Study On The Effect Of ?-Lipoic Acid On Autophagy In Parkinson's Disease Model And Its Mechanism

Parkinson's disease(PD)is a common type of motion disorder caused by substantia nigra-striatum pathway degeneration,often occurring in the elderly.The main cause of the disease is the degeneration of dopaminergic neurons in substantia nigra-striatum and the decrease in the biosynthetic capacity of residual neurons,which leads to the reduction of dopamine release in substantia nigra-striatum pathway.At present,the treatment of PD is limited to symptomatic remission,which could not prevent continuous degeneration of neurons.Finding effective drugs to prevent disease progression has always been a hot topic of international concern.Oxidative stress and autophagy play important roles in the pathogenesis of PD,and there is a complex network of interactions between them.Natural antioxidant alpha-lipoic acid(ALA)can protect neurons from oxidative stress,but its role in the regulation of autophagy in PD has not been reported.In this study,we will explore the interaction mechanism between oxidative stress and autophagy utilizing PD models in vitro and in vivo,and the underlying mechanism of autophagy regulation by ALA through ROS-AMPK/m TOR signaling pathways.Our study will help to understand the mechanism of interaction between oxidative stress and autophagy in PD,and provide theoretical and experimental supports for the development of safe and efficient drug therapy for PD.Objective1.To observe the effects of 6-hydroxydopamine(6-OHDA)on human neuroblastoma cells(SH-SY5Y)in concentration and time-course studies in order to determine the appropriate concentration and time range of the 6-OHDA for the in vitro model of PD,and to observe the effect of ALA on the survival rate,apoptosis rate and oxidative stress level of 6-OHDA-induced SH-SY5 Y cells.2.To observe the effect of ALA on the expression of autophagy-lysosomal pathway-related protein LC3-II,autophagy upstream regulatory protein mammalian rapamycin target protein(m TOR),and adenosine monophosphate-activated protein kinase(AMPK)in 6-OHDA-treated SH-SY5 Y cells.3.To observe the protective effects of ALA on neurons in 6-OHDA induced Parkinson's disease rats.Methods1.The appropriate concentration and time point of 6-OHDA treatment were chose according to the cytotoxic effects of 6-OHDA on SH-SY5 Y cells in concentration and time-course studies,respectively.Cell viability was measured by CCK-8 assay,apoptosis was detected by co-staining with Annexin V and PI,and ROS level was examined by fluorescent probe DCFH-DA.2.GFP-LC3 plasmid transfection was used to observe the localization of GFP-labeled LC3 protein in 6-OHDA-treated SH-SY5 Y cells;Western blot was used to analyze the protein expression of LC3-II,m TOR,p-m TOR,AMPK and p-AMPK.3.PD in vivo model was established by intracerebral injection of 6-OHDA.Behavioral tests were carried out after 3 weeks to choose the successful models of PD.Then,PD animals were exposed to ALA intervention.Behavioral tests were carried out after 2 weeks.Morphology of cells was observed by HE staining in substantia nigra,and TH content in substantia nigra was detected by immunohistochemistry.SPSS 22.0 software was used to analyze the results.Results1.6-OHDA induced cytotoxicity in SH-SY5 Y cells in a time-and concentrationdependent manner;compared with the control group,6-OHDA decreased the cell viability(P<0.01),increased the apoptosis rate(P<0.01),and increased the level of oxidative stress(P<0.01).Compared with the control group,ALA alone group showed no significant changes in the above indexes.Compared with 6-OHDA group,co-incubation with ALA and 6-OHDA increased cell viability(P< 0.05),decreased the rate of apoptosis(P<0.01),and attenuated the level of oxidative stress(P<0.01).2.Compared with the control group of SH-SY5 Y cells,the expression of LC3-II was higher in the 6-OHDA group(P<0.05),which was most pronounced in the 6-hour group;the expression of p-AMPK was higher(P<0.05)whereas p-m TOR was lower(P<0.05)12 hours after 6-OHDA treatment in SH-SY5 Y cells.There was no significant change in the above indexes in the ALA alone group;compared with the 6-OHDA group,the levels of both LC3-II and p-AMPK were decreased(P<0.05),while p-m TOR was increased(P<0.05)in the ALA+6-OHDA group.3.Compared with the control group,both decreased behavior ability and the loss of TH-positive neurons in substantia nigra were observed in the rats of 6-OHDA group(P<0.05);compared with 6-OHDA group,the behavior ability of rats in 6-OHDA + ALA group was significantly improved,and the decline in TH neurons in substantia nigra was reversed(P<0.05).However,no significant difference was observed in behavior ability and TH neuron number between low ALA group and high ALA group(P>0.05).Conclusion1.6-OHDA induced oxidative stress and autophagy in SH-SY5 Y cells,and decreased cell survival.ALA may attenuate 6-OHDA-induced cytotoxicity by inhibiting oxidative stress and autophagy mediated by AMPK/m TOR pathway.2.ALA protected neurons in 6-OHDA-Parkinson's disease rats.