The Role Of VDAC1 In A?_25-35 Induced Mitochondrial Apoptosis Of Hippocampal Neurons And The Intervention Of Cy3G

Objective:?1?To study the mechanism of mitochondrial apoptosis induced by beta-amyloid 25-35(A?_25-35)in the pathogenesis of AD and explore the key role of VDAC1 in the development of AD.?2?To explore the effective ways and related mechanisms of the neuroprotective effect of Cy3G.?3?To screen the differential expression of mi RNA in hippocampal neurons which were treated by A?_25-35 or Cy3G.Methods?1?Hippocampal neurons were isolated from newborn rat and incubated with A?_25-35?5?mol/L?to establish the alzheimer's cell model.?2?The appropriate concentration and method of Cy3G was screened with the colorimetric tetrazolium salt?MTT?assay;?3?Observe the protective effect of Cy3G on the damage ofhippocampusneurons induced by A?_25-35: the cell viability was analyzed by Lactate dehydrogenase?LDH?kit.The mitochondrial membrane potential andthe cell apoptosis rate were respectively detected by Rhodamine 123 and Hoechst 33342 staining.?4?The intracellular reactive oxygen species?ROS?was detected by DCFH-DA probe.The expression of apoptosisrelated protein including Bax,bcl-2,caspase-9,VDAC1 protein and VDAC1 oligomerizationwere detected by Western blot.?5?The differentially expression of mi RNAwasscreened by Gene chip.Next,to explore the specific characteristics and rules of the gene change before and after the stimulus,and analyze its downstream signal pathway.Results:?1?Compared with the normal control group,the survival rate of cells was significantly reduced after A?_25-35 treatment.Relatively,40?mol/L Cy3G and 5?mol/L A?_25-35 coincubation for 3h shows the best protection effect.?2?A?_25-35 leaded to LDH leakage,cell apoptosis rate increase and mitochondrial membrane potential decrease?P<0.05?,but Cy3G intervention antagonate the damage.?3?A?_25-35 improved the ROS level and the expression of apoptosis protein Bax,but the intervention of Cy3G effectively inhibit this expression?P<0.05?.The expression of VDAC1 and its oligomerizationalso be improved by A?_25-35.?4?A?_25-35 affect the the expression of mi R-135b-5p,mi R-153-3p and other mi RNAs.After an analysis of the biological differences of target m RNAs,the toxicity of A?_25-35 may be related to the cell apoptosis,oxidative stress and the complex interactions between proteins?protein homodimerization activity?.While the neuroprotective effect of Cy3G may be related to the negative regulation of RNA polymerase II promoter,MAPK cascade,protein dephosphorylation and positive autophagy regulation.Conclusion:?1?A?_25-35 may stimulate the oxidative stress,promote the Bcl-2 protein family to apoptosis direction,improve the expression of mitochondria VDAC1 protein and its oligomerization,start the apoptosis process,eventually lead to neuronal loss and AD develops further.?2?The neuroprotective effect of Cy3G may be due to its powerful antioxidant function and regulation of the expression of Bcl-2 family proteins.