Neuroprotective Effects Of Asiaticoside Against NMDA–induced Excitotoxicity And The Underlying Mechanism

OBJECTIVEGlutamate is an important excitatory neurotransmitter in the central nervous system(CNS). The accumulation of glutamate can excessively activate the N-methyl-D-aspartate(NMDA) receptors and cause excitotoxicity. NMDA subtype of glutamate receptor(NMDAR) mediates glutamate excitotoxicity due to its high calcium permeability.Calcium combines with calmodulin (CaM) within the cell, causing a cascade of reactions,such as reactive oxygen species (ROS) increased release, ultimately leading to apoptosisor necrosis. Excitotoxicity plays a key role in spinal cord injury, traumatic brain injury,stroke, and neurodegenerative diseases of the CNS. Asiaticoside (AS) is a triterpenoidproduct extracted from the plant Centella asiatica Umbelliferae, of the white crystalpowder. Treatment with AS has been found to anti-inflammatory, analgesic, liver-protect,anti-tumor, anti-oxidation, promote wound healing, nerve function recovering and othertreatments.However, little is known about the neuroprotective effects of AS onglutamate-induced excitotoxicity. Our study aimed to evaluate the neuroprotective effectsof AS against the excitotoxicity, as well as the possible underlying mechanisms.METHODS Primary cultured mouse cortical neuronsUnder sterile conditions, disassociated cortex tissues from embryonic14-15day-old(E14-15) mice were incubated with0.125%trypsin for10-15min at37oC. Then the corticeswere washed in DMEM supplemented with20%FBS to stop trypsin activity, and furtherdissociated by trituration. The single cell suspension was cultured on poly-L-lysine coatedplates with suitable density. It took7days for re-incubation, and the cells werecharacterized by immunohistochemistry staining for rabbit anti-MAP2antibody.Establishment of NMDA-induced injury modelPrimary cultured cortical neurons were cultured in96-well plates for7days, thenwere randomly divided into control group (Ctrl) and NMDA injury group, Neurons wereexposed to increasing concentrations of NMDA (50μM,100μM,200μM,400μM) for30min, then the medium were fully replaced by Neurobasal to re-incubation for24h.The cell viability were measured by MTT assay to choose a significant concentration ofNMDA for the injury model. Then neurons were expored to the chosen concentration ofNMDA for different times duration (15min,30min,60min), the cell viability weremeasured by MTT assay to ensure a significant time window.Cell viability by MTT assayPrimary cultured cortical neurons were cultured in96-well plates for7days, thenwere randomly divided into control group, model group (NMDA200μM), and AS group(0.1μM,1μM,10μM, and100μM). Cells were pretreated with AS for24h followedwith the administration of NMDA injury for30min, then the medium were fully replacedby Neurobasal to re-incubation for24h. The protective effects of AS against NMDA wereevaluated by MTT assay.Cell apoptosis by Hoechst/PI double stainingPrimary cultured cortical neurons were cultured in24-well plates for7days, thenwere randomly divided into control group, model group (NMDA200μM), and AS group(10μM).Cells were pretreated with AS for24h followed with the administration ofNMDA injury for30min, then the medium were fully replaced by Neurobasal tore-incubation for24h. The cells were stained with PI (10μg/ml) and Hoechst33258(10 μg/ml) for15min, and then fixed by4%formaldehyde for20min. Cells were observedunder a fluorescence microscope for the observation of apoptosis or necrosis.Expression of related proteins by Western blot assayPrimary cultured cortical neurons were cultured in6-well plates for7days, thenrandomly divided into different groups (as described above). Culture medium wasdiscarded after each treatment. Cells were collected and lysed by lysis buffer. Cellprotein was quantified by a BCA Kit rand the related protein expression levels weredetected by Western blot assay.Intracellular calcium concentration by calcium imagingPrimary cultured cortical neurons were cultured in35mm plates made especially forlaser scanning microscope. After7days, cultured cells were randomly divided intodifferent groups (as described above). The model group was not treated with NMDAtemporarily. Cultured cells were washed twice using Mg2+-free extracellular solution(ECS). Then, the neurons were incubated with2.5μM Fluo-3/AM at37oC. After30min,the cultures were returned to the original culture medium for an additional30min. Thedye-loaded cells were measured for fluorescence by using a confocal laser scanningmicroscope. The change of Ca2+concentration was estimated by the fluorescence ratio ofthe Fluo-3/AM-loaded neurons.RESULTS1. Primary cultured mouse cortical neurons The normal primary cultured neuronswere shown as the typical morphology of neurons, axons and dendrites, which connectedinto a network. This culture procedure yielded more than95%neurons, can be used forsubsequent experiments.2. Establishment of NMDA-induced injury model NMDA induced a time-andconcentration-dependent decrease in cell viability as measured by MTT assay. Anexposure to200μM NMDA for30min was used in subsequent experiments since cellinsult was significant in this paradigm (P <0.01). This model were used for subsequentexperiments.3. Pretreatment with AS increased the cell viability of neurons damaged by NMDA Pretreatment with AS significantly increased the cell viability of neuronsdamaged by NMDA in a concentration-dependent manner. Cell viability began to show asignificant difference compared with the control group, when the concentration of10μM(P <0.01). However, AS (10μM) alone showed no effect on cell viability (P>0.05).4. Pretreatment with AS decreased apoptosis or necrotic induced by NMDAThe results of Hoechst/PI double staining showed that NMDA injury induced a highcells mortality rate of49.9±2.8%(P <0.01, compared with control). AS pretreatmentsignificantly reduced the number of apoptotic or necrotic induced by NMDA (19.3±2.3%, P <0.01).5.1. Effects of AS on expression of apoptosis-related proteins Western blot resultsshowed that200μM NMDA treatment significantly reduced the levels of Bcl-2proteinexpression (P <0.01), and increased Bax protein levels (P <0.01), and Bax/Bcl-2ratio(P <0.01) compared with the control group. However, AS pretreatment significantlyreversed the levels of Bcl-2(P <0.01), Bax (P <0.05), and Bax/Bcl-2(P <0.01) inducedby NMDA.5.2. Effects of AS on expression of GluN2A-and GluN2B-containing NMDARs.GluN2B expression was markedly increased after exposure to NMDA (P <0.01),However, there was no difference of GluN2A subtype among three groups. Pretreatmentof AS (10μM) significantly reduced the over-expression of GluN2B induced by NMDAexposure (P <0.01), whereas it had no effect on GluN2A expression(P>0.05).6. Inhibition of NMDA-induced intracellular Ca2+overload by AS Real-timemonitoring of the concentration of calcium ions found that NMDA (200μM) evoked afast elevation of Ca2+concentration. A slow reduction was observed over the next fourminutes, after which the Ca2+concentration stayed at a relatively stable elevated stage.Pretreatment with AS (10μM) could attenuate the amplitude and speed of elevation inCa2+influx after NMDA exposure.CONCLUSIONOur results suggest that pretreatment with AS could protect cortical neurons fromNMDA induced excitotoxic injury. This is partially associated with downregulation of GluN2B-containing NMDARs, inhibiting GluN2B-containing NMDARs-mediatedintracellular calcium overload, and decreasing the level of Bcl-2expression.