电针神庭百会对脑缺血再灌注后认知障碍大鼠海马区自噬的影响
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摘要
目的:通过对比电针治疗前后对脑缺血再灌注后认知障碍大鼠海马区神经细胞自噬的变化,从而探讨电针神庭、百会对海马区神经细胞自噬的影响。方法:清洁级健康SD雄性大鼠50只,按照随机数字表分为电针组20只,模型组20只,假手术组10只。电针组和模型组采用改良Zea Longa线栓法建立大鼠脑缺血再灌注损伤模型,假手术组仅分离左侧的颈动脉,选用神经功能缺损症状评分标准,筛选出造模成功的大鼠。造模后2 h电针其神庭、百会穴,每次30 min,1次/d,电针治疗后3～5 d,通过Morris水迷宫实验完成对大鼠学习记忆能力的检测,造模后第7天断头取大鼠海马组织。选用神经功能缺损症状评分标准观察电针治疗前后大鼠神经功能情况,通过透电镜完成对海马神经细胞中自噬情况的观察,通过Western Blot、激光共聚焦显微镜完成对大鼠海马组织中LC3蛋白表达的定量分析。结果:(1)Morris水迷宫实验:与模型组相比较,电针组大鼠的逃避潜伏期缩短明显,穿越平台期的次数增多,路程缩短(P<0.05);(2)神经功能障碍评分:与假手术组大鼠相比,模型组及电针组大鼠均可见不同程度神经功能缺损;与模型组大鼠相比,电针组大鼠神经功能缺损症状明显减轻(P<0.05);(3)LC3蛋白:与假手术组大鼠相比,模型组及电针组大鼠LC3表达不同程度增多。与模型组相比,电针组大鼠LC3表达不同程度增加(P<0.05);(4)假手术组大鼠神经细胞未发生自噬。模型组可见自噬及凋亡形态结构。电针组大鼠与模型组大鼠相比,神经细胞自噬发生程度增高。结论:电针神庭、百会穴可明显改善大鼠神经功能缺损评分;能够有效的激活缺血区自噬,减轻大鼠海马神经细胞的损害,能提高自噬相关蛋白的表达水平,对神经细胞具有保护作用。
Objective:To detect electroacupuncture treatment of ischemia reperfusion rats hippocampus neural cells autophagy so as to discuss the impact on the hippocampus neural cellsautophagy. Methods: Fifty healthy male SD rats were randomly divided into electroacupuncture group(n=20), model group(n=20) and sham operated group(n=10). The model of cerebral ischemia reperfusion injury was established by modified Zea Longa suture method in electroacupuncture group and model group. The left carotid artery was isolated only in the sham operation group. On the second hour after modeling, the rats were treated with electroacupuncture for 30 min, once a day.And seven days after modeling, the animals were killed.We chose nerve function defect symptom scoring criteria to observethe nerve function and observed autophagy by electron microscopy. Western Blot and laser confocal microscopy were used to detected LC3 protein and gene expression. Results:(1)Morris water maze experiment:Compared with the model group,the electroacupuncture group' sescape latencywasobviously shortened,the numbers of crossing platform latencey was increased and the distance was shorter(P<0.05).(2)Nerve dysfunction score:Compared with the sham operation rats, the model and electroacupuncture groupshad visible different degrees of nerve function defect. The rats nerve dysfunction rating of the electroacupuncture group was obviously lower(P<0.05).(3)LC protein:Compared with the rats in the sham operation group, the expression of LC3 was increased in the model and the electroacupuncture groups. Compared with the rats in the model group, LC3 expression was increased in the electroacupuncture group(P<0.05).(4)No autophagy occurred in the nerve cells of rats in sham operation group. Model group had obvious autophagy and apoptosis morphology. Compared with rats in the model group, the incidence of autophagy increased.Conclusion: Electroacupuncture at Baihui(DU20) and Shenting(DU24) can significantly improve the neurological deficit scores of rats,,effectively activate autophagy in ischemic area, reduce rat hippocampal neuronal damage, improve the expression level of autophagy related protein, and has a protective effect on nerve cells.
Objective:To detect electroacupuncture treatment of ischemia reperfusion rats hippocampus neural cells autophagy so as to discuss the impact on the hippocampus neural cellsautophagy. Methods: Fifty healthy male SD rats were randomly divided into electroacupuncture group(n=20), model group(n=20) and sham operated group(n=10). The model of cerebral ischemia reperfusion injury was established by modified Zea Longa suture method in electroacupuncture group and model group. The left carotid artery was isolated only in the sham operation group. On the second hour after modeling, the rats were treated with electroacupuncture for 30 min, once a day.And seven days after modeling, the animals were killed.We chose nerve function defect symptom scoring criteria to observethe nerve function and observed autophagy by electron microscopy. Western Blot and laser confocal microscopy were used to detected LC3 protein and gene expression. Results:(1)Morris water maze experiment:Compared with the model group,the electroacupuncture group' sescape latencywasobviously shortened,the numbers of crossing platform latencey was increased and the distance was shorter(P<0.05).(2)Nerve dysfunction score:Compared with the sham operation rats, the model and electroacupuncture groupshad visible different degrees of nerve function defect. The rats nerve dysfunction rating of the electroacupuncture group was obviously lower(P<0.05).(3)LC protein:Compared with the rats in the sham operation group, the expression of LC3 was increased in the model and the electroacupuncture groups. Compared with the rats in the model group, LC3 expression was increased in the electroacupuncture group(P<0.05).(4)No autophagy occurred in the nerve cells of rats in sham operation group. Model group had obvious autophagy and apoptosis morphology. Compared with rats in the model group, the incidence of autophagy increased.Conclusion: Electroacupuncture at Baihui(DU20) and Shenting(DU24) can significantly improve the neurological deficit scores of rats,,effectively activate autophagy in ischemic area, reduce rat hippocampal neuronal damage, improve the expression level of autophagy related protein, and has a protective effect on nerve cells.
引文
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[13] 石秋艳,杨斌,孙原,等.Beclin-1、LC3-II 在缺血后处理大鼠再灌注中的表达及意义[J].中风与神经疾病杂志,2014,8(31):687-690.
[14] Zhao H,Sapolsky RM,Steinberg GK.Interrupting reperfusion asastroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats[J].J Cereb Blood Flow Metab,2012,26(9):1114-1121.
[15] 张巧梅,李立亚,高淅,等.电针对小鼠脑缺血后 Neurogenin2 表达和神经功能的影响[J].神经解剖学杂志,2013,29(3):321.
[16] 马璟曦,罗勇.电针合谷穴对大鼠局灶性脑缺血再灌注后脑内毛细血管密度及 CD34 表达的影响[J].中国康复理论与实践,2014,20(3):201.
[17] 刘延祥.自噬在电针治疗周围神经损伤修复中的作用研究[J].辽宁中医杂志,2013,24(8):2054.
[18] 高伶俐.电针神庭百会对缺血性脑卒中认知障碍大鼠自噬网络系统的影响[D].郑州:河南中医药大学,2016.
[19] 刘娇,冯晓东.电针神庭、百会穴配合康复训练治疗脑卒中后认知障碍的临床研究[J].中医学报,2013,28(4):608.
[20] Feng XD,Yang SL,Liu J,et al.Electroacupuncture ameliates cogni-tive impairment through inhibition of NF-k B-mediated neuronal cellapoptosis in cerebral ischemia-reperfusion injured rats[J].Mol Med Rep,2013,7:1516.
[2] 李梅,李晔,白洁.神经元自噬与缺血性脑卒中的相关研究[J].生命科学,2013,25(9):897-901.
[3] 孙金梅,张拥波,李继梅.自噬在缺血性卒中后的作用[J].中国卒中杂志,2013,8(4):311-315.
[4] 李忠仁.实验针灸学 [M].北京:中国中医药出版社,2007:360.
[5] Jin WY,Qiang X,Ke T,et al.Severe Global Cerebral Ischemia-Induced Programmed Necrosis of Hippocampal CA1 Neurons inRat is prevented by 3-Methyladenine:A Widely Used Inhibitor ofAutophagy[J].Neuropathol Exp Neurol,2011,70(4):314-322.
[6] Guo Z,Cao G,Yang H,et al.A combination of four active compounds alleviates cerebral ischemia-reperfusion injury in correlation with inhibition of autophagy and modulation of AMPK/m TOR and JNK pathways[J].J Neurosci Res,2014,92:1295-1306.
[7] Ravikumar B,Sarkar S,Davies JE,et al.Regulation of mammalian autophagy in physiology and pathophysiology[J].Physiol Rev 2010,90:1383-435.
[8] Wang P,Xue Y,Wei K,et al.ARRB1 /β-arrestin-1mediates neuro-protection through coordination of BECN1 dependent autophagy in cere-bral ischemia[J].Autophagy,2014,10(9):1535.
[9] 王燕梅.自噬参与低氧后处理对成年大鼠短暂全脑缺血的保护作用[D].广州:广州医学院,2010:312.
[10] Ding P,Wang D,Man HE.Short-term fasting introduced tolerance against focal cerebral ischemia by autophagy activation in rats [J].Chin J Neurosurg Dis Res,2013,1(3):201.
[11] Levine B.Cell biology:autophagy and cancer[J].Nature,2007,446:745-747.
[12] 高博.内质网应激诱导的自噬在脑缺血预适应和永久性缺血中的作用[D].苏州:苏州大学,2012:149-151.
[13] 石秋艳,杨斌,孙原,等.Beclin-1、LC3-II 在缺血后处理大鼠再灌注中的表达及意义[J].中风与神经疾病杂志,2014,8(31):687-690.
[14] Zhao H,Sapolsky RM,Steinberg GK.Interrupting reperfusion asastroke therapy:ischemic postconditioning reduces infarct size after focal ischemia in rats[J].J Cereb Blood Flow Metab,2012,26(9):1114-1121.
[15] 张巧梅,李立亚,高淅,等.电针对小鼠脑缺血后 Neurogenin2 表达和神经功能的影响[J].神经解剖学杂志,2013,29(3):321.
[16] 马璟曦,罗勇.电针合谷穴对大鼠局灶性脑缺血再灌注后脑内毛细血管密度及 CD34 表达的影响[J].中国康复理论与实践,2014,20(3):201.
[17] 刘延祥.自噬在电针治疗周围神经损伤修复中的作用研究[J].辽宁中医杂志,2013,24(8):2054.
[18] 高伶俐.电针神庭百会对缺血性脑卒中认知障碍大鼠自噬网络系统的影响[D].郑州:河南中医药大学,2016.
[19] 刘娇,冯晓东.电针神庭、百会穴配合康复训练治疗脑卒中后认知障碍的临床研究[J].中医学报,2013,28(4):608.
[20] Feng XD,Yang SL,Liu J,et al.Electroacupuncture ameliates cogni-tive impairment through inhibition of NF-k B-mediated neuronal cellapoptosis in cerebral ischemia-reperfusion injured rats[J].Mol Med Rep,2013,7:1516.