Hydroxysafflor Yellow A Protects Neurons From Excitatory Death Through Inhibition Of NMDA Receptors

ObjectiveIt has been known that excessive glutamate release and overactivation of NMDA receptors (NMDARs) cause excitatory neuronal damage in cerebral ischemia. Hydroxysafflor yellow A (HSYA), a water soluble compound extracted from Carthamus tinctorius L., was reported to be neuroprotective in ischemic animal model and in cultured cortical neurons. However, the underlying mechanism remains unclear. In present study we used whole-cell voltage-clamp recording, calcium imaging, cell viability test and western blot to examine neuronal protective effect of HSYA from the level of tissue, cell and molecular, and designed to provide a basis for clinical treatment of cerebral ischemia.Materials and Methodes1. The postnatal 4-6-week C57BL/6 mice were adopted and prepared acute hippcampal brain slices; Using whole-cell voltage-clamp recording, electric stimulate the SC-CA1 afferent nerve of hippacampal slices, pharmacology separate and record NMDAR-mediated EPSC in hippacapal CA1 PCs; recording the membrane deporlarization current caused by OGD in hippacapal CA1 PCs; recording AMAPR and NMDAR-mediated ischemia long-term potentiation which caused by OGD in hippacapal CA1 PCs; observing the effect of HSYA on them, respectively.2. Use primary cultured hippocampal CA1 neurons on day 12-16, detecte the cacium concentration of intraclluar in defferent conditions with Fluo-4 and the effect of HSYA on it.3. Use primary cultured hippocampal CA1 neurons on day 12-16, treat it with different conditions and stain with Hoechst. Then, observe the neuron apoptosis, necrosis and the protection effect of HSYA on them.4. Use western blot to observe the change of caspase-3 which is a important element in the progress of NMD A caused neuron apoptosis, meanwhile, observe the effect of HSYA on it.ResultWe found that HSYA inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDAR EPSCs), without affecting AMPA and GABAA receptors mediated currents. The inhibition of NMDAR EPSCs was showed a dose-dependent manner. HSYA also reduced the presynaptic glutamate transmitter release. Further more, HSYA reduced NMDAR-mediated membrane depolarization current evoked by oxygen-glucose-deprivation (OGD), and suppressed OGD-induced NMDAR-dependent long-term synaptic potentiation which was believed causing the severe reperfusion damage in the brain after ischemia. In addition, HSYA inhibited NMDA induced [Ca2+]i increase in the neurons, and reduced the incidence rate of apoptotic and necrotic cell death. Our data revealed that HSYA exerts the neuronal protective effect in different pathological condition through inhibiting NMDAR overactivation. This novel finding indicates that HSYA is a promising candidate in the treatment of brain ischemia.ConclutionHSYA neuroprotection is through inhibit NMDAR and it is feasible in cilinical treatment of cerebral ischemia.