Protective Effect Of Sarsasapogenin On Hyperglycemia Induced Neurotoxicity In Cultured Hippocampal Neurons

ObjectiveTo investigate whether Sarsasapogenin (Sar) can protect hippocampalneurons from hyperglycemia induced neurotoxicity.MethodsThe hippocampi of0~24h-old Sprague-Dawley (SD) rats were collectedunder sterile conditions, cut into pieces and digested with0.125%trypsin.Primary hippocampal neurons were cultured in dulbecco modified eaglemedium (DMEM)/F12medium supplemented with10%fetal bovine serum(FBS) and10%horse serum, after7d in culture, the cells were used for thetreatment. The experiment is divided into two parts, the first part of theobserved difference in concentrations of glucose treatment on hippocampalneurons of rats. The cultured hippocampal neurons were divided into fivegroups: control group; hyperglycemia treatment group; mannitol osmoticcontrol (25mmol·L-1mannitol+25mmol·L-1glucose) group; In control group,cultured hippocampal neurons were incubated with equal DMEM for48h. Inhyperglycemia treatment group, cultured neurons were incubated with glucose(30,50,100mmol L-1) for48h. In mannitol osmotic control (25mmol·L-1mannitol+25mmol·L-1glucose) group, cultured neurons were incubated withmannitol25mmol·L-1and glucose50mmol·L-1for48h. The cell viability wasassessed by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The protein expressions of synaptophysin were measured bywestern blot. The second part of was to investigate whether Sar can protecthippocampal neurons from glucose-induced injury, the experiment was divided into six groups: control group; hyperglycemia50mmol·L-1group;hyperglycemia50mmol·L-1+Sar (10,30,100μmol·L-1) group; Sar30μmol·L-1group; In hyperglycemia50mmol·L-1+Sar（10,30,100μmol·L-1） group, Sar（10,30,100μmol·L-1） was added to the neurons1h prior to incubation withglucose50mmol L-1. In Sar30μmol·L-1group, cultured hippocampal neuronswere incubated with Sar30μmol·L-1for48h. The neuronal apoptosis wasquantified by scoring the percentage of cells with apoptotic nuclearmorphology after Hoechst33258staining. The cultured cells were then fixedon7day and immunocytochemically stained with NeuN antibody andsynaptophysin antibody. The protein expressions of synaptophysin, activecaspase-3, and poly ADP-ribose polymerase (PARP) were measured bywestern blot.ResultsMorphological observation, just inoculated hippocampus neurons wereround, small volume, strong refraction, uniform distribution. After1d incubation,the majority of cells out of three or four short processes, almost all cellattached to the wall, which be repolymerized phenomenon some cells. After4d growth, neuronal cell bodies increases further, protrusions further increasedand obvious growth, network structure connected to form a sparse, neurons inmultipolar neurons. After7d incubation, cell volume continues to increase, thecell body was full, neurite growth thickening, more branches, forming a densenetwork. Cultured for7d in hippocampal neurons, NeuN immunofluorescencestaining of neuronal purity identification, indicated neuron purity above90%.MTT results show, the decrease of cell viability and synaptophysin proteinexpression in cultured hippocampal neurons were observed incubated withhigh glucose(50and100mmol·L-1) for48h compared with the normal controls.But the same osmotic mannitol group on neuronal activity and expression ofsynaptophysin were not affected. Sar(10,30,100μmol L-1) significantlyinhibited high glucose-induced decrease of synaptophysin protein expression.Hoechst33258nulear staining results showed, the apoptotic cell percentageswere increased from (8.144.18)%in the normal control group to(25.75.72)%in high glucose-treated group. Sar(10,30,100μmol L-1) wereadded to the neurons1h prior to incubation with high glucose，Sar inhibited high glucose-induced increase of the apoptotic cell percentages in dose-dependent manner, decrease to （19.415.35）%,（9.195.34）%and（8.484.9）%. The results of Western blotting showed that normal controlgroup cultured hippocampal neurons activated caspase-3expression is verysmall. Adding glucose after48h, can significantly increased activatedcaspase-3and the molecular weight89kDa PARP expression, but decreasedthe molecular weight116kDa PARP expressions. Sar (10,30,100μmol L-1)inhibited high glucose-induced changes on activated caspase-3and PARP.Conclusions1. High glucose may damage on cell viability and synaptophysinexpression of cultured hippocampal neurons.2. These results indicate that Sar can protect hippocampal neuronsagainst hyperglycemia neurotoxicity, this effect may be related to apoptosi ofhippocampal neurons against high glucose induced is closely.