Neuroprotective effects of Quercitrin on B-Amyloid-induced Neurotoxicity in rat Hippocampal Neuronal cultures

Abstract

The main objectives of the present study were to (1) characterize an in vitro model of Aβ-induced neurodegeneration in rat primary hippocampal neuronal cultures; (2) investigate the neuroprotective effects of quercitrin on Aβ-induced neurotoxicity in cultured neurons; and (3) elucidate the possible mechanisms of quercitrin that mediate neuroprotection in cultured neurons. In this connection, immature hippocampal neurons exposed to Aβ25-35 in primary culture were employed as the in vitro model of neurodegeneration. Neuronal cell injury and death were measured by using MTT reduction and LDH release assays. The production of ROS and lipid peroxidation levels, intracellular GSH levels, SOD and GPx activities were used to investigate the contribution of prooxidant and antioxidant mechanisms in Aβ25-35 -induced neurotoxicity. Caspase-3 activity, Bcl-2 and Bax protein contents, and cytochrome c release were used to investigate the contribution of apoptotic cell death. Quercitrin, a glycosidic member of phytoestrogens, was tested for its potential neuroprotective effects with this in vitro neurodegenerative model. Estrogen receptor antagonist, MEK and PI3K inhibitors, were also used to investigate the probable routes of its neuroprotective action. The present study showed that exposure to Aβ25-35 induced a concentration- and exposure time-dependent injury and death to cultured hippocampal neurons. Neither exposure to quercitrin nor exposure to 17β-estradiol at a concentration range of 0.001-100 μM for 72 hr induced cytotoxic effects to cultured hippocampal neurons. Instead, both compounds displayed a certain degree of neuroprotective effect as considered from the following evidence. Co-exposure but not pre-exposure with quercitrin or 17β-estradiol (50-100 μM) attenuated Aβ25-35 -induced neuronal injury and prooxidative status. Regarding antioxidative status, co-exposure with 17β-estradiol but not quercitrin significantly improved Aβ25-35 -induced reduction in cellular GSH content whereas co-exposure with quercitrin but not 17β-estradiol significantly improved Aβ25-35 -induced reduction in GPx activity. However, co-exposure to both compounds displayed no effects on SOD activity Neuroprotective effects of quercitrin and 17β-estradiol might involve inhibition of apoptotic cell death mechanisms because both compounds could attenuate Aβ25-35 -induced increases in cytochrome c release, caspase-3 activity and Bax protein expression. Moreover, both quercitrin and 17β-estradiol could counteract Aβ25-35 -induced decrease in Bcl-2 expression. However, neuroprotective effects of quercitrin and 17β-estradiol might not be related to ER-mediated mechanisms because a specific ER receptor antagonist failed to counteract their protective effects. In addition, their neuroprotection might not mediate through MAPK or PI3K signaling pathway because MEK inhibitor and PI3K inhibitor did not prevent quercitrin- and 17β-estradiol-induced neuroprotection. In conclusion, we firstly illustrate the potential neuroprotective effect of quercitrin on a neuronal cell culture model of neurodegeneration. This beneficial effect may involve inhibition of ongoing processes of apoptotic cell death. The underlying neuroprotective mechanisms of quercitrin may not relate to estrogen receptor-mediated mechanisms but, apparently, may involve with its antioxidant and free radical scavenging properties. Nevertheless, the exact mechanisms of quercitrin-induced neuroprotection and its potential therapeutic applications in neurondegenerative disorders are still unclear and warrant further investigation.