Effects Of ATP Sensitive Potassium Channel Opener On Neural NR2B Subunit Protein Expression Induced By Aβ_1-42

Objective:Though the initiating reasons and following molecular and cellular events contributing to the dysfunction and death of neurons in Alzheimer’s disease (AD) are not fully explained, the accumulation of amyloid β-peptide (AP) and oxidative stress, hyperphosphorylated tau, Ca2+disruption and impaired energy metabolism are related with.The abnormal deposition of Beta-amyloid peptides(AP) is its characteristic pathologic changes in the brain. Aβ1-42is the first precipitation and the most difficult to dissolve, which is the strongest cytotoxic substance. Existing studies have confirmed that AP has nerve toxicity and its toxicity mechanism is complex. More and more studies have confirmed that Aβ is closely related to overactivate the N-methyl-D-aspartate (NMDA) receptors (NMDARs) resulting in the elevation of intracellular Ca2+levels and excitotoxicity, which lead to neurodegeneration and synaptic dysfunction. NMDA receptors are critical for the ion glutamate receptors in the brain, which play an important role in the process of signal transfer between synaptic transmission and long-term potentiation. NR2B subunit is a regulating subunit of NMDA receptor and has a very important role in the structure and function of NMDA receptor. NR2B subunit adjusts NMDA receptor mediated excitatory postsynaptic potential and synaptic plasticity of the central nervous system; At the same time, ion channels of NMDA receptor containing NR2B show high permeability to Ca2+, which become a potential target for the treatment of NMDA receptors related diseases.ATP sensitive potassium channels (KTAP) is a heterogeneous polymer channel of the cell membrane and mitochondrial membrane, binding the cell metabolic state with the electrical activity together by feeling the change of intracellular ATP/ADP level. In recent years, studies have shown that the activation of ATP sensitive potassium channels in the central nervous system plays an important role in the pathogenesis of nerve protective effect. Mitochondrial KATP channels opener diazoxide (DIZ) can obviously reduce Aβ oligomers aggregation and Tau protein phosphorylation, and can improve the cognitive dysfunction of genetically modified AD rat model; Our initial experiments have confirmed that Diazoxide preconditioning can improve learning and memory of the establishment Wistar rat model by Aβ1-42and can not only enhance the expression of Bcl-2and but also reduce the expression of Caspase3, which hint to have resistance to apoptosis.While reversing over-expression of KATP subunits in cholinergic neurons caused by exposure to Aβ1-42, Diazoxide resisted neurotoxicity caused by Aβ1-42. Further study found that pathways of NF-κB or p38MAPK or PKC had a protective effect against excessive oxidative stress and destruction of the mitochondrial membrane potential and may be involved in the occurrence of Alzheimer’s disease. Study of effects of ATP sensitive potassium channel opener diazoxide on neural NR2B subunit protein expression induced by Aβ1-42may be providing a new theory basis for targeted therapy of AD.Methods:This experiment adopted the method of cell primary cultured, culturing cortex and hippocampus of cholinergic neurons of new borned big Wistar rats. Primary cultured neurons for up to7d, we adopted the method of immunofluorescence cytochemistry to identify NMDA receptor NR2B subunit of cortex and hippocampus neurons expression. Cells were randomly divided into two groups, each group was divided into four subgroups:the blank control group, simply Aβ1-42(2μmol/L) intervention group, Aβ1-42+DIZ intervention group, simply diazoxide intervention group (50μmol/L). Drug intervention methods of each group:when cells were cultured for7d, the control group was added the same amount of D-Hanks and culture medium. Aβ1.42(2umol/L) intervention group was given AP1.422μmol/L. APm2+DIZ intervention group:diazoxide first cultivated neurons for one hour, then added Aβ1-42(2μmol/L) to continue to be cultivated; Simply Diazoxide intervention group (50μmol/L):diazoxide first cultivated neurons for one hour, then added the same amount of D-Hanks and culture medium to continue to be cultivated. Four subgroups were treated by medications for24h and72h respectively. Using Western blot method to detect expression level of NR2B subunits changes when treated by Aβ1-42and Diazoxide for different times(24h and72h).Another group of four subgroups with experimental cells were randomly divided into four groups:Aβ1-42with different concentrations (0.2μM.5μM.10μM) treated with primary cultured neurons for72h, The expression level of NR2B subunit protein was determined by Western blotting.Results:(1). Cortex and hippocampus neuron immunofluorescence staining results showed that the neural nucleus were stained blue by DAPI and NR2B subunits protein were stained red.It can be seen that fluorescence intensity value of NR2B subunits was high in the cell membrane of cortex and hippocampus neuron and the dendrites and axons were also high but not as good as the cell membrane. We can conclude that NR2B subunits are expressed in the cell membrane, dendrites and axons of cortex and hippocampus neuron.(2).Being exposed to Aβ1-42(2μmol/L) for24h, compared with the control group, the expression level of NR2B subunit protein in the cultured neurons was no significant difference. Aβ1-42+DIZ(50μmol/L) and simple Aβ1-42intervention group compared with the control show no significant change in expressing level of NR2B subunit protein (p>0.05).(3).By applying different concentrations (0,2μM,5μM,10μM) of Aβ1-42primary cultured neurons after72h, with Aβ1-42concentration increasing, expression levels of NR2B subunits protein gradually increased, and had concentration dependence and statistical significance (p<0.05).(4).Being exposed to Aβ1-42(2βmol/L) for72h, the expression level of NR2B subunit protein was significantly decreased in Aβ1-42+DIZ group than in simple Aβ1-42interfere group (p<0.05). After exposure to Aβ1-42for72h, the expression level of NR2B subunit protein was obviously higher in simple Aβ1-42interfere group than in control group (p<0.01). Simple diazoxide interfere group in the expression level of NR2B subunit protein was no significant change compared with the control group (p>0.05).Conclusion:(1). NR2B subunits are expressed in the cortex and hippocampus neuron cell membrane, dendrites and axons in the primary cultured cortical and hippocampal cholinergic neurons.(2).Aβ1-42could up-grade the protein expression level of NR2B subunit in cultured primary neurons for72h, and ATP-sensitive potassium channel opener diazoxide could inhibit the increasing of protein expression level of NR2B subunit induced by Aβ1-42. it is indicated that diazoxide might affect Aβ1-42-induced cytotoxicity via NMDA receptor pathway.