CHIP Protection In MPP~+-Induced PD Model In Vitro

BackgroundParkinson’s disease(PD)is the second common neurodegenerative disease after Alzheimer’s disease.The disease is characterized by severe degeneration,deletion and inclusion of Lewy body(LB)in dopaminergic neurons within dense substantia nigra.The main clinical manifestations include resting tremor,muscle rigidity,slow movement,and posture instability.Mitochondrial function damage-and α-synuclein aggregation-leaded increased oxidative stress response has been found in PD patients with death.In recent decades,the study of PD has recieved more and more attention.Due to the interaction of environment and genetic,long-term oxidative stress and oxidative DNA damage,most of them have been considered to play an important role in the pathogenesis of PD.1-methyl-4-phenyl-1,2,3-tetrahydropyridine(MPTP)can make all kinds of significant mammalian striatum and nervous system dopamine reduction,damaging the striatal dopamine pathway,further resulting in symptoms similar to PD.MPTP is converted into 1-methyl-4-phenylpyridine ion(MPP~+)by the glial cells-produced monoamine oxidase in vivo,which enters mitochondrial to inhibit the complex Ⅰ enzyme activity,ROS production,mitochondrial dysfunction of oxidative respiratory chain,resulting in dopaminergic neuronal oxidative stress injury.MPP~+ has been widely used to simulate the performance of PD damage in vitro.However,the mechanism of MPP~+-induced dopaminergic neuronal death is not yet fully understood.CHIP(Carboxy terminus of HSP70 interacting protein),a ubiquitin ligase,is a kind of auxiliary chaperone protein,which plays an important role in protein folding,assembly,transport and degradation.CHIP has E3 ubiquitin ligase activity and,can ubiquitinate tau,alpha-synuclein,and polyglutamine protein(PolyQ),which are the proteins accumulated abnormally in neurodegenerative diseases.The CHIP can bind to HSP70,HSP90 or other molecular chaperones,degrade unfolded or misfolded proteins.Current studies have shown that CHIP is involved in maintaining normal cellular function and mitochondrial function and,CHIP dysfunction is leads to stressful intolerance and rapid death.In this study,we observed the protective effect of CHIP on MPP~+-induced injury of SH-SY5 Y cells by CHIP overexpression and CHIP shRNA knockdown in SH-SY5 Y cell line of human neuroblastoma cells,and discussed the possible molecular mechanism.Objective1.To construct MPP +-induced PD in vitro model;to screen out the optimal concentration of MPP +-induced SH-SY5 Y cell injury;2.To observe the protective effect of CHIP on MPP +-induced injury of SH-SY5 Y cells and to explore the possible molecular mechanism of protection of MPP +-induced SH-SY5 Y cells induced by CHIP.Methods1.The cell viability was measured by CCK8 method,and the LDH and ATP levels were measured by luminometer.2.The pcDNA-FLAG gene expression vector pcDNA-FLAG-CHIP,the expression of pEGFP-N3-CHIP-shRNA gene expression vector in CHIP expression were transfected into human embryonic blastocysts by eukaryotic cell transfection technique(Named Vector group,CHIP group,Scramble group,CHIP-shRNA group,respectively).3.Western blot was used to detect the expression level of Bax and Bcl-2 and the expression level of mitochondrial dynamin-related protein DRP1 in mitochondria.4.Mdivi-1 was used to inhibit the activity of DRP1 protein and detect the expression levels of DRP1,the LDH and ATP levels were measured by luminometer.Results1.Establishment of MPP~+ damage SH-SY5 Y Cell Model The results of CCK8 showed that the survival rate of normal SH-SY5 Y cells decreased in a dose-dependent manner with the increase of MPP~+ concentration.When the concentration of MPP~+ was maintain at 2 mM,the cell survival rate remained at about 60%.In order to make the cells in a state of injury without causing cell death,2m M was selected as a follow-up concentration.2.CCK-8 results showed that,compared with the control group,overexpressed of CHIP could reduce cell damage and increased cell viability.The cell viability was decreased after knockout CHIP compared with the control group.3.The contents of ATP and LDH showed that overexpression of CHIP could alleviate mitochondrial damage,increase the ATP content and decrease the LDH release.After knocking out CHIP,the mitochondrial ATP content decreased and the LDH release increased.(P < 0.05)4.Western Blot showed that the expression level of Bax /Bcl-2 ratio of endogenous apoptotic protein in CHIP group was significantly higher than that in CHIP-shRNA group(P < 0.05).5.Western Blot showed that the expression level of DRP1 protein was significantly decreased by overexpressed of CHIP compared with the control group.The expression level of DRP1 protein was significantly increased after knockout CHIP.(P < 0.05)Compared with CHIP-shRNA group without Mdivi-1,the content of ATP in CHIP-shRNA group increased significantly,the content of LDH was significantly decreased and the expression level of DRP1 was significantly decreased in Mdivi-1-treated CHP-shRNA group(P <0.05)Conclusions1.CHIP has protective effects on MPP +-induced Parkinson’s disease in vitro,and this protective effect may be related to the mitochondrial function of neuronal cells.2.The expression level of DRP1 in MPP +-induced cell injury is increased,and CHIP may play a neuroprotective role in the mitochondrial function by inhibiting the expression of mitochondrial DRP1 after MPP +-induced cell injury.