The Ameliorating Effect Of Tideglusib On Dopaminergic Cells Injury Induced By MPP~+/MPTP

Objective:Parkinson’s disease(PD)is the second most prevalent neurodegenerative disease.Currently,increasing dopamine concentrations or directly stimulating dopamine receptors as the drug therapeutics,are the main treatment measures of Parkinson’s disease,thereby improving and relieving symptoms.However,long-term use of such drugs cannot prevent or slow the progression of PD.Hence,there is an urgent need to seek new drugs with multiple potentials in alleviating the death of DA neurons in PD and slow the progression of PD.Glycogen synthase kinase 3β(GSK3β)is a potential therapeutic target.Inhibition of its activity in PD models can play a protective role,reduce apoptosis,increase cell vitality and so on.In this study,we used Tideglusib(Tide),a specific GSK-3β inhibitor,to explore its role in 1-methyl-4phenylpyridinium iodide(MPP+)and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)induced apoptosis in PD model.Methods:(1)Apoptosis of SH-SY5Y cells induced by MPP+was used as an in vitro model of PD,MTT assay was used to detect cell viability,and Hoechst staining and flow cytometry were used to detect cell apoptosis.Western blot was used to detect changes in phosphorylation levels of GSK-3β and tau proteins over time and the changes in phosphorylation levels of the two proteins with or without Tide treatment.(2)For the exploration of the pathway,we mainly used Western blot and immunofluorescence as detection methods to detect the effects of inhibition of GSK3β on the expression or activity of neurogenic gene Notch homologous protein 1(Notch-1),Hairy and enhancer of split 1(Hesl)and extracellular signal regulated kinase(ERK).(3)In the process of verifying the pathway,we used DAPT to inhibit the cleavage of Notch by inhibiting y-secretase,thereby inhibiting the activation of Notch-1 pathway.MTT was used to detect the effect of inhibition of this pathway on the viability of SHSY5Y cells induced by MPP+.Western blot was used to detect the expression of active form of apoptosis-associated cysteine peptidase 3(caspase-3).Calcein-propidium iodide(Calcein/PI)staining was used to observe the effect on cell death under the model conditions.(4)After siRNA knocked down the expression of Hes1 protein,cell viability was detected by MTT,the expression of apoptotic protein cleaved caspase 3 was detected Western blot,and the cell death rate was detected by Hoechst/PI to further explore the role of Notch-1 pathway in MPP+induced PD cell model.(5)After overexpress Hes1,detected cell viability by MTT assay and detected apoptotic proteins to explore the role of Notch-1/Hesl pathway in the efficacy of Tide by Western blot.(6)Using ERK protein inhibitor,SCH772984,blocked the phosphorylation of ERK,to explore the role of ERK protein activation in the efficacy of Tide.(7)Using behavioral experiments,immunofluorescence to detect the effect of Tide on the morphology and behavior of PD mice obtained by intraperitoneal injection of MPTP and using Western blot to detect apoptotic proteins and verify mechanism related proteins.Results:(1)Tide protected PD model from toxicity induced by MPP+in vitro,increased cell viability of SH-SY5Y cells,reduced apoptosis induced by MPP+,inhibited the activity of GSK-3β,reduced the production of phosphorylated tau protein.(2)In the mechanism study,it was found that Tide reduced the entry of Notch-1 into the nucleus,reduced the transcription and protein expression of Hesl,and increased the phosphorylation level of ERK.(3)Using DAPT and siRNA knocked down the expression of Hes1 to inhibit the activation of Notch-1.Inhibition of Notch 1 pathway decreased cell apoptosis induce by MPP+.At the same time,the use of ERK inhibition found that ERK inhibitors did not block the efficacy of Tide.(4)Overexpression of Hes1 reversed the protective effect of Tide on MPP+induced apoptosis,increase the expression of cleaved caspase 3 reduced by Tide,and reduced the expression of anti-apoptotic protein Bcl2.ERK inhibitors did not block the effectiveness of Tide.(5)Tide reduced the expression level of NICD/Hes1 in the substantia nigra and striatum of PD model mice.(6)Tide reduced the expression of the apoptotic protein Bax,and increased the expression of the anti-apoptotic protein Bcl2,increased the expression of TH in the substantia nigra and striatum,improved the dyskinesia of PD model mice caused by MPTP.Conclusion:Tide reduced the apoptosis of PD models induced by MPP+/MPTP by inhibiting the activation of GSK-3β/Notch-1/Hesl pathway.