The Protective Effect And Mechanism Of Autophagy Induced By VPA On The Treatment Of Cell Model Of Parkinson’s Disease

Backgroud:Pasrkinson’s Disease(PD)is presently considered as common neuro-degenerative disorder.It was first reported by James Parkinson in 1817.The key pathological features of PD is described as the apoptosis and loss of the dopaminergic neurons of the substantia nigra pars compacta and the formation of Lewy Body(LB)in the cytoplasm of DA neurons.Although the etiology and the mechanism of PD is still unrevealed clearly,recent study show that impaired autophagy may lead to the loss of DA neurons.Valproic Acid(VPA),as a Histonedeacetylases Inhibitor(HDACI),is a medication primarily used to treat epilepsy and bipolar disorder and to prevent migraine headaches.Recent studies show that VPA protects DA neurons through inhibition of the inflammatory reaction of microglia and stimulating the release of neurotrophic factors from astrocytes.Studies also show that VPA may induce autophagy in PD animal or cell models.While,whether the autophagy induced by VPA is protective or detrimental for PD is not known for now.Objectives:The aims of this study are:1)establish the in vitro PD cell model,and verify the protective role of VPA for the model;2)to investigate the autophagy level of PD cell model and how the VPA treatment influence the autophagy level;3)to explore the protective effect of autophagy induce by VPA and the mechanism underlying.Methods:The PD cell model was constructed by MPP+ treating neuroblastoma cell SH-SY5Y.In order to examine the protective effect of VPA,MTT and Hochest 33358 stain was used to test the cell viability and apoptosis rate after PD cell model was treated with VPA of concentration gradient.The autophagy level of PD model or after treatment with VPA was detected by western blot to examine the expression of LC3B and P62.Reactive oxygen species(ROS)and JC-1 was tested by flow cytometry analysis in different treatment groups.The apoptosis rate was examined by flow cytometry using Annexin-V PI in different groups.The expression of mitochondrial apoptosis pathway related protein(Bcl-2,Bax,Cyto-c,active-caspase3)were measured by western blot.Statistical analyses were performed using ANOVA and post hoc Fisher’s PLSD tests,with P<0.05 considered statistically significant.Results:The viability of the PD cell model constructed by MPP+ was correlated with the concentration and time of MPP+ treatment negtively.After the treatment of VPA within the therapeutic concentration,the cell viability of the model was enhanced and the apoptosis rate was decreased.We found the most protective concentration of VPA was 0.5mM,higher concentration of VPA did not show greater protective effect,but may even be detrimental to the cell model.Using western blot,we test the level of autophagy related proteins(LC3B,P62).We found autophagy flux was inhibited in the PD cell model.While,co-treated with VPA,the inhibition of autophagy flux was corrected.The flow cytometry analysis shows that the level of ROS increased and mitochondrial potentials reduced,while co-treated with VPA,the level of ROS decreased and mitochondrial potentials recovered.The flow cytometry were also used to test the apoptosis of each groups,we found the anti-apoptosis effect of VPA was reduced when the autophagy induction was inhibited by 3MA.MPP+ activated the mitochondrial apoptosis pathway by enhance Bax,and urge Cyto-c released from mitochondrial,and finally Caspase3 was activated into a-Caspase3,Which is a marker of apoptosis.We found co-treated with VPA,the mitochondrial apoptosis pathway activation was reduced,while the effect was vanished after inhibited the autophagy induced by VPA.Conclusion:Our findings suggest that VPA reversed autophagy flux inhibited by MPP+.VPA protected the mitochondrial function by reduce ROS and enhance mitochondrial potentials which function was dysregulated by MPP+.The autophagy induced by VPA play a role in the anti-apoptosis effect against MPP+.The protective effect of autophagy induced by VPA may depneds the timely clearance of impaired mitochondria by MPP+,and finally inhibit the mitochondrial apoptosis pathway.