Objective: To observe the effect of electroacupuncture(EA) at "Fengfu" (GV16) and "Taichong"(LR3) on the expression of tyrosine hydroxylase (TH), α-synuclein (α-syn) and microglial-related microglial (MG), Toll-like receptor 4（TLR4）/nuclear factor kappa B （NF-κB） signaling pathway in the substantia nigra (SN) of midbrain in Rotenone-induced Parkinson's disease (PD) rats, so as to explore its mechanisms underlying improvement of PD.
Methods: SD male rats were randomly divided into normal control, PD model and EA groups (n=12 in each group). The PD model was established by subcutaneous injection of rotenone (1 mg/kg) at the back of neck. EA (2 Hz, 1 mA) was applied to bilateral GV16 and LR3, once daily for 2 weeks. The rats' behavior(hair color, reaction capacity, locomotion and gait state)scores (0-10 points) were given and the autonomic movement state (trajectory of autonomous motion, total distance, average speed and duration of motion in 8 min) was detected by using open field tests. The immunoactivity of TH and α-syn in the SN tissue were determined by using immunohistochemistry staining, and the number of Iba-1-labelled microglia (MG) was detected by using immunofluorescence staining. The expression levels of TLR4 and NF-κB P65 proteins in the SN were detected by Western blot, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the SN were determined by using enzyme-linked immunosorbent assay.
Results: In comparison with the normal group, the behavioral score, α-syn immunoactivity, number of Iba-1 labelled microglia, expression of TLR4 and NF-κB P65 proteins and the contents of TNF-α and IL-6 in the SN were significantly increased (P<0.01), whereas the total distance, average speed, duration of motion of the autonomic movement in 8 min, and the TH immunoactivity were remarkably decreased in the model group (P<0.01). After EA intervention, compared with the model group, the increase of the behavioral score, α-syn immunoactivity, number of Iba-1-labelled microglia, expression of TLR4 and NF-κB P65 proteins, the contents of TNF-α and IL-6 in the SN, and the decrease of the total distance, average speed, duration of motion of the autonomic movement in 8 min, and the TH immunoactivity were reversed (P<0.05, P<0.01).
Conclusion: EA can improve the behavioral manifestations of PD rats, which may be associated with its functions in down-regulating the abnormal accumulation of pathological α-syn, TLR4/NF-κB signaling, inhibiting activities of microglia and in up-regulating the expression of TH in the SN of midbrain.
目的：观察电针“风府”“太冲”穴对鱼藤酮诱导的帕金森病(Parkinson’s disease，PD)大鼠中脑黑质酪氨酸羟化酶(TH)、α-突触核蛋白(α-syn)，小胶质细胞(MG)以及Toll样受体4(TLR4)/核因子(NF)-κB信号通路相关蛋白的影响，探讨电针治疗PD的可能作用机制。方法：SD大鼠随机分为正常组、模型组、电针组，每组12只。采取颈背部皮下注射鱼藤酮(1 mg/kg)制备PD大鼠模型。电针组电针“风府”和“太冲”，30 min/次，每日1次，连续治疗14 d。采用行为学评分和敞箱实验检测各组大鼠行为学改变，免疫组织化学法检测大鼠中脑黑质TH、α-syn的表达水平，免疫荧光标记法检测中脑黑质激活的MG数量，蛋白免疫印迹法检测大鼠中脑黑质TLR4、NF-κB P65，酶联免疫吸附法检测中脑黑质肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)等炎性因子水平。结果：与正常组比较，模型组大鼠行为学评分升高(P>0.01)，自主运动总路程、平均速度、运动持续时间减少(P>0.01)；中脑黑质TH表达减少(P>0.01)，α-syn表达、激活的MG数量、TLR4及NF-κB P65蛋白表达、TNF-α及IL-6含量增加(P>0.01)。与模型组比较，电针组行为学评分降低，自主运动总路程、平均速度、运动持续时间增加(P>0.01)；中脑黑质TH表达增多(P>0.01)，α-syn表达、激活的MG数量、TLR4及NF-κB P65蛋白表达、TNF-α及IL-6含量减少(P>0.01, P>0.05)。结论：电针能有效改善PD大鼠运动迟缓等行为学表现，减少α-syn的异常聚集，提高中脑黑质TH表达水平，其相关机制可能与抑制神经炎性反应及TLR4/NF-κB信号通路相关蛋白有关。.
Keywords: Electroacupuncture; Inflammatory factor; Microglia; Nuclear factor kappa B; Parkinson’s Disease; Toll-like receptor 4.