刮痧干预对帕金森病模型大鼠运动障碍及氧化应激水平的影响

Gua Sha Intervention on Motor Dysfunction and Oxidative Stress Levels in a Rat Model of Parkinson’s Disease

    摘要
  • 摘要:
    目的 观察刮痧对鱼藤酮诱导的帕金森病（PD）模型大鼠运动障碍及氧化应激水平的影响。
    方法 48只SD大鼠随机分为空白组、假手术组、模型组、药物组、假刮痧组、刮痧组，每组8只，通过向左侧黑质区注射1 μL鱼藤酮溶液（12 μg·μL-1）建立PD大鼠模型。空白组、假手术组、模型组不予以干预措施，药物组、假刮痧组、刮痧组在造模后第14天分别进行干预。药物组给予每只大鼠盐酸司来吉兰悬液0.5 mg·kg-1，连续灌胃23 d；刮痧组进行刮痧，隔日1次，共刮12次，造模后第36天刮痧结束；假刮痧组仅进行同刮痧组相同时间、固定姿势的抓取，不进行其他干预。干预结束后对各组大鼠进行旷场实验与步态检测；采用免疫荧光检测大鼠黑质区酪氨酸羟化酶（TH）荧光强度；Western blot检测黑质TH、α-Syn、Keap1、Nrf2蛋白表达；qRT-PCR检测大鼠黑质TH、α-Syn、Keap1、Nrf2、HO-1、NQO1表达；ELISA检测纹状体ROS及血清MDA、SOD、GSH-Px表达。
    结果 与假手术组相比，模型组运动旷场总距离显著缩短（P<0.001），中心区停留时间显著减少（P<0.01），进入中心区次数显著减少（P<0.001），步行周期延长，平均速度下降（P<0.01），四肢脚爪面积、步幅减小，黑质TH荧光强度降低（P<0.01），黑质TH蛋白、mRNA表达降低（P<0.001），α-Syn蛋白、mRNA表达升高（P<0.001）；纹状体ROS升高（P<0.05），血清MDA升高（P<0.01），血清SOD、GSH-Px降低（P<0.001），黑质GSH、GSH-Px降低（P<0.01）；黑质Keap1蛋白表达升高、Nrf2蛋白表达降低（P<0.001），黑质Nrf2、HO-1、NQO1 mRNA表达降低（P<0.05，P<0.01，P<0.001），黑质Keap1 mRNA表达升高（P<0.001），黑质G6PD、PGD蛋白表达降低（P<0.05，P<0.001）。与模型组相比，刮痧组旷场总距离增加（P<0.01），中心区停留时间（P<0.01），进入中心区次数增加，步行周期减少，平均速度增加（P<0.05），四肢脚爪面积、步幅增大，黑质TH荧光强度增强（P<0.01），黑质TH蛋白、mRNA表达升高（P<0.01，P<0.001），黑质α-Syn蛋白、mRNA表达降低（P<0.01，P<0.001）；纹状体ROS降低，血清MDA降低，血清SOD、GSH-Px升高，黑质GSH、GSH-Px升高（P<0.05）；黑质Keap1蛋白表达降低、Nrf2蛋白表达升高（P<0.001），黑质Nrf2、HO-1、NQO1 mRNA表达升高（P<0.05，P<0.01，P<0.001），Keap1 mRNA表达降低（P<0.01），黑质G6PD、PGD蛋白表达升高。刮痧组与药物组疗效相当。
    结论 刮痧可改善PD模型大鼠自主活动与步态障碍，其机制可能与激活Keap1/Nrf2氧化应激通路，减少氧化损伤，从而保护多巴胺能神经元有关。

     

    Abstract:
    OBJECTIVE To observe the effects of Gua Sha on motor dysfunction and oxidative stress levels in rotenone-induced Parkinson’s disease (PD) model rats.
    METHODS Forty-eight SD rats were randomly divided into the blank group, sham-operated group, model group, drug group, sham Gua Sha group, and Gua Sha group, with 8 rats in each group. A PD rat model was established by stereotactic injection of 1 μL rotenone solution (12 μg·μL-1) into the left substantia nigra. The blank group, sham-operated group, and model group received no intervention. The drug group, sham Gua Sha group, and Gua Sha group received corresponding interventions from d14 after modeling. The drug group was given selegiline hydrochloride suspension at 0.5 mg·kg-1 by gavage for 23 consecutive days. The Gua Sha group received Gua Sha intervention, once every other day for a total of 12 sessions, ending on d36 after modeling. The sham Gua Sha group was only subjected to the same duration of handling and fixation as the Gua Sha group, without any other intervention. After intervention, open field test (OFT) and gait analysis were performed in each group. Immunofluorescence (IF) was used to detect tyrosine hydroxylase (TH) fluorescence intensity in the substantia nigra. Western blot was used to detect the protein expression of TH, α-Syn, Keap1, and Nrf2 in the substantia nigra. qRT-PCR was used to detect the mRNA expression of TH, α-Syn, Keap1, Nrf2, HO-1, and NQO1 in the substantia nigra. ELISA was used to detect striatal ROS and serum MDA, SOD, and GSH-Px levels.
    RESULTS Compared with the sham-operated group, the model group showed a significantly shortened total distance in the OFT (P<0.001), significantly reduced time spent in the central zone (P<0.01), and significantly fewer entries into the central zone (P<0.001). The gait cycle was prolonged, while the mean speed (P<0.01), paw area, and stride length were decreased. TH fluorescence intensity in the substantia nigra was decreased (P<0.01), TH protein and mRNA expression were decreased (P<0.001), and α-Syn protein and mRNA expression were increased (P<0.001). Striatal ROS was increased (P<0.05), serum MDA was increased (P<0.01), and SOD and GSH-Px were decreased (P<0.001). GSH and GSH-Px in the substantia nigra were decreased (P<0.01). Keap1 protein expression in the substantia nigra was increased, while Nrf2 protein expression was decreased (P<0.001). The mRNA expression of Nrf2, HO-1, and NQO1 in the substantia nigra was decreased (P<0.05, P<0.01, P<0.001), Keap1 mRNA expression was increased (P<0.001), and G6PD and PGD protein expression in the substantia nigra was decreased (P<0.05, P<0.001). Compared with the model group, the Gua Sha group showed an increased total distance in the OFT (P<0.01), increased time spent in the central zone (P<0.01), and increased entries into the central zone. The gait cycle was shortened, while the mean speed (P<0.05), paw area, and stride length were increased. TH fluorescence intensity in the substantia nigra was enhanced (P<0.01), TH protein and mRNA expression were increased (P<0.01, P<0.001), and α-Syn protein and mRNA expression were decreased (P<0.01, P<0.001). Striatal ROS decreased, serum MDA decreased, serum SOD and GSH-Px increased, and substantia nigra GSH and GSH-Px increased (P<0.05). Keap1 protein expression in the substantia nigra was decreased, while Nrf2 protein expression was increased (P<0.001). The mRNA expression of Nrf2, HO-1, and NQO1 in the substantia nigra was increased (P<0.05, P<0.01, P<0.001), while Keap1 mRNA expression was decreased (P<0.01). G6PD and PGD protein expression in the substantia nigra was increased. The therapeutic effect of the Gua Sha group was comparable to that of the drug group.
    CONCLUSION Gua Sha can improve spontaneous activity and gait dysfunction in PD model rats, and its mechanism may be related to activation of the Keap1/Nrf2 oxidative stress pathway, reduction of oxidative damage, and protection of dopaminergic neurons.

     

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