The Regulation Of Proanthocyanidins On SH-SY5Y Cell Injury And Apoptosis Induced By Aβ_25-35 Through Wnt/β-catenin Signaling Pathway

Alzheimer’s disease（AD）,characterized by memory impairment,executive dysfunction,personality and behavior changes,is the most common type of dementia in the elderly.Its pathological features are the deposition of extracellular amyloid beta plaques,the formation of senile plaques（SP）,tau protein hyperphosphorylation and the loss of neuronal cells.Wnt signaling pathway is a more in-depth study of the mechanism in recent years.It is very important in the development of embryonic nervous system,and is also related to the proliferation,differentiation,migration,synaptogenesis and axonal growth of adult neurons.Proanthocyanidins（PC）is a kind of plant polyphenols,which has a variety of biological activities.This study observed the effect of Aβ_25-35on Aβsecretion,tau protein phosphorylation and apoptosis on human neuroblastoma cells SH-SY5Y and observed the changes of the corresponding indicators after using proanthocyanidins.At the same time,β-catenin,cyclin D1 and p-GSK-3β（Ser9）,the related proteins of the classical Wnt signaling pathway,were detected.In order to explore the effect and mechanism of proanthocyanidins,the levels of the above proteins were measured simultaneously after using Wnt3a or Dkk1.This study provided experimental basis for the control and prevention of Alzheimer’s disease and the expansion of the application scope of proanthocyanidins.In the experiment research,this paper has included two parts.PartⅠ:Protective effects of proanthocyanidins on injury and apoptosis of SH-SY5Y cells induced by Aβ_25-35Objective:SH-SY5Y cells induced by Aβ_25-35were used to investigate the effects of proanthocyanidins on Aβ_1-42production,tau protein phosphorylation and apoptosis.Methods:（1）The control group and the proanthocyanidins groups（0.1,1.0,2.5,5.0,7.5,10.0μg/m L）were established.The cells were intervened with different concentrations of proanthocyanidins.MTT assay was used to determine the cell viability.（2）Control group and different concentrations of Aβ_25-35groups（1.0,5.0,15.0,20.0,25.0μmol/L）were established.SH-SY5Y cells were treated by Aβ_25-35.MTT assay was used to determine the cell viability and the subsequent dose.（3）Set up control group,Aβ_25-35group,proanthocyanidins（1.0,2.5,5.0,7.5,10.0μg/m L）+Aβ_25-35（15.0μmol/L）groups.PC intervention group pretreated with Aβ_25-35for 2h.The medium containing PC was replaced and cultured for 24h.The cell viability was evaluated by MTT.The secreted Aβ_1-42was detected by ELISA.The expression of BACE1,tau,p-tau,caspase-3,Bcl-2 and Bax proteins were measured by Western blot.Results:（1）Proanthocyanidins（0.1,1.0,2.5,5.0,7.5,10.0μg/m L）had no effect on cell viability of SH-SY5Y cells（P>0.05）.（2）The cell viability was decreased by Aβ_25-35（15.0,20.0,25.0μmol/L）（P<0.05）,and 15.0μmol/L Aβ_25-35was selected for the follow-up experiments to establish cell injury model.（3）Compared with the control group,the cell viability was decreased in Aβ_25-35（15.0μmol/L）group.Compared with Aβ_25-35（15.0μmol/L）group,proanthocyanidins（2.5,5.0,7.5,10.0μg/m L）could increase the cell viability（P<0.05）,and improve the toxicity of Aβ_25-35in cells.（4）Compared with the control group,the expression of p-tau,BACE1and caspase-3 proteins,the secretion of Aβ_1-42increased and Bcl-2/Bax decreased in Aβ_25-35（15.0μmol/L）group（P<0.05）.While PC could reverse the toxic effect of Aβ_25-35.（5.0,7.5μg/m L）PC intervention could reduce the expression of p-tau and BACE1 proteins（P<0.05）.（1.0,2.5,5.0,7.5μg/m L）PC intervention could make the expression of caspase-3 and Aβ_1-42decreased,while Bcl-2/Bax increased（P<0.05）.PartⅡ:Regulation of proanthocyanidins on injury and apoptosis of SH-SY5Y cells induced by Aβ_25-35through Wnt/β-catenin signaling pathwayObjective:To explore the protective mechanism of proanthocyanidins on SH-SY5Y cells injury and apoptosis induced by Aβ_25-35.Methods:（1）The cells were divided into control group,Aβ_25-35（15.0μmol/L）group,proanthocyanidins（1.0,2.5,5.0,7.5μg/m L）+Aβ_25-35（15.0μmol/L）groups,the expression levels ofβ-catenin,cyclin D1,p-GSK-3β（Ser9）and GSK-3βproteins were measured by Western blot.（2）The cells were divided into control group,Aβ_25-35（15.0μmol/L）group,proanthocyanidins（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group,Dkk1（100.0 ng/m L）group,Dkk1（100.0 ng/m L）+proanthocyanidins（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group.The expression levels of BACE1,tau,p-tau,caspase-3,Bcl-2,Bax,β-catenin,cyclin D1,p-GSK-3β（Ser9）and GSK-3βproteins were measured by Western blot.The secreted Aβ_1-42was detected by ELISA.（3）The cells were divided into control group,Aβ_25-35（15.0μmol/L）group,proanthocyanidins（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group,Wnt3a（100.0 ng/m L）group,Wnt3a（100.0 ng/m L）+Aβ_25-35（15.0μmol/L）group.The expression of BACE1,tau,p-tau,caspase-3,Bcl-2,Bax,β-catenin,cyclin D1,p-GSK-3β（Ser9）and GSK-3βproteins were measured by Western blot.Results:（1）Compared with the control group,15.0μmol/L Aβ_25-35decreased the expression ofβ-catenin,cyclin D1 and p-GSK-3β（ser9）proteins（P<0.05）.（2）Compared with the Aβ_25-35（15.0μmol/L）group,（1.0,2.5,5.0,7.5μg/m L）proanthocyanidins could increase the expression level ofβ-catenin and cyclin D1proteins and（2.5,5.0,7.5μg/m L）proanthocyanidins could increase the expression of p-GSK-3β（ser9）protein exposed to Aβ_25-35（P<0.05）.（3）Compared with the PC（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group,the expression level ofβ-catenin,cyclin D1,p-GSK-3β（ser9）proteins and Bcl-2/Bax expression decreased（P<0.05）,the reduction of the p-tau,BACE1,caspase-3 and Aβ_1-42proteins expression increased（P<0.05）in Dkk1（100.0 ng/m L）+PC（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group.（4）Compared with the Aβ_25-35（15.0μmol/L）group,the expression ofβ-catenin,cyclin D1,p-GSK-3β（ser9）and Bcl-2/Bax increased while the expression of BACE1,p-tau and caspase-3proteins decreased（P<0.05）in Wnt3a（100.0 ng/m L）+Aβ_25-35（15.0μmol/L）group and PC（7.5μg/m L）+Aβ_25-35（15.0μmol/L）group.Conclusion:（1）Aβ_25-35（15.0,20.0,25.0μmol/L）could decrease the cell viability,while proanthocyanidins（0.1,1.0,2.5,5.0,7.5,10.0μg/m L）had no significant toxic effect on the viability of SH-SY5Y cells in the experimental dose.PC（2.5,5.0,7.5,10.0μg/m L）could reverse the reduction of cell viability caused by Aβ_25-35.（2）Proanthocyanidins could inhibit Aβ_1-42production,tau protein hyperphosphorylation and apoptosis on SH-SY5Y induced by Aβ_25-35.（3）Proanthocyanidins could increase the expression of Wnt/β-catenin signaling pathway related proteinsβ-catenin,cyclin D1 and p-GSK-3β（ser9）proteins.（4）The regulation effect of PC on cell injury and apoptosis decreased after inhibiting the Wnt/β-catenin signaling pathway.PC may regulate the cell injury and apoptosis on SH-SY5Y induced by Aβ_25-35through Wnt/β-catenin signaling pathway.The effect of proanthocyanidins was similar with Wnt3a,an activator of Wnt/β-catenin signaling pathway.PC may resist the cytotoxicity caused by Aβ_25-35on SH-SY5Y through regulating Wnt/β-catenin signaling pathway positively.