkgolide B on the cytotoxic action of Aβ (1–42) were tested. Aβ-induced apoptosis in SH-SY5Y cells was evaluated by DNA
Conflict of interest
There are no conflicts of interest.
Acknowledgements
Many thanks for support from the Medical Sciences Research Foundation of Guangdong Province (No. B2007035).
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