The Study Of Diazoxide,Cyclosporine A To Antagonize Apoptosis Induced By Aβ_1-42 On Cholinergic Neurons

Alzheimer's disease(AD) is one of the most common neurodegenerative disorders affecting many elderly people worldwide.AD,characterized by progressive cognitive impairments and neuronal loss,is the most common form of dementia.The neuropathological hallmarks of AD include extracellular senile plaques and intracellular neurofibrillary tangles(NFTs).beta-Amyloid protein(Aβ) forms the core of plaques.The 40-42 amino acids beta-amyloid peptide(Aβ_1-40,42) derived from proteolysis of amyloid precursor protein(APP) is the major component of the senile plaque found in Alzheimer's disease brains.Recently,it has become more clearly that Aβmay play a pivotal role in the pathogenesis of AD.Although the mechanism of Aβ-induced cell damage is unclear,apoptotic pathways may be involved.In vitro studies show Aβis neurotoxic leading to increased generation of reactive oxygen species(ROS),mitochondrial dysfunction,impairment of Na⁺/K⁺ ATPase,and ultimately leading to apoptosis.ATP-sensitive potassium(K_ATP) channels with different pharmacological properties are present on the plasmalemmal membrane and on the inner mitochondrial membrane.Considerable interest has been generated recently from studies in which activation of mitochondrial ATP-sensitive potassium channels(MitoK_ATP) has been shown to be protective against anoxic and chemical stresses in brain.The immunosuppressant Cyclosporine A has been shown to exert potent neuroprotective effects,possibly via the inhibition of mitochondrial permeability transition pore (MPTP) formation.MPT is a phenomenon induced by an increase in intracellular calcium and characterized by the opening of the MPT pore.Studies have shown that cyclosporine A has the ability to block mitochondrial permeability transition pore opening.Administration of Cyclosporine A has been demonstrated to reduce cell death following ischaemia-reperfusion injury.The permeability transition pore(MPTP) and the ATP-dependent potassium (MitK_ATP) channel of mitochondria are known to play key roles in mitochondrially mediated apoptosis.We investigated modulation of the mitochondria permeability transition pore(MPTP) and the mitochondria ATP-dependent potassium(MitK_ATP) channel,either as single elements or in combination,had what effect in vitro Alzheimer's disease models.ObjectiveTo investigate the toxic effects of Aβ_1-42 in cultured primary rat basal forebrain cholinergic neurons.To investigate effects of diazoxide(Mitochondria ATP-sensitive potassium channel opener),cyclosporine A and in combination to antagonize apoptosis induced by beta-Amyloid protein(Aβ_1-42) on cultured primitive rat basal forebrain cholinergic neurons.MethodsPrimary rat basal forebrain cholinergic neurons were cultured and evaluated by Immunocytochemistry.The cells were randomly divided into 3 groups:the control goup,the Aβ_42-1(24h) goup and Aβ_1-42(72h) goup.The cell morphology were observed by inverted microscope,changes of cell apoptosis were determined by MTT.The cell apoptosis model was induced by Aβ_1-42(2μmmol/L),then use 500μmol/L diazoxide,20μmol/L cyclosporine A and the two to interfere it.The cells were randomly divided into 5 groups:the control goup,the Aβ_1-42 goup,Aβ_1-42+ Diazoxide goup,Aβ_1-42+ diazoxide +Cyclosporine A group;Each group was divided into 2 subgroups(24h and 72h).The cell morphology were observed by inverted microscope, changes of cell apoptosis were determined by MTT,the expression of target proteins (bcl-2,bax,cytochrome C,caspasce-3 and Cleaved Caspase-3) was determined by Western blot when the neurons were interfered by the drugs for different times(24, 72h).ResultsFor identification of cholinergic neurons,cells grown for 7days in proliferation growth medium.Then immunocytochemistry using anti-Choactase was carried out. Over 90%of the cells were cholinergic neuronsBeing exposed to Aβ_1-42 for 72h,the cell activity remarkably decreased,the expression of Bcl-2 decreased and the expression of cytochrome C,caspasce-3 and Cleaved Caspase-3 increased.When the cell apoptosis model were exposed to diazoxide,cyclosporine A and the two for 72h,the expression of Bcl-2 increased obviously,the expression of Cytochrome C,Caspasce-3 and Cleaved Caspase-3 decreased,the cell activity increased.Being exposed to Aβ_1-42 for 72h,the neurons began apoptosis. ConclusionBeing exposed to Aβ_1-42 for 72h the expression of Bcl-2 decreased and the expression of cytochrome C,caspasce-3 and Cleaved Caspase-3 increased and were induced apoptosis.Diazoxide,cyclosporine A and in combination could protect neurons from apoptosis induced by Aβ_1-42.