Effect of electroacupuncture on the MMP-2/9 pathway mediated blood-brain barrier permeability in mice with Parkinson's disease

探讨电针对帕金森病 (PD) 小鼠血脑屏障 (BBB) 通透性的影响及对基质金属蛋白酶 (MMPs) 的调控作用. 方法: 将48只C57/BL6小鼠随机分为正常组 (NC组) ,PD模型组 (PD组) ,电针组 (EA组) 和EA+SB-3CT抑制剂组 (EA+SB-3CT组) , 每组各12只.对于PD组,EA组和EA+SB-3CT组, 采用鱼藤酮灌胃4周建立PD模型, 其中EA+SB-3CT组于鱼藤酮灌胃1 h后, 予以MMP-2/-9抑制剂SB-3CT (25 mg/kg/d) 腹腔注射一次.造模成功后, EA组和EA+SB-3CT组均于“风府”双侧“太冲” “足三里”接受电针刺激, 刺激强度1 mA, 频率2 Hz, 每次30 min, 每日1次, 连续2周.采用敞箱实验观察各组小鼠行为学变化, 免疫组化法测定黑质中酪氨酸轻化酶 (TH) 含量, 伊文思蓝染色法检测BBB通透性, 以及蛋白免疫印记法检测脑组织中ZO-1,occludin,claudin-1,MMP-2和MMP-9蛋白表达. 结果 :与NC组比较, PD组小鼠行为学评分较高 ( P <0.05) , 自主运动的总时间较短,总路程较短,平均速度较低 ( P <0.05) , 黑质TH表达较少 ( P <0.05) , 脑组织伊文思蓝含量较高 ( P <0.05) , ZO-1,occludin,claudin-1蛋白表达均较低 ( P <0.05) ,MMP-2和MMP-9蛋白的表达均较高 ( P <0.05) .与PD组比较, EA组小鼠行为学评分较低 ( P <0.05) , 自主运动的总时间较长,总路程较长,平均速度较高 ( P <0.05) , 黑质TH表达较高 ( P <0.05) , 脑组织伊文思蓝含量较低 ( P <0.05) , ZO-1,occludin蛋白均表达较高,MMP-2和MMP-9蛋白的表达较低 ( P <0.05) .与EA组比较, EA+SB-3CT组小鼠脑组织伊文思蓝含量较低 ( P <0.05) , ZO-1,occludin蛋白表达均较高 ( P <0.05) ,MMP-2和MMP-9蛋白的表达较低 ( P <0.05) . 结论: 电针可有效改善PD小鼠的运动功能障碍, 减轻多巴胺能神经元的损伤, 发挥神经保护作用.同时, EA通过上调紧密连接蛋白ZO-1和occludin的表达水平, 下调基质金属蛋白酶MMP-2和MMP-9的表达水平, 有效改善PD小鼠的BBB通透性.EA发挥神经保护作用机制可能通过改善MMP-2/-9通路介导的血脑屏障通透性所产生的. To investigate the effects of electroacupuncture (EA) on the blood-brain barrier permeability and the regulation of matrix metalloproteinases (MMPs) in the mice with Parkinson's disease (PD). Forty-eight C57BL/6 mice were randomly assigned into the normal control (NC) group, the PD model (PD) group, the EA group and the EA + SB-3CT inhibitor group (EA + SB-3CT), with 12 mice in each group. In this experiment, the PD model was established by intragastric administration (IG) with rotenone for 4 weeks in the PD group, EA group and EA+SB-3CT group. In the EA + SB-3CT group, 1 h after IG with rotenone, the mice were intraperitoneally injected with MMP-2/9 inhibitor, SB-3CT (25 mg/kg/d). After successfully modeled, in the EA group and EA + SB-3CT group, EA was conducted at “Fengfu (GV16)” and bilateral “Taichong (LR3)” and “Zusanli (ST36)”, at 1 mA and 2 Hz for 30 min each time, once a day, for consecutive 2 weeks. The behavioral changes of the mice were observed in each group using the open field test, the level of tyrosine hydroxylase (TH) in the substantia nigra was determined by immunohistochemistry, the permeability of the blood-brain barrier was detected by Evans blue staining, and the protein expression of ZO-1, ocludin, claudin-1, MMP-2 and MMP-9 in the substantia nigra was detected by Western blotting. Compared with the NC group, the behavioral scores increased ( P < 0.05), while total time of locomotion, total distance and average speed were reduced ( P < 0.05) in the PD group. The expression of TH in the substantia nigra decreased ( P < 0.05), Evans blue level in the brain tissue increased ( P < 0.05), and the protein expression of ZO-1, occludin and claudin-1 was lower ( P < 0.05), whereas MMP-2 and MMP-9 expression was higher ( P < 0.05) in the PD group. Compared with the PD group, behavioral scores decreased ( P < 0.05), while the total time of locomotion, total distance and average speed increased ( P < 0.05) in the EA group. Additionally, TH expression in the substantia nigra was elevated ( P < 0.05), Evans blue level in the brain tissue was lower ( P < 0.05), the protein expression of ZO-1, occludin and claudin-1 was up-regulated ( P < 0.05), and MMP-2 and MMP-9 expression was down-regulated ( P < 0.05) in the EA group. Compared with the EA group, Evans blue level was reduced ( P < 0.05), the protein expression of ZO-1 and occludin was up-regulated ( P < 0.05), and MMP-2 and MMP-9 expression was further down-regulated ( P < 0.05) in the EA+SB-3CT group. EA can effectively ameliorate the motor dysfunction of PD mice, reduce the damage of dopaminergic neurons, and play a neuroprotective role. EA can effectively improve the blood–brain barrier permeability in PD mice by up-regulating the expression of tight junction proteins, ZO-1 and occludin, and down-regulating the expression of matrix metalloporteinases, MMP-2 and MMP-9. The neuroprotective role of EA may be obtained by improving the blood-brain barrier permeability mediated by MMP-2/9 pathway.