OBJECTIVES: 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 stimulation at GV16, LR3 and ST36, while the sham EA group received shallow needling 1 mm away from the above acupoints without electrical stimulation. The motor ability of mice in each group was evaluated using an open field experiment. 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, LC3Ⅱ 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 (<0.01)；the positive expressions of TH and LC3 were decreased (<0.01), while the positive expression of α-syn increased (<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 LC3Ⅱ mRNA and protein in the midbrain substantia nigra were decreased (<0.01), while the expression levels of α-syn and P62 mRNA and protein were increased (<0.01,<0.05). Compared with the model group, the mice in EA group showed a significant increase in the total exercise distance, time, movement speed and times of crossing central region (<0.01,<0.05)；the positive expressions of TH and LC3 were increased (<0.01,<0.05), while the positive expression of α-syn was decreased (<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 LC3Ⅱ mRNA and protein in the midbrain substantia nigra were increased (<0.01,<0.05), while the mRNA and protein expression levels of α-syn and P62 were decreased (<0.01)；the sham EA group showed an increase in the total exercise distance and time(<0.05), with an increase in the positive expression of TH (<0.05) and a decrease in the positive expression of α-syn (<0.05)；some mitochondria exhibited swelling, and no autophagic vacuoles were observed；the protein expression levels of TH, SIRT3, Parkin and LC3Ⅱ were increased (<0.01,<0.05), and the expression levels of P62 mRNA, α-syn mRNA and protein were decreased (<0.01,<0.05), and LC3Ⅱ mRNA expression was increased (<0.05). In comparison to the sham EA group, the EA group showed an extension in the total exercise time (<0.01), the positive expression and mRNA expression levels of α-syn were decreased (<0.01,<0.05), while the expression levels of TH, SIRT3, PINK1, Parkin mRNA and SIRT3 protein were increased (<0.05). P P P P P P P P P P P P P P P P P P P P P P P P P P
CONCLUSIONS: EA at GV16, 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.
