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 (P<0.01)；the positive expressions of TH and LC3 were decreased (P<0.01), while the positive expression of α-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 LC3Ⅱ 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 significant 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 LC3Ⅱ 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 expression 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 LC3Ⅱ 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 LC3Ⅱ 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).
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.
目的: 观察电针“风府”“太冲”“足三里”对帕金森病（PD）小鼠中脑黑质沉默调节蛋白3（SIRT3）及其下游PTEN诱导的假定激酶1（PINK1）、PARK2基因编码蛋白（Parkin）介导的线粒体自噬的影响，探讨电针治疗PD的可能机制。方法: C57BL/6小鼠随机分为对照组、模型组、电针组、假电针组，每组12只。采用腹腔注射1-甲基-4-苯基-1，2，3，6-四氢吡啶复制PD小鼠模型。电针组小鼠给予针刺“风府”“太冲”“足三里”，每天1次，每次20 min，连续12 d。以旷场实验评估各组小鼠的运动能力；免疫组织化学法检测各组小鼠中脑黑质区酪氨酸羟化酶（TH）、α-突触核蛋白（α-syn）阳性表达；透射电镜观察黑质区神经元超微结构；免疫荧光染色法检测小鼠黑质自噬轻链蛋白3（LC3）的阳性表达；实时荧光定量PCR法检测小鼠黑质TH、α-syn、SIRT3、PINK1、Parkin和线粒体自噬受体蛋白P62、Beclin-1、LC3Ⅱ mRNA表达水平；Western blot法检测小鼠黑质TH、α-syn、SIRT3、PINK1、Parkin、P62、Beclin-1、LC3Ⅱ蛋白表达水平。结果: 与对照组比较，模型组小鼠运动总距离、运动总时间、运动速度和进入中央区域的次数均下降（P<0.01）；TH、LC3阳性表达减少（P<0.01），α-syn阳性表达增加（P<0.01）；部分线粒体肿胀，线粒体嵴断裂减少，溶酶体数量减少；中脑黑质TH、SIRT3、PINK1、Parkin、Beclin-1、LC3Ⅱ mRNA及蛋白表达水平降低（P<0.01），α-syn和P62 mRNA及蛋白表达水平升高（P<0.01，P<0.05）。与模型组比较，电针组小鼠运动总距离、运动总时间、运动速度和进入中央区域的次数增加（P<0.01，P<0.05），TH、LC3阳性表达增加（P<0.01，P<0.05），α-syn阳性表达减少（P<0.01），线粒体数量增多，出现自噬小体，肿胀减轻，中脑黑质TH、SIRT3、PINK1、Parkin、Beclin-1、LC3Ⅱ mRNA及蛋白表达水平升高（P<0.01，P<0.05），α-syn、P62 mRNA及蛋白表达水平降低（P<0.01）；假电针组运动总距离、总时间增加（P<0.05），TH阳性表达增加（P<0.05），α-syn阳性表达减少（P<0.05），部分线粒体肿胀，未见自噬体出现，TH、SIRT3、Parkin、LC3Ⅱ蛋白表达水平升高（P<0.01，P<0.05），P62 mRNA、α-syn mRNA及蛋白表达水平降低（P<0.01，P<0.05），LC3Ⅱ mRNA表达水平升高（P<0.05）。与假电针组比较，电针组运动总时间延长（P<0.01），α-syn阳性表达及mRNA表达水平下降（P<0.01，P<0.05），TH、SIRT3、PINK1、Parkin mRNA及SIRT3蛋白表达水平升高（P<0.05）。结论: 电针“风府”“太冲”“足三里”可改善PD小鼠运动能力，其机制可能是通过激活SIRT3/PINK1/Parkin通路，增强线粒体自噬，促进PD小鼠中脑黑质TH表达，减少α-syn聚集，从而发挥神经保护作用。.
Keywords: Electroacupuncture; Mitophagy; PARK2 gene coding protein; PTEN induced putative kinase 1; Parkinson’s disease; Silencing regulatory protein 3.