Neuroprotective effects of flavonoids on hypoxia-, glutamate-, and oxidative stress–induced retinal ganglion cell death

PURPOSE: This study was conducted to investigate the effect of flavonoids, a major family of antioxidants contained in foods, on retinal ganglion cell (RGC) death induced by hypoxia, excessive glutamate levels, and oxidative stress. Moreover, to assess the structure-activity relationships of flavonoids, three types of flavonoids with different numbers of hydroxyl groups and varieties of sugar chains were studied. METHODS: Three kinds of flavonoids—nicotiflorin, rutin, and quercitrin—were used. The death of neonatal rat purified RGCs was induced by hypoxic conditions (5% O(2), 5% CO(2), 37 °C) for 12 h, 25 µM glutamate over three days, or oxidative stress by depleting antioxidants from the medium for 24 h. RGC survival rates were calculated under each condition and compared with vehicle cultures. Modification of cell death signaling after stress-induced apoptosis and necrosis by flavonoids was assessed using caspase-3 and calpain immunoreactivity assays. RESULTS: Under hypoxic and glutamate stress, both nicotiflorin and rutin significantly increased the RGC survival rate at 1 nM or higher, while quercitrin increased it at 100 nM or higher. Under oxidative stress, nicotiflorin, rutin, and quercitrin also significantly increased the RGC survival rate at 1 nM, 0.1 nM, and 100 nM or higher, respectively. Rutin significantly inhibited the induction of caspase-3 under both hypoxia and excessive glutamate stress, as well as blocking the induction of calpain during oxidative stress. CONCLUSIONS: Nicotiflorin and rutin showed neuroprotective effects on hypoxia-, glutamate- or oxidative stress–induced RGC death at concentrations of 1 nM or higher. The presence of a specific sugar side chain (rutinoside) may enhance neuroprotective activity.