Protective Effect Of Sarsasapogenin On Tau Hyperphosphorylation And Neurotoxicity Induced By Okadaic Acid In Mouse Hippocampus

ObjectiveTo investigate whether Sarsasapogenin（Sar）has protective effect on tau protein hyperphosphorylation and neurotoxicity caused by okadaic acid（OA）and its mechanism.To further clarify the neuroprotective effect of Sar on OA-mediated learning and memory impairment,glial cell response,and neuronal damage and its mechanism.In order to further study the role of Sar on neurodegenerative diseases such as Alzheimer’s disease（AD）with pathological changes of tau protein,and provide theoretical basis for exploring new clinical prevention and treatment.MethodsC57BL/6 adult male mice that weighed 17-23 g were randomly divided into five groups（n=8）:control（vehicle injection）,OA（okadaic acid intra-hippocampal injection）,Sar30+OA（i.g.30 mg/kg/d and i.h.）,Sar100+OA（i.g.100 mg/kg/d and i.h.）,and Sar100（i.g.100 mg/kg/d）.Mice in the Sar30+OA group,Sar100+OA group,and Sar100 group were given Sar30 mg/kg or 100mg/kg for 6 weeks,and control and OA group were injected distilled water by gavage.On the 31^stday of administration,the right hippocampus of each mouse was injected with OA（150 ng）or normal saline.From 36^thday,mice started Morris Water Maze（MWM）exeriment for six days.After MWM experiment,the morphology of vertebral neurons in the hippocampal CA1 region of mice was observed using Nissl staining.Elisa was performed to detect PP2A activity in mouse hippocampus.Western blot was used to detect tau5,p-tau at Ser396sites,CDK5,and S9 sites p-GSK 3βand synapse-related proteins SNAP25,SYP,and PSD95 protein expression levels.IHC staining was performed to observe the glial cell markers Iba-1 and GFAP in the hippocampal CA1,CA3,and DG regions of mice.ResultsMWM test showed that intra-hippocampal injection of OA could induced spatial learning and reference memory impairments in mice.The mice in OA group exhibited significantly higher mean escape latencies than the controls in training trial on day 3,4,and 5（P<0.01）.Compared with the OA-injected group,the escape latencies in the mice treated with Sar（30 mg/kg or 100 mg/kg）were significantly shortened on day 4 and 5（P<0.05）.In probe trial,OA induced the target quadrant entries,crossing platform times and percentage of time spent in target quadrant were significantly reduced（P<0.01）.The swimming trajectory of the mice was chaotic,without trending.Compared with OA group,the platform quadrant entries,times of crossing the platform and the percentage of time spent in the target quadrant were increased significantly in the Sar30+OA group and the Sar100+OA group（P<0.05）,and the mice swimming trajectory maps had clear trend.The results of Nissl staining showed that the number of neural cells in the hippocampal CA1 region was significantly reduced in the OA group,and cells were sparsely distributed compared with those in the sham-operated group.However,the hippocampal neuron loss was reduced by the treatment with Sar（30 mg/kg or 100 mg/kg）.Immunohistochemical staining showed that OA significantly upregulated the expression of Iba-1 and GFAP in various areas of the hippocampus,indicating the high activation of microglia and astrocytes.Sar（100 mg/kg）treatment could effectively inhibit the upregulation of Iba1 and GFAP level induced by OA-injected.Western blot results showed that OA down-regulated the expression of SNAP25,SYP and PSD95,indicating that OA induced neuronal synaptic function damage.Sar inhibited the reduction of OA-induced synaptic protein levels and protected synaptic function.Furthermore,OA decreased PP2A activity and up-regulated the expression levels of protein kinases CDK5 and GSK 3β,and then over-expressed the level of phosphorylated tau protein.Sar reduced over-expression of phosphorylated tau protein by increasing PP2A activity and down-regulating the expression of protein kinases CDK5 and GSK 3β.Conclusions1.Sarsasapogenin has protective effects on synaptic plasticity damage,glial cell activation and spatial learning and memory impairment in mice induced by OA mediated tau hyperphosphorylation.2.It is suggests that the sapogenin of Sarsasapogenin may play a protective role in OA mediated neurotoxicity by up-regulating the activity of PP2A and down-regulating the activity of Cdk5 and GSK3β,reducing the level of over expression of phosphorylated tau protein.