Experimental Study Of Salidroside Protecting Dopaminergic Neurons Through The Dj-1/akt Pathway

Objective:To investigate whether Sal can exert neuroprotection in vitro and in vivo through the DJ-1/Akt antioxidant pathway.Experiment 1:To establish the damage model of PD-MN9D cells induced by MPP~+and screen out the optimal concentration of MPP~+for apoptosis of MN9D cells and the optimal protective concentration of Sal for MPP~+induced PD cells.Experiment 2,3:PD-MN9D cells were transfected with siAkt and siDJ-1,respectively,to explore the protective mechanism of Akt and DJ-1 on cell model induced by Sal.Experiment 4:To establish the PD model of C57BL/6 mice induced by MPTP,and to investigate the effects of Sal on the behavior and neurohistology of the mouse model.Methods:Experiment 1:CCK-8 kit was used to detect cell viability,and the optimal concentration of MPP~+and Sal was screened out respectively;Four groups was established:(1)blank control group,(2)Sal group,(3)MPP~+group,(4)Sal+MPP~+group;Hoechst staining and flow cytometry were used to detect the morphological changes and apoptotic levels of cells in different treatment groups;The protein expression levels of DJ-1,Akt,p-Akt,GSK3?,p-GSK3?and cleaved-caspase3 were detected by Western blotting.Experiment 2,3:PD-MN9D cells were transfected with siAkt and siDJ-1,respectively,four groups was established:(1)blank control+scramble siRNA group,(2)MPP~++scramble siRNA group,(3)MPP~++Sal+scramble siRNA group,(4)MPP~++Sal+scramble siRNA+SiAkt/siDJ-1 group;Hoechst staining and flow cytometry were used to detect the morphological changes and apoptotic levels of cells in different treatment groups;The protein expression levels of DJ-1,Akt,p-Akt,GSK3?,p-GSK3?were detected by Western blotting.Experiment 4:Animal grouping and model preparation:40 C57BL/6 mice were randomly divided into 4 groups,10 mice in each group,Four groups was established:(1)normal control group:continuous intraperitoneal injection of saline for 12 days,once a day;(2)Sal group:After 5 days of continuous intraperitoneal injection of normal saline,Sal(45mg/Kg)was intraperitoneally injected for 7 days,once a day;(3)MPTP model group:MPTP model group:MPTP(30mg/Kg)was injected intraperitoneally for 5 consecutive days,then saline was injected intraperitoneally for 7 days,once a day.(4)Sal+MPTP group:MPTP(30mg/Kg)was injected intraperitoneally for 5 consecutive days,then Sal(45mg/Kg)was injected intraperitoneally for 7 days,once a day;The mice in each group were treated according to the experimental design,The rod climbing test and suspension test were used to detect the behavior of mice.The brain tissues of the tested mice were fixed and frozen sections were prepared,and immunohistochemical method was used to detect tyrosine hydroxylase(TH)and dopamine transporter(DAT)immunofluorescence staining,counting the number of positive neurons,and observing the effect of Sal on the number of TH and DAT in MPTP-induced mouse model.The protein expression levels of DJ-1,Akt,p-Akt,GSK3?,p-GSK3?and cleaved-caspase3 were detected by Western blotting.Results:Experiment 1:Cells treated with different concentrations of MPP~+(100,200,300,400,500 uM)for 24 hours showed a dose-dependent decrease in cell viability,300uM MPP~+was used as the optimal concentration for 24h treatment of MN9D cells for subsequent cell experiments;After pretreatment with Sal at different concentrations for24 hours,the cells were treated with 300 uM MPP~+for 24 hours,and sifted out that 60uM Sal could significantly reduce the cell viability induced by MPP~+;After Hoechst staining,Sal pretreatment significantly reduced MPP~+induced cell morphology,nuclear fragmentation and chromatin condensation(P<0.05);Flow cytometry showed that Sal pretreatment could significantly reduce the ratio of MPP~+induced apoptosis(P<0.01);After 24 hours of Sal pretreatment,the expression levels of DJ-1,p-Akt/Akt,p-GSK3?/GSK3?increased(P<0.05)and Cleaved-caspase 3 decreased(P<0.05)compared with MPP~+group.Experiment 2,3:After siAkt and siDJ-1 were transfected into PD-MN9D cells respectively,both siAkt and siDJ-1 decreased the protective effects of Sal on MPP~+induced cell morphology,nuclear fragmentation and chromatin condensation(P<0.05);After siAkt transfection,Sal inhibited the expression of p-Akt/Akt,p-GSK3?/GSK3?induced by MPP~+after siAkt transfection(P<0.05),but did not inhibit the expression of DJ-1(P>0.05).After siDJ-1 transfection,Sal inhibited the expression of DJ-1,p-Akt/Akt,p-GSK3?/GSK3?induced by MPP~+(P<0.05).Experiment 4:After intraperitoneal injection of MPTP,the mice began to show tail erection,arch back,unstable gait,slow movement and decrease,the symptoms increased with the days of administration,after intraperitoneal injection of Sal,the symptoms gradually improved.In rod climbing and suspension experiments,compared with MPTP group,the time of rod climbing was shortened(P<0.01)and the suspension score was increased(P<0.05)after Sal treatment.Immunofluorescence staining showed that the number of TH and DAT positive cells in MPTP group was significantly lower than that in control group(P<0.01),after Sal treatment,the number of TH and DAT positive cells increased significantly(P<0.05).Westen Blot technique was used to detect the expression of related proteins in the middle brain tissue of mice treated with different groups:Compared with the control group,the expression levels of DJ-1,p-Akt/Akt,p-GSK3?/GSK3?in the middle brain tissue of MPTP model mice decreased,and the expression levels of Cleaved-caspase3 increased(P<0.05).After Sal treatment,the expression levels of DJ-1,p-GSK3?/GSK3?increased(P<0.05),and the expression levels of p-Akt/Akt increased(P<0.05).The expression level of Cleaved-caspase 3decreased with the increase of the expression level,but there was no statistical significance(P>0.05).Conclusion:Experiment 1:Sal can reduce MPP~+induced cell viability,inhibit apoptosis,mitigate apoptotic changes such as nuclear fragmentation,increase the expression levels of DJ-1,p-Akt/Akt,p-GSK3?/GSK3?,inhibit the expression of Cleaved-caspase3 and play a neuroprotective role.Experiment 2,3:DJ-1 and Akt,as well as the regulated antioxidant enzymes,participated in the protective effect of Sal on MPP+induced apoptosis.SiAkt did not significantly affect the expression of DJ-1 after transfection,while siDJ-1 significantly affected the expression of Akt after transfection.This suggests that DJ-1 may be an upstream regulator of Akt,regulating Akt and other anti-apoptotic proteins,and plays a role in the regulation of oxidative stress and neuron protection.Important role.Experiment 4:Sal can significantly alleviate the motor disorder symptoms,improve behavioral abnormalities,reduce the number of TH and DAT neurons in the substantia nigra of mice and protect DA neurons.Sal may play a neuroprotective role in MPTP-induced mouse model through DJ-1/Akt pathway.