电针、加味芍药甘草汤及针药结合对脑卒中肢体痉挛模型大鼠海马突触超微结构及可塑性蛋白的影响

Used Alone or in Combination on the Synaptic Ultrastructure and Plasticity Protein in Hippocampus of Stroke Model Rats with Limb Spasticity

目的比较电针"曲池""阳陵泉"、加味芍药甘草汤及针药结合三种疗法缓解脑卒中肢体痉挛的疗效及可能作用机制。方法将54只SD大鼠随机分为空白组、假手术组、模型组、针药组、中药组、电针组,每组9只。采用改良Zea-Longa线栓法+内囊注射N-甲基-D-天冬氨酸受体制备脑卒中肢体痉挛大鼠模型。造模后电针组大鼠行双侧"阳陵泉""曲池"电针刺激,每次30min,每日1次,持续5天;中药组给予浓度为10g/ml加味芍药甘草汤水煎剂1ml/100g灌胃,每日1次,持续5天;针药组先给予加味芍药甘草汤灌胃,再行电针,方法同上;模型组和假手术组大鼠进行相同束缚及等量生理盐水灌胃;空白组不做任何干预。各组大鼠予行为学评分评价神经功能损伤情况,电镜下观察海马突触的超微结构,检测大鼠大脑海马中突触素(SYN)及突触后致密物95(PSD95)mRNA与蛋白表达。结果与空白组和假手术组比较,模型组大鼠行为学评分升高(P<0.01);与模型组比较,各干预组行为学评分降低(P<0.05),各干预组组间比较差异无统计学意义(P>0.05)。镜下观察:模型组较空白组、假手术组突触数量减少明显,囊泡数量减少密度不均,前后膜及间隙发生融合界限模糊,突触后细胞终末胞质变性溶解显著,突触后致密带的密度也下降;各干预组突触结构明显缓解,且针药组突触细胞囊泡明显成形,界限清晰,间隙规则。与空白组、假手术组比较,模型组大鼠海马SYN、PSD95 mRNA相对表达量与蛋白表达均降低(P<0.01);与模型组比较,各干预组大鼠海马SYN、PSD95 mRNA相对表达量与蛋白表达升高(P<0.05或P<0.01),且针药组表达优于中药组、电针组(P<0.05或P<0.01)。结论电针"曲池""阳陵泉"、加味芍药甘草汤及针药结合三种疗法均可以缓解脑卒中肢体痉挛的神经损伤情况,其机制可能通过提高海马可塑性蛋白表达从而改善海马突出超微结构,且以针药结合效果最优。

Objective To compare the effect of acupuncture at‘Quchi’(LI 11)and‘Yanglingquan’(GB 34)acupoints,the modified Shaoyao Gancao Decoction(芍药甘草汤),and the combination of two therapies on relieving limb spasticity after stroke,and to explore the possible mechanism.Methods Fifty-four SD rats were randomly divided into a blank group,a sham operation group,a model group,a combination therapy group,an herbal medicine group,and an electroacupuncture group,with nine rats in each group.The stroke rats model with limb spasticity was developed by modified Zea-Longa thread embolization and internal capsule syringe of N-methyl-D-aspartate receptor.After modeling,the rats in electroacupuncture group were given electroacupuncture at bilateral‘Quchi’and‘Yanglingquan’acupoints,30 minutes each time,once daily,for 5 days.The herbal medicine group was administered with 10 g/ml modified Shaoyao Gancao Decoction,once daily,for 5 days.The combination therapy group was given electroacupuncture and modified Shaoyao Gancao Decoction,in the same way with electroacupuncture group and herbal medicine group.The rats in model group and sham operation group were all blinding and received equal amount of normal saline by gavage.No intervention was implemented in the blank group.The behavioral scores were used to assess the neurological damage;the ultrastructure of hippocampal synapses was observed under electron microscope;and the mRNA and protein expression of synaptophysin(SYN)and postsynaptic compact 95(PSD95)in the hippocampus of rats were detected.Results There was significantly higher behavioral score in the model group compared to blank group and sham operation group(P<0.01),as well as significantly lower score in all treatment groups compared to model group(P<0.05);the comparison between six groups was not statistically significant(P>0.05).Compared to blank group and sham operation group,the number of synapses significantly decreased in model group;the number of vesicles was reduced and the density was uneven;the front and back membranes and gaps were blurred;the terminal cytoplasm of postsynaptic cells was degenerated and dissolved significantly;and the density of the postsynaptic density zone also decreased.In all treatment groups,the structure of synapses was significantly relieved;and the synaptic cell vesicles in combination treatment group were clearly formulated with clear boundaries and regular gaps.The mRNA and protein expression of SYN and PSD95 were significantly lower in model group than blank group and sham operation group(P<0.01),and were significantly higher in all treatment groups than model group(P<0.05;P<0.01);the increase was more significant in combination treatment group compared to herbal medicine group and electroacupuncture group(P<0.05;P<0.01).Conclusion Electroacupuncture at‘Quchi’and‘Yanglingquan’acupoints and modified Shaoyao Gancao Decoction,used alone or in combination,can alleviate the neurological damage caused by stroke limb spasticity,through improving synaptic ultrastructure and increasing plasticity protein expression;and the combination of electroacupuncture and herbal medicine is the most effective.