The Protective Effect Of Nouring Spleen Yin Recipe On Aβ-Induced Neurotoxicity In Primary Cultured Rat Hippocampal Neurons

Objective: Alzheimer's disease (AD) is a neurodegenerative disorder that affects cognitive function. Many recent researches have focused on the toxic effect of amyloid-β-peptide (Αβ) which plays a central role in the pathogenesis of AD.Αβis considered to cause progressive synaptic degeneration and neuronal loss, thereby resulting in cognitive dysfunction and behavioral abnormalities in AD.Αβis directly toxic to neurons or potentialize neuronal vulnerability to excitatory neurotoxins, resulting in extensive neuronal loss and synapse dysfunction. Synapses are highly dynamic"plastic"structures, and corresponding dynamic changes in the efficiency of synaptic transmission are thought to represent the physic- ological basis of learning and memory. Dendritic spines are the major sites of synapse formation. The development and morphology changes of spines are considered as strong candidates for the cellular mechanism regulating synaptic activity. It was reported that serum-inducible kinase (SNK) was induced by synapse activity and targeted spine-associated Rap guanosine triphosphatase (GTPase) activating protein (SPAR). By the ubiquitin- proteasome pathway SPAR was degraded. SPAR is a multidomain postsynaptic protein that controls dendritic spines shape by regulating actin arrangement as well as the signalof small GTPase Rap. Actin dynamics and Rap activity are both regulated by synaptic activity and involved in synaptic plasticity. Understanding the mechanisms that control the actin cytoskeleton of dendritic spines may help to reveal the cellular basis of the synaptic plasticity that underlies learning and memory. There are no reports about what role does the toxic ofΑβplay in SNK-SPAR pathway and the dysfunction of synapse. What's more, N-methyl-D-aspartate Receptors (NMDARs) are involved in a wide variety of processes in the central nervous system (CNS), including synaptogenesis, synaptic plasticity, memory and learning.Αβcould cause extracellular accumulation of glutamate and destabilize calcium homeostasis. We presume whether or not there is close relationship betweenΑβ-induced neurotoxicity, SNK-SPAR pathway and NMDAR.Principle of nouring spleen yin recipe (Zibu Piyin Recipe, ZBPYR)is to nourish spleen yin, it is a prescription designed specially for deficiency of spleen-yin. ZBPYR serum was gained by the method of serum pharma- cology. Previous researches and clinic experiments reported that ZBPYR had significant effect on brain protection and could protect neurons from excitatory injury in vitro. However, the molecular protective mechanism of ZBPYR for synapses and dendritic spines has not been elucidated clearly.The objective of this study is to observe the relationship between toxicity induced byΑβ, SNK-SPAR pathway and NMDAR in primary cultured rat hippocampal neurons, and to explore the mechanism of the protective effect of ZBPYR serum on neurons againstΑβtoxicity, and to supply a new therapy method for AD and other neurodegenerative disorders.Methods: 1. The primary rat hippocampal neurons were cultured.2. Small interfering RNA (siRNA) targeting for SNK was transfected by Lipofectamine2000 into neurons and then we detected the expression of SNK and SPARmRNA with RT-PCR method.3.Αβ1-40 peptide was added to 1×PBS and incubated in the incubator for 72h, 37℃, and then aggregated fibrillarΑβ1-40 was obtained.4. RT-PCR was used to detect SNK,SPAR mRNA expression by exposure to 5μMΑβ1-40 for different time intervals.5. ZBPYR serum was gained by the method of serum pharmacology; RT-PCR was used to detect SNK, SPAR, NR1, NR2A and NR2B mRNA expression by exposure toΑβ1-40 and with different concentration of ZBPYR serum pretreatment.Results: 1. After siRNA was transfected into neurons, SNK mRNA was down-regulated, and SPAR mRNA was up-regulated. 2. After exposed toΑβ1-40 for different time intervals, we found SNK mRNA was up-regulated and SPAR mRNA was down-regulated compared with the control group, and the changes of mRNA expression were significant at 2h.3. After exposed toΑβ1-40 for 2h, we found SNK mRNA was up-regulated, and SPAR, NR1, NR2A, NR2B mRNA were down-regulated compared with the control group.4. While with ZBPYR serum pretreatment, we observed SNK mRNA was down-regulated and SPAR, NR1, NR2A, NR2B mRNA were up- regulated compared withΑβ1-40 group, and the 2%ZBPYR serum had the best effect.Conclusions: 1. siRNA could down-regulate the expression of SNKmRNA with corresponding change of SPARmRNA expression. It suggested that SNK played an important role in the SNK-SPAR pathway.2.Αβ1-40 could induce the changes of SNK and SPAR mRNA expression and these changes had time dependent. It suggestedΑβ-induced neurotoxicity was related to SNK-SPAR pathway.3. ZBPYR serum could protect neurons againstΑβneurotoxicity, and 2%ZBPTR serum had the best effect. What's more, the protective effect of ZBPYR serum on neurons againstΑβtoxicity was performed by preventing over-activation of NMDAR and blocking of SNK-SPAR pathway.