Genistein ameliorates β-amyloid peptide (25–35)-induced hippocampal neuronal apoptosis

https://doi.org/10.1016/j.freeradbiomed.2003.10.018Get rights and content

Abstract

β-Amyloid protein (Aβ), a major component of senile plaques of Alzheimer's disease (AD) brain, causes elevation of the intracellular free Ca2+ level and the production of robust free radicals, both of which contribute greatly to the AD-associated cascade including severe neuronal loss in the hippocampus. Genistein, the most active molecule of soy isoflavones, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of genistein against 25–35-induced apoptosis in cultured hippocampal neurons, as well as the underlying mechanism. 25–35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, is associated with an increase in intracellular free Ca2+ level, the accumulation of reactive oxygen species (ROS), and the activation of caspase-3. All these phenotypes induced by Aβ25–35 are reversed by genistein. Our results further show that at the nanomolar (100 nM) level, genistein protects neurons from Aβ25–35-induced damage largely via the estrogen receptor-mediated pathway, and at the micromolar (40 μM) level, the neuroprotective effect of genistein is mediated mainly by its antioxidative properties. Our data suggest that genistein attenuates neuronal apoptosis induced by Aβ25–35 via various mechanisms.

Introduction

In Alzheimer's disease (AD), the intracellular and extracellular deposits of filamentous proteins are correlated with the neuronal dysfunction eventually leading to dementia [1]. Amyloid β-peptide (Aβ) principally constitutes the extracellular deposit in senile or diffusive plaques and in cerebral vasculatures in AD brain. Substantial evidence indicates that Aβ contributes significantly to the pathological cascades in AD by various mechanisms, such as by generating reactive oxygen species (ROS), elevating intracellular free Ca2+, and other cytotoxic stimuli [2], [3], [4]. Additional indicators for the association of neuronal injury in AD with oxidant stress include the expression of a heme oxgenase type 1 (HO-1) and the increased expression and nuclear localization of the p50 subunit of NF-κB in neurons adjacent to senile plaques and in the dystrophic neuritis contiguous to Aβ amyloid deposits [5], [6], [7].

In AD, the neocortical area, amygdala, hippocampus, and parahippocampus show a high degree of susceptibility to neuronal degeneration [8], [9]. It is documented that Aβ25–35 induces massive Ca2+ influx [10] and free radical production that eventually lead to apoptosis. Caspases play a major role in the late phase of ROS production [12] and caspase-3 activation is required for the cell death resulting from a variety of apoptotic inducers [11].

Genistein (4′,5,7-trihydroxyisoflavone), the most active component of soy isoflavones, showing an affinity to estrogen receptors (ERs) [13], possesses antioxidative activity [14], [15], [16], [17], increases cellular reduced glutathione [18], inhibits protein tyrosine kinase (PTK) [19], [20], and has other physiological functions [21], [22]. The soy isoflavones exert protective action against several chronic diseases such as atherosclerosis [23], the diseases associated with postmenopausal estrogen deficiency, and hormone-dependent breast and prostate cancers [24]. It has been reported that in the nervous system, genistein suppresses Aβ25–35-induced ROS overproduction in isolated rat brain synaptosomes [25]. Linford and Dorsa showed that soy isoflavones attenuate primary neuronal apoptosis by activation of ERs [26], [27]. In this study, we evaluated the protective effect of genistein on cultured hippocampal neurons against Aβ-induced apoptosis and demonstrated that genistein inhibits the elevation of intracellular free Ca2+ and the production of oxidant free radicals caused by Aβ25–35. We also demonstrated that genistein suppresses DNA fragmentation and the activation of caspase-3 induced by Aβ25–35 via the ER-associated mechanism.

Section snippets

Materials

25–35, purchased from Bachem (Torrance, CA, USA), was dissolved in sterile double-distilled water at a concentration of 1 mg/ml as a stock solution and stored at −20°C. The stock solution was aged at 37°C for 2–4 days before use. Genistein was purchased from Sigma Chemical Company (St. Louis, MO, USA) and was dissolved in DMSO and stored at −20°C in dark. Hoechst 333258, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT), trypsin, penicillin, streptomycin, and 2,7-dichlorofluoroescin diacetate

Genistein ameliorates aged Aβ25–35-induced loss of hippocampal neuronal cell viability

Cell viability was expressed as the MTT conversion rate. The effect of genistein at different concentrations on Aβ-induced loss of hippocampal neuronal cell viability is depicted in Fig. 1. Treatment with 25 μg/ml aged Aβ25–35 for 24 h decreased the viability of hippocampal neuronal cells about 43.3% relative to controls. Genistein at concentrations of 0.1 and 40 μM rescued the aged Aβ25–35-induced decrease in viability rate by 7.2 and 13.9%, respectively. The ER antagonist ICI182,780

Discussion

In the present study, we have demonstrated that exposure of hippocampal neurons to genistein prevents aged Aβ25–35-induced apoptosis in several aspects. Genistein did not have a marked effect on cell viability measured by MTT conversion. However, genistein dramatically blocked several aged Aβ25–35-induced apoptotic signals including intracellular Ca2+ level, ROS production, DNA fragmentation, and caspase-3 activity. Genistein, at 40 μM, cause a 63% reduction in ROS production induced by Aβ25–35

Acknowledgements

This work was supported by a grant from the National Natural Science Foundation of China. We thank Qiangliu for technical assistance.

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