Neuroprotective Effects And Mechanisms Of Phenolic Alkaloids From Menispermum Dauricum Rhizome On Oxygen-Glucose Deprivation Injury In Primary Rat Cortical Cultures And Brain Slices

Neuroprotective Effects and Mechanisms of Phenolic Alkaloids from Menispermum dauricum Rhizome on Oxygen-Glucose Deprivation Injury in Primary Rat Cortical Cultures and Brain SlicesAIMWe explored the protective effects of Phenolic alkaloids from Menispermum dauricum rhizome (PAM) on oxygen-glucose deprivation (OGD) induced injury in primary rat cortical cultures and brain slices. To clarify the mechanism of its action, we have systematically investigated the effect of PAM on extracellular glutamate content, intracellular reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and the expression of excitatory amino acid transporters (EAATs).METHODS AND RESULTSPart I Protective effect and mechanisms of PAM on oxygen-glucose deprivation/reoxygenation injury in primary rat cortical culturesOxygen and glucose deprivation in primary rat cortical cultures was used to mimic ischemia-reperfusion injury. Cell survival was determined by measuring 3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazoliumbromide (MTT) reduction, and cell membrane integrity was determined by lactate dehydrogenase (LDH) leakage. At the end of the OGD period, extracellular glutamate content in the culture medium was estimated by high-performance liquid chromatography (HPLC) analysis. ROS generation and MMP in rat cortical cultures was examined by using flow cytometry. PAM (0.1,1,10μg/mL) were selected according to the results of the pilot test. Administration time was selected at the beginning of the OGD and lasted till the end of reoxygenation.The results suggested that PAM protected rat primary cortical cultures against OGD-reoxygenation (OGD-R) induced cytotoxicity. PAM decreased extracellular glutamate content and markedly reduced intracellular ROS generation. In addition, PAM stabilized mitochondrial membrane potential.PartⅡProtective effects and mechanisms of PAM on oxygen-glucose deprivation/reoxygenation injury in rat brain slicesOxygen and glucose deprivation in rat brain slices was used to mimic ischemia-reperfusion injury. PAM was added at the start of OGD and maintained during reoxygenation period. Infarction was assessed by dyeing after 2,3,5-triphenyltetrazolium chloride (TTC) staining at reoxygenation for 4 h. To investigated the possible mechanisms of action of PAM, the expression of EAAT2 (glial glutamate transporter-1, GLT-1) and EAAT3 (excitatory amino acid carrier 1, EAAC1) were observed by western blot.The results suggested that PAM (0.3,3,30μg/mL) protected rat brain slices against OGD-R induced toxicity. OGD-R significantly decreased the expression of GLT-1 and increased the expression of EAAC1 on cortical and hippocampus slices. Although PAM had no significant effect on the expression of EAAC1 in hippocampus slices, but PAM markedly prevented the effects induced by OGD-R on protein expression of GLT-1 and EAAC1 in cortical slices. CONCLUSIONSPAM markedly protected rat primary cortical cultures and brain slices against OGD-R induced toxicity. Our findings revealed that the possible mechanisms of action of PAM protected against brain ischemia injury involves regulation of GLT-1, EAACl expression, extracellular glutamate content, ROS generation and MMP stabilization.