Objective: To observe the effect of "Tiaoshen Tongluo" acupuncture (TTA) at "Dingzhongxian" (MS5) and right "Dingpangxian" (MS8) on neurological injury, muscle tension and neurotransmitters through nuclear transcription factor E2 related factor 2 (Nrf2)/reactive oxygen species (ROS) signaling pathway in spastic rats after stroke, so as to explore its mechanisms underlying relief of post-stroke spasm (PSS).
Methods: A total of 90 male SD rats were randomly divided into 6 groups, i.e. sham operation, PSS model, medication, non-acupoint acupuncture, TTA, TTA+ML385 groups, with 15 rats in each group. The PSS model was established by middle cerebral artery occlusion. After modeling, rats of the medication group were treated by gavage of baclofen (0.4 mg/kg), once daily for 7 days. For rats of the non-acupoint acupuncture group, the spot about 10 mm above the iliac crest and below the armpit of the affected side was needled, and for those of the TTA group and TTA+ML385 group, EA stimulation (1 mA, 2 Hz/15 Hz) was applied to MS5 and right MS8 for 10 min, once daily for 7 consecutive days. Intraperitoneal injection of ML385 ［ a specific nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg］ was given to rats of the TTA+ML385 group before TTA was performed. The rats' neurological deficit score (0-4 points) was evaluated by referring to Zea Longa's methods and the muscular spasm degree of the quadriceps femoris of the left hindlimb (0-4 points) assessed by using Ashworth scale (MAS). The muscular tension of the left quadriceps femoris was measured by using a tension sensor, and Hoffman (H)-reflex response and M and H waves of electromyogram of the muscle between the metatarsals of the left foot were measured using an electrophysiological recorder. The cerebral infarction volume was measured after 2,3,5-triphenyltetrazolium chloride (TTC) staining. The contents of γ-aminobutyric acid (GABA), glycine (Gly), glutamic acid (Glu) and aspartic acid (Asp) of the right cortical infarct area were detected by using high performance capillary electrophoresis, and the contents of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) were detected by fluorescence spectrophoto-metry, as well as the level of ROS in the right cerebral cortical infarction tissues was detected by dihydroethidium staining. The expression levels of Nrf2 and heme oxygenase-1 (HO-1) proteins in the infarcted cerebral area were detected using Western blot.
Results: Compared with the sham operation group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu and Asp and ROS level were significantly increased (P<0.001), whereas the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, cerebral Nrf2 and HO-1 protein expression levels were apparently decreased (P<0.001) in the model group. In comparison with the model group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu, Asp and ROS levels were decreased (P<0.001), and the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, Nrf2 and HO-1 protein expressions were increased (P<0.001, P<0.01) in both the medication and TTA groups. No significant differences were found between the non-acupoint group and model group, and between the medication and TTA groups in all the indexes mentioned above (P>0.05). After administration of ML385, the effects of TTA in reducing neurological deficit score, MAS score, Hmax/Mmax, percentage of cerebral infarct volume, Glu, Asp, ROS, and up-regulating H-reflex threshold, GABA, Gly, 5-HT, DA, NE, Nrf2 and HO-1 levels were eliminated (P<0.001，P<0.05,P<0.01).
Conclusion: TTA can improve neurological behavior and muscle spasm in rats with PSS, which may be associated with its functions in regulating the levels of neurotransmitters in the cortical infarcted area by activating the Nrf2/ROS signaling pathway.
目的：观察调神通络针刺法通过核转录因子E2相关因子2(Nrf2)/活性氧(ROS)通路对脑卒中后痉挛大鼠神经功能、肌张力及神经递质的影响，探讨其作用机制。方法：SD大鼠分为假手术组、模型组、西药组、非穴位针刺组、调神通络针刺组、调神通络针刺+ML385组，每组15只。采用大脑中动脉栓塞法制备脑卒中后痉挛大鼠模型。造模后，西药组大鼠给予巴氯芬灌胃（0.4 mg/kg）；非穴位针刺组选择大鼠患侧肋下髂嵴上10 mm的固定点进行针刺；调神通络针刺组于顶中线、右侧顶旁线给予调神通络法针刺；调神通络针刺+ML385组大鼠在行调神通络针刺前30 min按30 mg/kg剂量给予腹腔注射Nrf2抑制剂ML385。针刺干预均每次10 min，每日1次，各组均干预7 d。用神经功能缺损评分和改良Ashworth量表(MAS)评分评估大鼠神经功能和痉挛程度；电生理记录仪检测大鼠肌张力和H反射；用TTC染色法测定大鼠脑梗死体积百分比；用高效毛细管电泳法检测大鼠大脑皮层梗死区组织氨基酸类神经递质γ-氨基丁酸(GABA)、甘氨酸(Gly)、谷氨酸(Glu)、天冬氨酸(Asp)含量；荧光分光光度法检测大脑皮层梗死区组织单胺类神经递质5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)含量；二氢乙锭染色检测大鼠大脑皮层梗死区组织ROS水平；Western blot法检测大鼠大脑皮层梗死区组织Nrf2、血红素氧合酶-1(HO-1)蛋白表达。结果：与假手术组比较，模型组大鼠神经功能缺损评分、MAS评分、Hmax/Mmax比值、脑梗死体积百分比、Glu含量、Asp含量、ROS水平均升高(P<0.001)，肌肉反应对张力传感器的作用力、诱发H反射的刺激阈值，GABA、Gly、5-HT、DA、NE含量及Nrf2、HO-1蛋白表达水平均降低(P<0.001)；与模型组比较，非穴位针刺组各指标差异均无统计学意义；与模型组、非穴位针刺组比较，西药组、调神通络针刺组大鼠神经功能缺损评分、MAS评分、Hmax/Mmax比值、脑梗死体积百分比、Glu含量、Asp含量、ROS水平均降低(P<0.001)，肌肉反应对张力传感器的作用力、诱发H反射的刺激阈值，GABA、Gly、5-HT、DA、NE含量及Nrf2、HO-1蛋白表达水平均升高(P<0.001，P<0.01)；与调神通络针刺组比较，调神通络针刺+ML385组大鼠神经功能缺损评分、MAS评分、Hmax/Mmax比值、脑梗死体积百分比、Glu含量、Asp含量、ROS水平均升高(P<0.001，P<0.05)，肌肉反应对张力传感器的作用力、诱发H反射的刺激阈值，GABA、Gly、5-HT、DA、NE含量及Nrf2、HO-1蛋白表达水平均降低(P<0.001，P<0.05，P<0.01)。结论：调神通络针刺法可通过激活Nrf2/ROS通路，调节皮层梗死区神经递质含量，从而改善脑卒中后痉挛大鼠神经功能损伤和肌肉痉挛。.
Keywords: Muscular tone; Neurological function; Neurotransmitter; Nuclear transcription factor E2 related factor 2/reactive oxygen species pathway; Post-stroke spasm; “Tiaoshen Tongluo” acupuncture.