摘要
目的:观察不同频率电针对脊髓损伤后大鼠运动功能的影响,探讨其相关机制。方法:健康雄性SD大鼠随机分为假手术组、模型组、2 Hz/100 Hz电针组、100 Hz电针组、2 Hz电针组,每组20只。脊髓打击器打击大鼠胸(T)10节段脊髓制备急性脊髓损伤模型。术后1 d开始各电针组分别用不同频率电针刺激大鼠T9、T11节段的'夹脊'穴,每天1次,连续7 d。用Basso Beattie Bresnahan(BBB)评分、斜板实验、足迹印记实验观察大鼠运动功能;尼氏染色、Fluoro-Jade B(FJB)染色观察大鼠脊髓形态和神经元凋亡情况;Western blot法检测脊髓微管相关蛋白1轻链3(LC3)和自噬相关蛋白Beclin 1及活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)蛋白表达情况。结果:与假手术组比较,模型组大鼠BBB评分和斜板角度明显降低(P<0. 001);大鼠后足脚掌无法着地,基本无运动能力,呈拖行步态;脊髓可见空洞,尼氏体模糊不清,数量较少;FJB阳性神经元数量明显增多(P<0. 001);Beclin 1和Caspase-3蛋白表达增加(P<0. 05,P<0. 001)。电针3 d后,与模型组比较,2 Hz/100 Hz电针组BBB评分与斜板角度明显升高(P<0. 001);电针7 d后,与模型组比较,2 Hz/100 Hz电针组和100 Hz电针组大鼠BBB评分与斜板角度均明显升高(P<0. 001,P<0. 05)。与模型组比较,2 Hz/100 Hz电针组与100 Hz电针组足印较清晰;脊髓尼氏体分布较均匀,着色较深;FJB阳性神经元数量减少(P<0. 001);LC3-Ⅱ/Ⅰ和Beclin 1蛋白表达升高(P<0. 01,P<0. 001);Caspase-3蛋白明显降低(P<0. 001)。与模型组比较,2 Hz电针组大鼠脊髓FJB阳性神经元数量减少(P<0. 05);LC3-Ⅱ/Ⅰ降低(P<0. 05)。与2 Hz/100 Hz电针组比较,100 Hz电针组步态不协调,脊髓Beclin 1蛋白降低(P<0. 05)。结论:2 Hz/100 Hz及100 Hz电针都能有效促进脊髓损伤后大鼠运动功能的恢复,其中2 Hz/100 Hz的效果更好,其机制可能与促进受损神经细胞的自噬,减少神经元凋亡有关。
ObjectiveTo investigate the effect of different frequencies of electroacupuncture(EA)on motor function and expression of autophagy-related proteins microtubule-associated protein light chain 3(LC3),etc.in spinal cord injury(SCI)rats,so as to reveal its underlying mechanisms and provide a reference for clinical application.MethodsA total of 100 male SD rats were randomly divided into sham control,model,2 Hz-EA,100 Hz-EA and2 Hz/100 Hz-EA groups(n=20 in each group).EA(2 Hz,100 Hz or 2 Hz/100 Hz,0.4-0.5-0.6 mA increased by0.1 mA every10 min)was applied to'HuatuoJiaji'(EX-HN1,T9 and T11)for 30 min,once a day for 7 days.The rat’s hindlimb motor function was assessed by using Basso,Beattie and Bresnahan(BBB)locomotor rating scale,inclined plane test and plantar imprinting test,separately.The histological changes and neuronal apoptosis of the spinal cord tissue were observed by Fluoro-Jade B(FJB)and Nissl staining,respectively.The expression of LC3,Beclin 1 and cleaved Caspase-3 proteins in the spinal cord was detected by Western blot.ResultsAfter modeling,the BBB scores and the angles of inclined plane on day 1,3 and 7 were significantly decreased(P<0.001),and the number of FJB positive cells,and the expression levels of Beclin 1 and cleaved Caspase-3 proteins were considerably increased in the model group compared with the sham control group(P<0.001,P<0.05).After EA intervention,the BBB score and the angles of inclined plate on day 3 in the 2 Hz/100 Hz-EA group(rather than in the 2 Hz-and 100 Hz-EA groups),the BBB score and the angles of inclined plate on day 7 in both 2 Hz/100 Hz and 100 Hz-EA groups(rather than in the 2 Hz-EA group),and the expression levels of Beclin 1 and LC3-Ⅱ/Ⅰin both 2 Hz/100 Hz and 100 Hz-EA groups(rather than in the 2 Hz-EA group)on day 7 were obviously increased(P<0.001,P<0.01,P<0.05),while the number of FJB-positive neurons in the 3 EA groups,and the expression of cleaved Caspase-3 protein in both 2 Hz/100 Hz and 100 Hz-EA groups and the LC3-Ⅱ/Ⅰin the 2 Hz-EA group were obviously decreased relevant to the model group(P<0.001,P<0.05).The expression of Beclin 1 in the 100 Hz-EA group was obviously decreased relevant to the 2 Hz/100 Hz-EA group(P<0.05).Nissl staining displayed appearance of cavities and fuzzy shape of Nissl bodies with light coloration in the injured spinal cord of model group,which was milder in both 2 Hz/100 Hz-EA and 100 Hz-EA groups.Plantar imprinting tests showed dragging gait prints in the model group due to disability in movement,and relatively distinct foot imprints in both 2 Hz/100 Hz and 100 Hz-EA groups.ConclusionBoth 100 Hz and 2 Hz/100 Hz-EA can effectively promote the recovery of hindlimb locomotor function of SCI rats,which may be related to its function in promoting autophagy of damaged nerve cells and in reducing neuronal apoptosis.
作者
罗慧颖
曾志威
于小波
何菁
曹靖
徐波
LUO Hui-ying;ZENG Zhi-wei;YU Xiao-bo;HE Jing;CAO Jing;XU Bo(Graduate School,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Department of Anesthesiology,General Hospital of Southern Theater Command of People’s Liberation Army,Guangzhou 510010;School of Basic Medical Sciences,Zhengzhou University,Zhengzhou 450001)
出处
《针刺研究》
CAS
CSCD
北大核心
2019年第9期625-631,共7页
Acupuncture Research
基金
国家自然科学基金项目(No.61773130、81671091)
全军科技计划项目(No.CWH17J024)
广州市科技计划项目(201904010389)
关键词
电针
不同频率
脊髓损伤
自噬
凋亡
Electroacupuncture
Different frequencies
Spinal cord injury
Autophagy
Apoptosis
作者简介
第一作者:罗慧颖,博士研究生,研究方向:慢性疼痛。E-mail:luohuiying1017@163.com;通信作者:曹靖,博士,教授,博士生导师,研究方向:慢性疼痛。E-mail:caojing73@126.com;通信作者:徐波,博士,主任医师,博士生导师,研究方向:精确麻醉。E-mail:xubo333@hotmail.com
