| Objective:The purpose of this study was to screen the neuroprotective activity ingredients from15phenolic compounds isolated from Gastrodia elata Blume by duplicating oxygen-glucose deprivation and reintroduction(OGD/R) model on PC12cell, and then the activities of these screened ingredients would be verified on cortical neurons OGD/R model. Subsequently, we would make preliminarily study on their neuroprotective mechanisms.Methods:1. We chose earle’s solution containing sodium dithionite (Na2S2O4) without glucose to cause PC12cell’s oxygen and glucose deprivation, then give normal medium to cause reintroduction. On this model, the active ingredients from15phenolic compounds isolated from Gastrodia elata Blume could be screened by observing the cells morphology and testing the survival rate(MTT assay).2. We used18～19days-pregnant rats to acquire cortical neurons by primary isolation technology. Observing the effects of active components on the morphology structure of cortical neurons’OGD/R model under invert microscope. The survival rate of neurons was analyzed by MTT assay.3. The viability of lactate dehydrogenase(LDH) in intracellular and extracellular was tested by LDH kits. Then the leak rate of LDH was calculated to evaluate the effect of phenolic compounds from Gastrodia elata Blume on the cortical neurons’OGD/R model.4. The concentrations of intracellular calcium([Ca2+]i) of cortical neurons was measured by fluorescent probe to explore whether the phenolic compounds from Gastrodia elata Blume could inhibit the nerve intracellular calcium overload. Results:1. The phenolic compounds had different effects on PC12cell OGD/R model. Some phenolic compounds could improve the morphological structure of model cells. MTT assay showed that compounds1#、3#、4#、8#、11#and13#could improve the cells survival rate, however, compounds2#、5#、6#and9#could decrease the cells survival rate. Compounds7#、12#and14#at low dose could improve but high dose decrease the cells survival rate. Compounds10#and15#had no effects.2. After damaged by OGD/R, the neuronal body refraction indexes were decreased, synapse retraction or even fracture, eventually shrinkage and loss observed under invert microscope. Neuronal morphology structure was improved in the model group treated by nimodipine and active components of Gastrodia elata Blume.3. Compared with the model group,MTT assay showed that nimodipine2.5、5μg/ml dose and compounds8#、11#、12#increased neuronal survival rate significantly(P<0.05;P<0.01), but the other components showed weak effects.4. The LDH leak rate of cortical neurons increased significantly (P<0.01) after damaged by OGD/R. Compared with the model group, the active compounds of8#、11#、12#from Gastrodia elata Blume reduced the leak rate of LDH in different degrees(P<0.05;P<0.01).5. Compared with the normal group, the [Ca2+]i of model group increased significantly (P<0.01); Compared with the model group, nimodipine2.5μg/ml and5μg/ml dose8#12.5μg/ml dose、11#25μg/ml and50μg/ml dose、12#6.25μg/ml and12.5μg/ml dose could reduce the [Ca2+]i significantly (P<0.01).Conclusions:Phenolic components1#、3#、4#、8#、11#、12#and13#could improve the morphological structure of PC12cells and cortical neurons damaged by OGD/R, increase the survival rate of cells,8#、11#and12#strong effects,could reduce the leak of LDH, Therefore, these components had neuroprotective effects in vitro, and which mechanism was related with inhibiting nerve intracellular calcium overload. |
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