| ObjectiveParkinson’s disease(PD)is a common and refractory neurodegenerative disease.Impaired autophagy is one of the key pathogenic mechanisms of PD.Nuclear factor erythroid-2 related factor 2(Nrf2)is an important regulator of autophagy and is involved in the pathogenesis of PD.Based on the regulation of autophagy by Nrf2 signaling,this study aimed to investigate the ameliorative effects of electroacupuncture(EA)at Baihui(GV20)and Taichong(LR3)points on nerve injury in PD mice and its potential mechanism.Methods1.Wild-type C57 mice were randomly divided into 18 mice in the normal control group,and the remaining mice were injected intraperitoneally with MPTP 30 mg/kg once a day for 7 days to establish the PD mouse model.The success of the model establishment was evaluated by using behavioral experiments.The MPTP-induced PD mice were then randomly divided into three groups:MPTP group,MPTP+EA and MPTP+Madopar,with 18 mice in each group.The control group and MPTP group were kept normally;EA group was continuously acupunctured at "Baihui" and"Taichong" points for 20 min/day for 14 days;MPTP+Madopar group was given positive drug 62.5 mg/kg/d for 7 days;the latter 7 days were increased to 125 mg/kg/d.At the end of all interventions,changes in motor function were examined in each group of mice using rotarod and pole tests.The expression of DAergic neuronal marker tyrosine hydroxylase(TH)and pathological product α-synuclein(α-syn)in mouse substantia nigra-striatal was detected by immunohistochemistry and Western Blotting,and the expression of Nrf2 signal in mouse substantia nigra-striatal was detected by Western Blotting and RT-qPCR.The levels of autophagy proteins such as Agt7,p62 and LC3 were detected by WB to investigate the neuroprotective effect of EA and the regulation of autophagy and Nrf2 by EA.2.Using gene identification techniques to screen out male pure-sibling Nrf2 knockout(Nrf2 KO)mice.Nrf2 knockout(Nrf2 KO)mice were randomly divided into 10 mice in the control groups,and the remaining mice were injected with MPTP 30 mg/kg intraperitoneally once daily for 7 days to establish PD mouse model,and the success of the model was judged by behavioral observation.After successful modeling,Nrf2 KO PD mice were randomly divided into MPTP group and MPTP+electroacupuncture group,with 10 mice in each group.The behavioral changes were detected by rotarod and pole tests,and the expression of TH and α-syn in the substantia nigra-striata was detected by Western Blotting and immunohistochemistry,and the expression of Keapl,Agt7,p62 and LC3 was detected by WB.Results1.In WT mice,behavioral results showed that motor coordination was diminished in the MPTP group compared with normal controls(P<0.05)and motor impairment was improved in the electroacupuncture group of PD mice(P<0.05);and there was no significant difference between the electroacupuncture and drug groups(P>0.05).IHC and WB showed that TH expression was decreased and α-syn expression was increased in the brain of MPTP-induced PD mice(P<0.05).Compared with the MPTP group,TH expression was increased and α-syn expression was decreased in the MPTP+EA group and MPTP+madopar group(P<0.05),and TH expression was not statistically significant between the two groups(P>0.05).RTqPCR and WB showed that total-Nrf2 expression was decreased and Keapl protein expression was increased in the MPTP group(P>0.05).WB results showed that the expression of Atg7 and LC3 was decreased and p62 protein was increased in MPTP group(P>0.05).EA could reverse the expression of these proteins(P<0.05);WB results showed that EA could up-regulate nuclear-Nrf2 expression,suggesting that EA activates Nrf2(P<0.05).2.In Nrf2 KO mice,behavioral experiments showed that:the motor ability of mice in the electroacupuncture group was not improved compared with the MPTP group(P>0.05);in immunohistochemical and WB experiments,the damage of DAergic neurons in the MPTP+electroacupuncture group was not alleviated compared with the MPTP group,and no significant difference was seen in the expression of α-syn between the two groups(P>0.05).The expression of autophagic proteins such as Agt7,p62,and LC3 was decreased in Nrf2 KO mice compared with WT mice control group(P<0.05);there was no significant change in Agt7,p62,and LC3 after electroacupuncture(P>0.05).Conclusions1.Electroacupuncture ameliorates motor dysfunction,alleviates MPTP-induced DAergic neuronal damage and reduces α-syn expression in PD mice,which has the effects of improvement on nerve injury in PD mice.2.Electroacupuncture can exert neuroprotective effects by modulating autophagy levels via activating Nrf2. |
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