基于PINK1/Parkin信号通路的早期运动训练对缺血性脑卒中大鼠的脑保护作用及机制

关键词：早期运动训练  缺血性脑卒中  PINK1/Parkin信号通路  线粒体自噬  脑保护

Keywords:early sports training  ischemic stroke  PINK1/Parkin signaling pathway  mitochondrial autophagy  brain protection

摘要：

      摘要 目的：探讨早期运动训练对缺血性脑卒中大鼠PINK 1/Parkin信号通路的影响。 方法：雄性SD大鼠，模型制备前3d进行跑台适应性训练，取适应良好的60只入组。随机选取12只入假手术组，余组建立大脑中动脉缺血模型，分为模型组、运动Ⅰ、Ⅱ、Ⅲ组。运动组分别在术后24h、72h、7d进行跑台训练，30min/次，1次/d，6次/周，干预2周。评估神经功能缺损体征，检测局部脑血流量，TTC染色评估脑梗死体积，分析步态，透射电镜观察神经元结构，Western Blot法和RT-qPCR法检测Atg5、Beclin-1、PINK 1、Parkin表达。 结果：与同时点假手术组比较，模型组、运动组mNSS评分增高、rCBF降低、脑梗死体积增加（P＜0.01）；步行平均速度、四肢平均压强（MI）及最大接触面积（MCA）均降低、Atg5、Beclin-1、PINK 1、Parkin表达增高（P＜0.01）；神经元结构损坏严重。与模型组比较，21d时Ⅰ、Ⅱ组mNSS评分降低（P＜0.01）；运动Ⅰ、Ⅱ组rCBF升高，运动组脑梗死体积减小（P＜0.01，P＜0.05）；运动Ⅰ、Ⅱ组右肢和运动Ⅲ组右后肢（RH）的MI及左后肢（LH）的MCA提高（P＜0.01，P＜0.05），Ⅰ组LF、右肢及Ⅱ组RH的MCA提高（P＜0.01，P＜0.05）；运动组神经元结构有所改善；运动Ⅰ、Ⅱ组四因子表达均升高，Ⅲ组Atg5、Beclin-1、PINK 1蛋白及mRNA表达增高（P＜0.01，P＜0.05），Parkin蛋白表达增高（P＜0.05）。组内各时间点比较，模型组、运动组mNSS评分降低（P＜0.01，P＜0.05）；Ⅰ组rCBF最高、脑梗死体积缩小显著（P＜0.01），其中Ⅱ组脑梗死体积小于Ⅲ组（P＜0.05）；Ⅰ组LH、右肢MI和LH、RH的MCA高于Ⅱ、Ⅲ组（P＜0.01，P＜0.05）；神经元整体改善程度以Ⅰ组最佳；Ⅰ组Atg5、Beclin-1、PINK 1蛋白和四因子mRNA表达均高于其他两组（P＜0.01，P＜0.05），其中Ⅱ组Atg5、Beclin-1、PINK 1蛋白和Atg5 mRNA表达高于Ⅲ组（P＜0.05）。 结论：早期运动训练可调控MCAO大鼠缺血半暗区PINK1/Parkin信号通路，调节线粒体自噬，促进神经元修复，提高脑血流量，改善运动功能。

Abstract:

      Abstract Objective：To investigate the effect of early exercise training on the PINK1/Parkin signaling pathway in ischemic stroke rats. Method: Male SD rats underwent treadmill adaptability training 3 days before model preparation, and 60 well-adapted rats were included in the study. Twelve rats were randomly assigned to the sham operation group, while the remaining rats were subjected to middle cerebral artery occlusion (MCAO) to establish the ischemic model. The MCAO rats were divided into a model group and three exercise groups (Ⅰ, Ⅱ, and Ⅲ). The exercise groups received treadmill training at 24h, 72h, and 7d after surgery, respectively, with each session lasting 30min, once daily, 6 times per week, for a total of 2 weeks. Signs of neurological impairment were assessed, local cerebral blood flow (rCBF) was detected, infarct volume was evaluated using TTC staining, gait was analyzed, neuronal structure was observed by transmission electron microscopy, and the expressions of Atg5, Beclin-1， PINK1，and Parkin were detected by Western Blot and RT-qPCR. Result: Compared with the sham operation group, the model group and exercise groups showed increased mNSS scores, decreased rCBF, and larger cerebral infarction volumes (P<0.01). The mean walking speed，MI and MCA of limbs were decreased, while the expressions of Atg5, Beclin-1, PINK1, and Parkin were increased (P<0.01). Neuronal structures were severely damaged. Compared with the model group, the mNSS scores of exercise groupsⅠandⅡdecreased at 21 days （P<0.01）. The rCBF in exercise groups Ⅰ and Ⅱ increased, and the cerebral infarction volume in all exercise groups decreased （P<0.01，P<0.05）. The MI and MCA of the right hindlimb (RH) in exercise groups I and II, as well as the MCA of RH in exercise group Ⅲ, were increased （P<0.01，P<0.05). The MCA of the left forelimb (LF), right limb, and RH in exercise groups I and II also increased （P<0.01，P<0.05). Neuronal structures in the exercise groups showed improvement. The expressions of all four factors (Atg5, Beclin-1, PINK1, and Parkin) were elevated in exercise groups I and II, while exercise group III exhibited increased protein and mRNA expressions of Atg5, Beclin-1, and PINK1 (P<0.01，P<0.05), along with elevated Parkin protein expression (P<0.05). Within-group comparisons revealed that the mNSS scores of the model group and exercise groups decreased （P<0.01，P<0.05). Exercise group I demonstrated the highest rCBF and the most significant reduction in infarct volume (P<0.01), with group II showing a smaller infarct volume than group Ⅲ （P<0.05）.The MI of the left hindlimb (LH) and right limb, as well as the MCA of LH and RH in group I, were higher than those in groupsⅡand Ⅲ（P<0.01，P<0.05）. Neuronal improvement was the most pronounced in group I. The The protein expressions of Atg5, Beclin-1 and PINK 1, as well as the mRNA expressions of all four factors in groupⅠ,were higher than those in the other two groups（P<0.01, P<0.05）， Group II exhibited the higher protein expressions of Atg5, Beclin-1, and PINK1, as well as the higher Atg5 mRNA expression, compared to group Ⅲ (P<0.05）. Conclusion：Early exercise training can regulate the PINK1/Parkin signaling pathway in the ischemic penumbra of MCAO rats， regulate mitochondrial autophagy, promote neuronal repair, increase cerebral blood flow, and improve motor function.