电针神庭、百会穴对脑缺血再灌注大鼠学习记忆能力及自噬相关蛋白表达的影响
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摘要
目的:观察电针神庭、百会穴后对缺血再灌注大鼠的学习记忆能力的影响,和自噬相关蛋白表达的数值分析。方法:线栓法制备SD大鼠脑缺血再灌注模型。将造模成功的45只SD大鼠采用随机数字表法分成3组,即电针组(n=15)、模型组(n=15)和假手术组(n=15)。通过神经行为学评分评估各组大鼠脑神经损伤程度。电针组大鼠模型制备成功后行电针神庭、百会穴干预治疗14 d。Morris水迷宫实验进行行为学观测;2,3,5-氯化三苯基四氮唑(TTC)染色法观察脑梗死的区域及脑梗死的体积;RT-PCR检测PI3K、AKT、Beclin-1的表达水平。结果:神经功能缺损评分:电针组评分明显低于模型组和假手术组,差异具有统计学意义(P<0.05);Morris水迷宫实验:与模型组相比,电针组穿越逃生平台的次数明显增多,差异具有统计学意义(P<0.001),其逃避潜伏期明显降低,差异具有统计学意义(P<0.001);TTC染色:电针组脑梗死体积较模型组和假手术组明显缩小,差异具有统计学意义(P<0.001);RT-PCR法:与模型组相比,电针组大鼠皮质及海马区PI3K、AKT、Beclin-1蛋白表达水平升高,差异显著(P<0.05)。结论:电针神庭、百会穴可明显改善脑缺血再灌注大鼠的学习及记忆能力,且可调控自噬相关蛋白PI3K、AKT、Beclin-1的表达,从而达到保护大鼠脑组织的作用,同时可从一方面说明细胞自噬调控机制在具有认知障碍的脑卒中患者中发挥着至关重要的作用。
Objective:To observe the effects of electroacupuncture at Shenting(DU24) and Baihui(DU20) on learning and memory ability of rats with ischemia reperfusion and the expression of autophagy related proteins. Methods:Forty-five MCAO models were divided into electroacupuncture group(n=15), ischemia control group(n=15) and sham control group(n=15) after the models were successed. Neurobehavioral scores were used to assess the degree of brain damage in rats. The electroacupuncture group received electroacupuncture at Shenting(DU24)and Baihui(DU20)for 14 days. The learning and memory ability were tested by Morris water maze. The area of cerebral infarction and the volume of cerebral infarction were detected by TTC. The expression levels of PI3 K, AKT and Beclin-1 were detected by RT-PCR. Results: Neurological impairment score: The score of the electroacupuncture group was significantly lower than that of the ischemia control group and the sham control group(P<0.05). Morris water maze experiment: Compared with the ischemia control group, the frequency of the electroacupuncture group through the escape platform increased significantly(P<0.001). The escape latency was significantly reduced(P<0.001). TTC:The volume of cerebral infarction in the electroacupuncture group was significantly smaller than that in the ischemia control group and the sham control group(P<0.001). RT-PCR: Compared with ischemia control group, the expressions of PI3 K, AKT and Beclin-1 protein in the cortex and hippocampus of the electroacupuncture group were increasedand the difference was significant(P<0.05). Conclusion:Electroacupuncture at Shenting(DU24) and Baihui(DU20) can significantly improve the ability of learning and memory among the MACO rats,and can regulate autophagy related protein expressions of PI3 K, AKT and Beclin-1,which can protect the brain of MACO rats. At the same time, it can be seen that the autophagy regulation mechanism plays a vital role in the patients with cognitive impairment.
Objective:To observe the effects of electroacupuncture at Shenting(DU24) and Baihui(DU20) on learning and memory ability of rats with ischemia reperfusion and the expression of autophagy related proteins. Methods:Forty-five MCAO models were divided into electroacupuncture group(n=15), ischemia control group(n=15) and sham control group(n=15) after the models were successed. Neurobehavioral scores were used to assess the degree of brain damage in rats. The electroacupuncture group received electroacupuncture at Shenting(DU24)and Baihui(DU20)for 14 days. The learning and memory ability were tested by Morris water maze. The area of cerebral infarction and the volume of cerebral infarction were detected by TTC. The expression levels of PI3 K, AKT and Beclin-1 were detected by RT-PCR. Results: Neurological impairment score: The score of the electroacupuncture group was significantly lower than that of the ischemia control group and the sham control group(P<0.05). Morris water maze experiment: Compared with the ischemia control group, the frequency of the electroacupuncture group through the escape platform increased significantly(P<0.001). The escape latency was significantly reduced(P<0.001). TTC:The volume of cerebral infarction in the electroacupuncture group was significantly smaller than that in the ischemia control group and the sham control group(P<0.001). RT-PCR: Compared with ischemia control group, the expressions of PI3 K, AKT and Beclin-1 protein in the cortex and hippocampus of the electroacupuncture group were increasedand the difference was significant(P<0.05). Conclusion:Electroacupuncture at Shenting(DU24) and Baihui(DU20) can significantly improve the ability of learning and memory among the MACO rats,and can regulate autophagy related protein expressions of PI3 K, AKT and Beclin-1,which can protect the brain of MACO rats. At the same time, it can be seen that the autophagy regulation mechanism plays a vital role in the patients with cognitive impairment.
引文
[1] 金昊,柳维林,林冰冰,等.电针百会、神庭穴对脑缺血再灌注大鼠学习记忆能力及海马5-羟色胺1A受体表达的影响[J].中国康复理论与实践,2017,23(10):1145-1151.
[2] 李忠仁. 实验针灸学[M].北京:中国中医药出版社,2007:360.
[3] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
[4] 金昊,柳维林,林冰冰,等.电针百会、神庭穴对脑缺血再灌注大鼠学习记忆能力及海马5-羟色胺1A受体表达的影响[J].中国康复理论与实践,2017,23(10):1145-1151.
[5] 高玲莉,冯晓东.电针神庭、百会穴对脑缺血再灌注后认知障碍大鼠学习记忆能力及自噬相关基因和蛋白表达的影响[J].康复学报,2016,26(4):17-22.
[6] 孙李晴.大鼠脑缺血再灌注后CathepsinL与细胞自噬的相关研究[D].长沙:南华大学,2015:1-26.
[7] Luo TF,Park Y,Sun X,et al.Protein Misfolding,Aggregation,and Autophagy After Brain Ischemia[J].TRANSLATIONAL STROKE RESEARCH,2013,4:581-588.
[8] Kim MS,Quan W, Lee MS.Role of hypothalamic autophagy in the control of whole body energy balance[J].REVIEWS IN ENDOCRINE & METABOLIC DISORDERS,2013,14:377-386.
[9] 孟明,梁红格,方皓,等.PI3K/Akt/mTOR 通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.
[10] Beaulieu M,Sotnikova TD,Yao WD,et al. Lithium antagonizes dopamine dependent behaviors mediated by an akt/glycogen synthase kinase 3 signaling cascade[J].Proc Natl Acad Sci USA,2004,101(14):5099-5104.
[11] Song G,Ouyang G ,Bao S. Theactivationof Akt/PKB signalingpathwayandcellsurvival[J].Cell Mol Med,2005,9(1):59-71.
[12] Isabelle V,Vojo D.The role of PI3P phosphatases in the regulation of autophagy[J].FEBS Letters,2010,584(7):1313-1318.
[13] Matsui T,Tao J,Monte F,et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo[J].Circulation,2001,104(3):30-335.
[14] 王和峰,翟纯刚,庞文会,等.PI3K/Akt/mTOR 信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[J].中国病理生理杂志,2013,29(3):390-397.
[15] Shioda N, Morioka HM,Fukunaga k. Bis(1-oxy-2-pyridinethiolato)oxovana dium(IV)enhances neurogenesis via phosphatidylinositol 3-kinase/Akt and extracellular signal regulated kinase activation in the hippocampal subgranular zone after mouse focal cerebral ischemia[J]. Neuroscience,2008,155(3):876-887.
[16] Vanderlaag K, Su Y, Frankel AE, et al. 1,1- Bis(3’-indolyl)-1-(p-substituted phenyl) methanes induce autophagiccell death in estrogen receptor negative breast cancer[J]. BMCCancer, 2010, 10: 669.
[17] 黄凤媛,李菲,黄贵华,等. 自噬基因Beclin1 启动子细胞自噬模型的建立[J]. 广州中医药大学学报, 2015, 32(2): 325-329.
[2] 李忠仁. 实验针灸学[M].北京:中国中医药出版社,2007:360.
[3] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
[4] 金昊,柳维林,林冰冰,等.电针百会、神庭穴对脑缺血再灌注大鼠学习记忆能力及海马5-羟色胺1A受体表达的影响[J].中国康复理论与实践,2017,23(10):1145-1151.
[5] 高玲莉,冯晓东.电针神庭、百会穴对脑缺血再灌注后认知障碍大鼠学习记忆能力及自噬相关基因和蛋白表达的影响[J].康复学报,2016,26(4):17-22.
[6] 孙李晴.大鼠脑缺血再灌注后CathepsinL与细胞自噬的相关研究[D].长沙:南华大学,2015:1-26.
[7] Luo TF,Park Y,Sun X,et al.Protein Misfolding,Aggregation,and Autophagy After Brain Ischemia[J].TRANSLATIONAL STROKE RESEARCH,2013,4:581-588.
[8] Kim MS,Quan W, Lee MS.Role of hypothalamic autophagy in the control of whole body energy balance[J].REVIEWS IN ENDOCRINE & METABOLIC DISORDERS,2013,14:377-386.
[9] 孟明,梁红格,方皓,等.PI3K/Akt/mTOR 通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.
[10] Beaulieu M,Sotnikova TD,Yao WD,et al. Lithium antagonizes dopamine dependent behaviors mediated by an akt/glycogen synthase kinase 3 signaling cascade[J].Proc Natl Acad Sci USA,2004,101(14):5099-5104.
[11] Song G,Ouyang G ,Bao S. Theactivationof Akt/PKB signalingpathwayandcellsurvival[J].Cell Mol Med,2005,9(1):59-71.
[12] Isabelle V,Vojo D.The role of PI3P phosphatases in the regulation of autophagy[J].FEBS Letters,2010,584(7):1313-1318.
[13] Matsui T,Tao J,Monte F,et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo[J].Circulation,2001,104(3):30-335.
[14] 王和峰,翟纯刚,庞文会,等.PI3K/Akt/mTOR 信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[J].中国病理生理杂志,2013,29(3):390-397.
[15] Shioda N, Morioka HM,Fukunaga k. Bis(1-oxy-2-pyridinethiolato)oxovana dium(IV)enhances neurogenesis via phosphatidylinositol 3-kinase/Akt and extracellular signal regulated kinase activation in the hippocampal subgranular zone after mouse focal cerebral ischemia[J]. Neuroscience,2008,155(3):876-887.
[16] Vanderlaag K, Su Y, Frankel AE, et al. 1,1- Bis(3’-indolyl)-1-(p-substituted phenyl) methanes induce autophagiccell death in estrogen receptor negative breast cancer[J]. BMCCancer, 2010, 10: 669.
[17] 黄凤媛,李菲,黄贵华,等. 自噬基因Beclin1 启动子细胞自噬模型的建立[J]. 广州中医药大学学报, 2015, 32(2): 325-329.