电针对缺血性学习记忆障碍大鼠氧自由基及凋亡相关蛋白表达的影响

Effects of Electroacupuncture Intervention on Oxygen Free Radicals and Expression of Apoptosis-related Proteins in Rats with Ischemic Learning and Memory Disorder

目的:观察电针对脑缺血大鼠学习记忆障碍的影响,从氧自由基与细胞凋亡的角度探讨电针治疗该病的机制。方法:用Morris水迷宫实验筛选学习记忆良好的健康雄性SD大鼠60只,随机分为假手术组、模型组、药物组、电针组,每组15只。采用永久性结扎双侧颈总动脉方法制备缺血性学习记忆障碍模型。造模成功后第7天进行干预。电针组采用"智三针"通电治疗,每日1次,每次30min,共3周。药物组给予安理申灌胃,剂量为0.03mg/kg,每日1次,共3周。在不同时间点用Morris水迷宫检测大鼠的学习记忆能力;TUNEL法检测海马区细胞凋亡;免疫组化法检测海马区Bcl-2、Bax及Caspase-3蛋白表达的情况;化学比色法测量血清及海马超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-Px)活性及丙二醛(MDA)含量。结果:与假手术组比较,造模后模型组大鼠学习记忆能力明显降低(P<0.01);治疗14d时,两治疗组大鼠的学习记忆能力较模型组明显升高(P<0.01);治疗21d时,电针组大鼠学习记忆能力提高的程度优于药物组(P<0.05)。与假手术组相比,模型组大鼠海马凋亡细胞数目明显增多(P<0.01),两治疗组较模型组明显减少(P<0.01)。与假手术组比较,模型组Bcl-2、Bax和Caspase-3蛋白表达明显升高(P<0.01);两治疗组Bcl-2蛋白表达较模型组升高(P<0.01),且电针组Bcl-2蛋白表达较药物组升高明显(P<0.01),两治疗组Bax和Caspase-3蛋白表达较模型组降低(P<0.01)。与假手术组比较,模型组大鼠血清及海马组织SOD、GSH-Px活性明显降低(P<0.01),MDA含量明显升高(P<0.01);两治疗组与模型组比较,SOD、GSH-Px活性明显升高(P<0.01),MDA含量明显降低(P<0.01);电针组血清中3项指标及海马GSH-Px活性的改善程度较药物组明显(P<0.01,P<0.05)。结论:电针"智三针"可降低缺血性学习记忆障碍大鼠血清及海马内氧自由基含量,进而抑制细胞凋亡,改善学习记忆能力。

Objective To observe the effect of electroacupuncture（EA）therapy on levels of oxygen free radicals（OFR）and hippocampal apoptosis-related protein expression in ischemic learning-memory disorder rats so as to investigate its mechanisms underlying improvement of ischemic learning-memory impairment.Methods A total of 60 SD rats were randomly divided into sham operation（sham）,model,medication,and EA groups,with 15 rats in each group.The learning-memory disorder model was made by occlusion of bilateral carotid arteries.EA（2-3Hz,2mA）was applied to＂Zhi San Zhen＂[＂Shenting＂（GV 24）and bilateral＂Benshen＂（GB 13）]for 30 min,once a day for 3weeks.The rats of the medication group were treated by lavage of Aricept（0.03mg·kg-1·d-1）,once daily for 3weeks.The rats＇ learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining and the expression of hippocampal Bcl-2,Bax and Caspase-3proteins was detected by immunohistochemistry.Serum and hippocampal superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）activity and malondialdehyde（MDA）contents were detected by chemical colorimetric analysis.Results Compared with the sham group,the escape latencies（place-navigation）after modeling were evidently prolonged,and the times of target-platform crossing in 90sec（spatial probe test）considerably reduced in the model group（P0.01）,suggesting an impairment of learning-memory ability.After the treatment for 21 d,the increased escape latency and the reduced target-platform crossing time in both EA and medication groups were reversed in comparison with the model group（P0.01）,suggesting an improvement of memory ability,and the effect of the EA group was significantly superior to that of the medication group（P0.05）.Compared with the sham group,the number of apoptotic cells in hippocampal CA 1-CA 3regions,and the expression levels of hippocampal Bcl-2,Bax and Caspase-3proteins,and serum and hippocampal MDA contents were significantly increased in the model group（P0.01）,while serum and hippocampal SOD and GSH-Px levels obviously decreased in the model group（P0.01）.After the treatment for 21 days,compared to the model group,the number of the apoptotic cells,the expression levels of hippocampal Bax and Caspase-3proteins,and the contents of serum and hippocampal MDA were notably decreased in the EA and medication groups（P0.01）,whereas,Bcl-2protein expression levels,and serum and hippocampal SOD and GSH-Px activity were notably up-regulated in the EA and medication groups（P0.01）.The effects of EA group were obviously superior to those of medication group in increasing hippocampal Bcl-2immunoactivity,serum SOD and GSH-Px and hippocampal GSH-Px activity and in down-regulating serum MDA level（P0.01,P0.05）.Conclusion Electroacupuncture intervention can improve learning-memory ability in ischemic learning-memory disorder rats which may be associated with its effects in reducing blood and hippocampal OFR contents and hippocampal cellular apoptosis.