Effects of electroacupuncture on mitophagy mediated by SIRT3/PINK1/Parkin pathway in Parkinson's disease mice
Objective To observe the effects of electroacupuncture(EA)at"Fengfu"(GV16),"Taichong"(LR3),and"Zusanli"(ST36)on mitophagy mediated by silencing regulatory protein 3(SIRT3)/PTEN induced putative kinase 1(PINK1)/PARK2 gene coding protein(Parkin)in the midbrain substantia nigra of Parkinson's disease(PD)mice,and to explore the potential mechanisms of EA in treating PD.Methods C57BL/6 mice were randomly divided into the control,model,EA,and sham EA groups,with 12 mice in each group.The PD mouse model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).The EA group received EA stimu-lation at GV16,LR3 and ST36,while the sham EA group received shallow needling 1 mm away from the above acu-points without electrical stimulation.The motor ability of mice in each group was evaluated using an open field experi-ment.Immunohistochemistry was used to detect the expression of tyrosine hydroxylase(TH)and α-synuclein(α-syn)in the substantia nigra of mice.The ultrastructure of neurons in substantia nigra was observed by transmission electron microscope(TEM).Immunofluorescence was used to detect the expression of the autophagy marker autophagy-associated protein light chain 3(LC3).The expression levels of TH,α-syn,SIRT3,PINK1,Parkin,P62,Beclin-1,LC3 Ⅱ mRNA and protein were detected by PCR and Western blot.Results Compared with the control group,mice in the model group showed a decrease in the total exercise distance,time,movement speed and times of crossing central region(P＜0.01);the positive expressions of TH and LC3 were decreased(P＜0.01),while the positive expression ofα-syn increased(P＜0.01),accompanied by mitochondrial swelling,mitochondrial cristae fragmentation and decrease,and decreased lysosome count;the expression levels of TH,SIRT3,PINK1,Parkin,Beclin-1,and LC3 Ⅱ mRNA and protein in the midbrain substantia nigra were decreased(P＜0.01),while the expression levels of α-syn and P62 mRNA and protein were increased(P＜0.01,P＜0.05).Compared with the model group,the mice in EA group showed a signifi-cant increase in the total exercise distance,time,movement speed and times of crossing central region(P＜0.01,P＜0.05);the positive expressions of TH and LC3 were increased(P＜0.01,P＜0.05),while the positive expression of α-syn was decreased(P＜0.01),accompanied by an increase in mitochondrial count,appearance of autophagic va-cuoles,and a decrease in swelling,the expression levels of TH,SIRT3,PINK1,Parkin,Beclin-1 and LC3 Ⅱ mRNA and protein in the midbrain substantia nigra were increased(P＜0.01,P＜0.05),while the mRNA and protein expression levels of α-syn and P62 were decreased(P＜0.01);the sham EA group showed an increase in the total exercise distance and time(P＜0.05),with an increase in the positive expression of TH(P＜0.05)and a decrease in the positive expres-sion of α-syn(P＜0.05);some mitochondria exhibited swelling,and no autophagic vacuoles were observed;the protein expression levels of TH,SIRT3,Parkin and LC3 Ⅱ were increased(P＜0.01,P＜0.05),and the expression levels of P62 mRNA,α-syn mRNA and protein were decreased(P＜0.01,P＜0.05),and LC3 Ⅱ mRNA expression was increased(P＜0.05).In comparison to the sham EA group,the EA group showed an extension in the total exercise time(P＜0.01),the positive expression and mRNA expression levels of α-syn were decreased(P＜0.01,P＜0.05),while the expression levels of TH,SIRT3,PINK1,Parkin mRNA and SIRT3 protein were increased(P＜0.05).Conclusion EAatGV16,LR3,and ST36 can exert neuroprotective function and improve the motor ability of PD mice by activating the SIRT3/PINK1/Parkin pathway to enhance the expression of TH and reduce α-syn aggregation in the substantia nigra of PD mice.
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