目的 探究电针治疗通过调控磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）、蛋白激酶B（protein kinase B，AKT）、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）影响帕金森大鼠的行为学反应和脑组织自噬机制。 方法 将60只大鼠随机分为对照组、帕金森组、电针组、西罗莫司组、电针+PI3K激活组，每组12只。制备帕金森大鼠模型，斜板实验、旷场实验评估大鼠运动行为学功能；免疫组化检测黑质内酪氨酸羟化酶（tyrosine hydroxylase，TH）、α-突触核蛋白（α-synuclein，α-syn）表达；提取脑组织线粒体，荧光素酶标记法检测ATP合成酶活性，线粒体呼吸检测仪检测线粒体活性；Western blot检测脑组织PI3K、AKT、mTOR、p-PI3K、p-AKT、p-mTOR及自噬相关蛋白微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）B-II/LC3B-I、Beclin1表达水平。 结果 与对照组相比，帕金森组大鼠斜板停留最大角度、跨格次数、站立次数、脑组织RCR、ATP合成酶活性、黑质内TH平均光密度显著降低，α-syn、Beclin1蛋白表达量、LC3B-II/LC3B-I和PI3K、AKT、mTOR蛋白磷酸化程度显著升高（P<0.05）；与帕金森组相比，电针组、西罗莫司组大鼠斜板停留最大角度、跨格次数、站立次数、脑组织RCR、ATP合成酶活性、Beclin1蛋白表达量、LC3B-II/LC3B-I、黑质内TH平均光密度显著升高，α-syn蛋白表达量和PI3K、AKT、mTOR蛋白磷酸化程度显著降低（P<0.05）；与电针组相比，电针+PI3K激活组大鼠斜板停留最大角度、跨格次数、站立次数、脑组织RCR、ATP合成酶活性、Beclin1蛋白表达量、LC3B-II/LC3B-I、黑质内TH平均光密度显著降低，α-syn蛋白表达量和PI3K、AKT、mTOR蛋白磷酸化程度显著升高（P<0.05）；与电针组相比，西罗莫司组各项指标无显著差异（P>0.05）。 结论 电针治疗可通过抑制PI3K、AKT、mTOR蛋白磷酸化，激活自噬，改善大鼠运动行为功能，从而治疗帕金森临床症状。
Abstract
Objective To explore the effect of electroacupuncture participating in the behavioral response and brain autophagy of Parkinson's rats by regulating phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR). Methods Sixty rats were randomly separated into a control group, a Parkinson's group, an electroacupuncture group, a sirolimus group, an electroacupuncture + PI3K activation group, with 12 rats in each group. The Parkinson's rat model was prepared, and the motor behavioral function of the rats was evaluated by the inclined plate test and the open field test. The expression of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in substantia nigra was measured by immunohistochemistry. Brain tissue mitochondria were extracted, ATP synthase activity was measured by luciferase labeling method, and mitochondrial activity was measured by mitochondrial respiration detector. The expression levels of PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR and autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3) B-II/LC3B-I, and Beclin1 in brain tissue were measured by Western blot. Results Compared with the control group, the Parkinson's group showed a significant decrease in the maximum angle of sloping plate stay, times of crossings lattice, times of standing, RCR in brain tissue, ATP synthase activity, and the average optical density of TH in the substantia nigra, and a significant increase in α-syn, Beclin1 protein expression, LC3B-II/LC3B-I and PI3K, AKT, mTOR protein phosphorylation (P<0.05). Compared with the Parkinson's group, the EA group and the sirolimus group showed significant increase in the maximum angle of sloping plate stay, times of crossings lattice, times of standing, RCR in brain tissue, ATP synthase activity, Beclin1 protein expression, LC3B-II/LC3B -I, and average optical density of TH in the substantia nigra, and significant decrease in the expression of α-syn protein and the protein phosphorylation of PI3K, AKT and mTOR (P<0.05). Compared with the electroacupuncture group, the rats in the electroacupuncture + PI3K activation group had a significant decrease in the maximum angle of sloping plate stay, times of crossings lattice, times of standing, RCR in brain tissue, ATP synthase activity, Beclin1 protein expression, LC3B-II/LC3B -I, and average optical density of TH in the substantia nigra, and a significant increase in the expression of α-syn protein and the protein phosphorylation of PI3K, AKT and mTOR (P<0.05). Compared with the electroacupuncture group, there was no significant difference in the indicators in the sirolimus group (P>0.05). Conclusions Electroacupuncture can inhibit the phosphorylation of PI3K, AKT and mTOR proteins, activate autophagy, and improve the motor behavioral function of rats, so as to treat the clinical symptoms of Parkinson's disease.
关键词
帕金森 /
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电针治疗 /
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磷脂酰肌醇3-激酶 /
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蛋白激酶B /
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哺乳动物雷帕霉素靶蛋白
Key words
Parkinson's disease /
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Electroacupuncture /
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Phosphatidylinositol 3-kinase /
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Protein kinase B /
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Mammalian target of rapamycin
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