Cyanidin-3-glucoside inhibits glutamate-induced Zn2+ signaling and neuronal cell death in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of reactive oxygen species

Abstract

Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. However, effects of C3G on glutamate-induced [Zn2+]i increase and neuronal cell death remain unknown. We studied the effects of C3G on glutamate-induced [Zn2+]i increase and cell death in cultured rat hippocampal neurons from embryonic day 17 maternal Sprague–Dawley rats using digital imaging methods for Zn2+, Ca2+, reactive oxygen species (ROS), mitochondrial membrane potential and a MTT assay for cell survival. Treatment with glutamate (100µM) for 7min induces reproducible [Zn2+]i increase at 35min interval in cultured rat hippocampal neurons. The intracellular Zn2+-chelator TPEN markedly blocked glutamate-induced [Zn2+]i increase, but the extracellular Zn2+ chelator CaEDTA did not affect glutamate-induced [Zn2+]i increase. C3G inhibited the glutamate-induced [Zn2+]i response in a concentration-dependent manner (IC50 of 14.1±1.1µg/ml). C3G also significantly inhibited glutamate-induced [Ca2+]i increase. Two antioxidants such as Trolox and DTT significantly inhibited the glutamate-induced [Zn2+]i response, but they did not affect the [Ca2+]i responses. C3G blocked glutamate-induced formation of ROS. Trolox and DTT also inhibited the formation of ROS. C3G significantly inhibited glutamate-induced mitochondrial depolarization. However, TPEN, Trolox and DTT did not affect the mitochondrial depolarization. C3G, Trolox and DTT attenuated glutamate-induced neuronal cell death in cultured rat hippocampal neurons, respectively. Taken together, all these results suggest that cyanidin-3-glucoside inhibits glutamate-induced [Zn2+]i increase through a release of Zn2+ from intracellular sources in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of ROS, which is involved in neuroprotection against glutamate-induced cell death.