Diazoxide Antagonizes The Protein Expression Of Na⁺-K⁺-ATPase β Subunit Induced By Aβ_1-42 In Neurons

Objective:Alzheimer’s disease (AD) is the most common neurodegenerative disesae in eldely people, clinical manifestations of hypomnesis, progressive smart recession, accompanied by personality changes. Neurofibrillary tangles and extracellular senile plaques together with neuronal loss and cortical atrophy are characteristic neuropathological features of the disease. Although the mechanism of AD is unclear, the amyloid neurotoxic cascade was always emphasized by most of studies. Aβ has neurotoxicity and its toxicity mechanism is complex. The amyloid neurotoxic cascade was always emphasized by most of studies. Oxidative stress, Ca2+overload and Glutamate cocentration increased are related with Aβ1-42. Aβ1-42 is the easier aggregation, precipitation and the most difficult to dissolve, it might lead to impairment of Na+-K+-ATPase and ultimately apoptosis.Na+-K+-ATPase of the brain tissue locates in the cell membrane. It is a material basis of maintaining neuronal excitatory transmission and intracellular environment by transporting ions against the concentration gradient after hydrolyzing ATP. Na+-K+-ATPase is adimermainly consisting of a-subunits and β-subunits. The a-subunitis for enzyme catalytic reaction; the β-subunit is ahighlygly cosylated plasma membrane protein with a molecular weight of nearly 60kDa, having tissue selectivity. At present, more research is of a-subunit while research of regulatory subunit β is less. However, some research shows that the expression and organizational adaptability of P-subunitare strong. Na+-K+-ATPase activity depends on the mRNA expression of the β-subunit. The β-subunit is also a necessary part to maintain the Na+-K+-ATPase stability and the correct positioning of membrane.Excessive oxidative stress and toxic effects of free radicals play an important role in the pathogenesisof AD. Aβ produces a large amount offree radicals which can directlyoxidate the amino acid residues of the enzyme protein, inactivate the enzyme and affect the Na+-K+-ATPase activity, further leading to nervecell Na+accumulation and K+loss, cell ion imbalanceand internal environment disorder. Research shows that monosialotetrahexosyl ganglioside (GM1)contributes to maintain the Na+-K+-ATPase activityon the cell membrane of the central nervous system, balance intracellular ion, mitigate nervecell swelling and prevent the accumulation of intracellular Ca2+. It can also suppress neurotoxic effects of excitatory amino acids, reduce damage effect of free radical to nerve cells and thus play an important role on neuroprotection. The ATP sensitive potassium channels (KATP channel) diazoxide can suppress Aβ oligomers gathering and diazoxide preconditioning can resist Aβ1-42 neurotoxic. However, there has not yet been reported whether diazoxide pretreatment relates to the influence of Aβ on the expression of β-subunit of the Na+-K+-ATPase. Therefore, this study aims to investigate the influence of Aβ 1-42 and diazoxide pre-intervention on the expression of β-subunit of the nerve cell Na+-K+-ATPase.Methods:This experiment adopted the method of cell primary cultured, culturing cortex and hippocampus of cholinergic neurons of new borned 24h big Wistar rats. Cells were randomly divided into 4 groups:the control group, the AP1-42 group, the diazoxide+Aβ1-42 group, the diazoxide. Drug intervention methods of each group: when cells were cultured for 7d, the diazoxide+Aβ1-42 group, diazoxide first cultivated neurons for one hour, then added Aβ1.42 (2μmol/L) to continue to be cultivated; the Aβ1-42 group, the same amout of D-Hanks first cultivated neurons for one hour, then given Aβ1.42 (2μmol/L). Simply diazoxide intervention group (50μmol/L):diazoxide first cultivated neurons for one hour, then added the same amount of culture medium to continue to be cultivated, the control group was added the same amount of D-Hanks and culture medium. Four groups were treated by medication for 24h and 72h respectively. Na+-K+-ATPase P-subunit was determined by immunofluorescence and immunoblotting.Results:(1)Immunofluorescence staining results showed that the neural nucleus were stained blue by DAPI and Na+-K+-ATPase β-subunit protein were stained green. We can conclude that Na+-K+-ATPase β-subunits are expressed in the cortex and hippocampus neurons.(2)Immunofluorescence showed:being exposed to Aβ1-42 for 72h, the expression of Na+-K+-ATPase β-subunit was significantly reduced; Compared with Aβ1-42 group, the Na+-K+-ATPase β-subunit were significantly up-regulated in the pretreatment with the diazoxide+Aβ1-42 group for 72h.(3)Immunoblotting showed:Compared with control group and Aβ1-42 group, the protein expression level of Na+-K+-ATPase β-subunit of both diazoxide+Aβ1-42 group and the diazoxide group significantly reduced for 24h(P<0.01). Being exposed to Aβ1-42 for 72h, the protein expression of Na+-K+-ATPase β-subunit was significantly decrease.(P<0.05) Compared with the Aβ1-42 group, the of Na+-K+-ATPase β-subunit was significantly increased in the diazoxide+Aβ1-42 group for 72h(P<0.05).Conclusion:(1)Aβ1-42 could reduced the protein expression level of Na+-K+-ATPase β-subunit in cultured primary neurons for 72h, it is indicated that Aβ might affect Na+-K+-ATPase β-subunit.(2)Immunofluorescence and Immunoblotting showed:the diazoxide+Aβ1-42 group for 72h, diazoxide could significantly reverse the expression of Na+-K+-ATPase β-subunit induced by Aβ1-42.However, the diazoxide+Aβ1-42 group for 24h, diazoxide cloud not affect it.(3)Immunoblotting showed:Diazoxide could reduce the protein expression level of Na+-K+-ATPase β-subunit.Significance:It is indicated Na+-K+-ATPase β-subunitin maybe a new theoretical basis for research aims of the prevention and treatment of AD.