目的 研究定志小丸对血管性痴呆（VD）小鼠 PTEN 诱导假定激酶 1（PINK1）/E3 泛素连接酶 PARK2 （PARKIN）/肌细胞增强因子 2D（MEF2D）线粒体自噬通路的影响。方法 采用双侧颈总动脉夹闭再灌注方法复 制 VD 小鼠模型。将 ICR 小鼠随机分为假手术组、模型组、盐酸多奈哌齐组（1 mg·kg-1 ，灌胃给药）、PINK1 抑制 剂组（20 mg·kg-1 环孢菌素 A，腹腔注射给药）及定志小丸低、中、高剂量组（0.71、1.43、2.56 g·kg-1 ，灌胃给 药），每组 6 只，每天 1 次，连续给药 4 周。采用 Morris 水迷宫实验检测小鼠学习记忆能力；ELISA 法检测海 马组织乙酰胆碱酯酶（AchE）、活性氧（ROS）、白细胞介素 1β（IL-1β）、肿瘤坏死因子 α（TNF-α）、谷胱甘肽过 氧化物酶 4（GPX4）、紧密连接蛋白 1（ZO-1）水平及血清神经元特异性烯醇化酶（NSE）、基质金属蛋白酶 9 （MMP9）、中枢神经特异性蛋白（S100β）水平；qRT-PCR 及 Western Blot 法检测海马组织中 PINK1、PARKIN、 MEF2D、Bcl-2 同源结构域蛋白（Beclin-1）、微管相关蛋白 1 轻链 3β（LC3B）、螯合体 1（p62）mRNA 及蛋白表 达水平；HE 及尼氏染色法观察海马组织病理变化。结果 与假手术组比较，模型组小鼠的逃逸潜伏期显著延 长（P＜0.01），穿越平台次数显著减少（P＜0.01）；海马组织 AchE、ROS、IL-1β、TNF-α 水平显著升高（P＜ 0.01），GPX4、ZO-1 水平显著降低（P＜0.01）；血清 NSE、MMP9、S100β 水平显著升高（P＜0.01）；海马组织 中 PINK1、PARKIN、Beclin-1、LC3B mRNA 及蛋白表达显著上调（P＜0.01），MEF2D、p62 mRNA 及蛋白表达 显著下调（P＜0.01）；海马 CA1 区神经元排列疏松，细胞核呈三角形或不规则形状，或出现溶解现象，细胞出 现较为明显的变性；海马 CA1 区的尼氏体数量和层数明显减少，排列较为紊乱，甚至出现空泡结构。与模型 组比较，各给药组小鼠的逃逸潜伏期显著缩短（P＜0.01），穿越平台次数显著增加（P＜0.01）；海马组织 AchE、 ROS、IL-1β、TNF-α 水平显著降低（P＜0.01），GPX4、ZO-1 水平显著升高（P＜0.01）；血清 NSE、MMP9、 S100β 水平显著降低（P＜0.01）；海马组织中 PINK1、PARKIN、Beclin-1、LC3B mRNA 表达显著下调（P＜ 0.01），MEF2D、p62 mRNA 表达显著上调（P＜0.01）；小鼠海马 CA1 区神经元排列更为紧密，核固缩现象有所 缓解，发生变性的细胞数量减少；海马 CA1 区的尼氏体数量均有所增加，细胞层数损失减少，排列相对紧密 有序。与模型组比较，定志小丸各剂量组及 PINK1 抑制剂组小鼠海马组织中 PINK1、PARKIN 蛋白表达显著下 调（P＜0.05，P＜0.01）；定志小丸各剂量组的 Beclin-1 蛋白表达显著下调（P＜0.01），p62 蛋白表达显著上调 （P＜0.05，P＜0.01）；定志小丸中、高剂量组及 PINK1 抑制剂组的 MEF2D 蛋白表达显著上调（P＜0.01）；定志 小丸中、高剂量组的 LC3B 蛋白表达显著下调（P＜0.01）。结论 定志小丸对 VD 小鼠具有海马神经元保护作 用，能降低氧化应激和炎症反应，其机制可能与调控 PINK1/PARKIN/MEF2D 线粒体自噬通路相关。

Objective To study the effect of Dingzhi Xiaowan Decoction （DZXW） on PTEN-induced mitophagy pathway of PTEN-induced putative kinase 1（PINK1） /E3 ubiquitin-protein ligase PARK2 （PARKIN） /myocyte enhancer factor 2D （MEF2D） in vascular dementia （VD） mice. Methods VD mouse model was replicated by bilateral common carotid artery occlusion and reperfusion. ICR mice were randomly divided into sham operation group， model group， Donepezil Hydrochloride group （1 mg·kg-1 ， intragastric administration）， PINK1 inhibitor group （20 mg·kg-1 cyclosporine A，intraperitoneal injection） and DZXW low-，medium- and high- dose groups （0.71， 1.43，2.56 g·kg-1 ，intragastric administration），with 6 mice in each group，once a day for 4 consecutive weeks. Morris water maze test was used to detect the learning and memory ability of mice. The levels of acetylcholinesterase （AchE），reactive oxygen species（ROS），interleukin-1β（IL-1β），tumor necrosis factor-α（TNF-α），glutathione peroxidase 4 （GPX4） and zonula occludens-1 （ZO-1） in hippocampus and the levels of neuron-specific enolase （NSE），matrix metalloproteinase 9 （MMP9） and central nervous system-specific protein （S100β） in serum were detected by enzyme-linked immunosorbent assay （ELISA） . The mRNA and protein expression levels of PINK1， PARKIN，MEF2 D，Bcl-2 interacting protein （Beclin-1），microtubule-associated protein 1 light chain 3 beta （LC3B） and sequestosome 1 （p62） in hippocampus were detected by qRT-PCR and Western Blot. HE and Nissl staining were used to observe the pathological changes of hippocampus. Results Compared with the sham operation group，the escape latency of the model group was significantly prolonged （P＜0.01），and the times of crossing the platform was significantly reduced （P＜0.01）. The levels of AchE，ROS，IL-1β and TNF-α in hippocampus were significantly increased （P＜0.01），and the levels of GPX4 and ZO-1 were significantly decreased （P＜0.01）. The levels of serum NSE，MMP9 and S100β were significantly increased（P＜0.01） . The mRNA and protein expressions of PINK1，PARKIN，Beclin-1 and LC3B in hippocampus were significantly up-regulated（P＜0.01），while the mRNA and protein expressions of MEF2D and p62 were significantly down-regulated （P＜0.01） . The neurons in the hippocampal CA1 area were loosely arranged，the nucleus was triangular or irregularly shaped，or dissolved，and the cells showed obvious degeneration. The number and layers of Nissl bodies in the hippocampal CA1 region were significantly reduced，the arrangement was disordered，and even vacuoles appeared. Compared with the model group， the escape latency of mice in each administration group was significantly shortened （P＜0.01），and the times of crossing platforms was significantly increased （P＜0.01） . The levels of AchE， ROS， IL-1β and TNF- α in hippocampus were significantly decreased （P＜0.01），and the levels of GPX4 and ZO-1 were significantly increased （P＜0.01） . The levels of serum NSE， MMP9 and S100β were significantly decreased （P＜0.01） . The mRNA expressions of PINK1，PARKIN，Beclin-1 and LC3B in hippocampus was significantly down-regulated （P＜0.01）， and the mRNA expressions of MEF2D and p62 were significantly up-regulated （P＜0.01） . The neurons in the hippocampal CA1 region of mice were more closely arranged，the phenomenon of nuclear pyknosis was alleviated，and the number of cell degeneration was reduced. The number of Nissl bodies in the hippocampal CA1 region increased，the loss of cell layers decreased，and the arrangement was relatively tight and orderly. Compared with the model group，the protein expressions of PINK1 and PARKIN in hippocampus of mice in each dose group of DZXW and PINK1 inhibitor group was significantly down-regulated（P＜0.05，P＜0.01） . The protein expression of Beclin-1 in each dose group of DZXW was significantly down-regulated（P＜0.01），and the protein expression of p62 was significantly up-regulated （P＜0.05，P＜0.01）. The protein expression of MEF2D was significantly up-regulated in the medium- and high- dose groups of DZXW and the PINK1 inhibitor group（P＜0.01） . The protein expression of LC3B in the medium- and high- dosed DZXW groups was significantly down-regulated（P＜0.01） . Conclusion DZXW has a protective effect on hippocampal neurons in VD mice and can reduce oxidative stress and inflammatory response. The mechanism may be related to the regulation of PINK1/PARKIN/MEF2D mitophagy pathway.

R285.5

国家自然科学基金项目（81904104）；广州市科学技术局项目（2023A03J0744）；广东省科技计划项目（2023B1212060062）；广州中医药 大学校院联合科技创新基金项目（GZYZS2024G14）；广东省中医院中医证候全国重点实验室项目（QZ2023ZZ31）；广东省中医院广东省中医急症 研究重点实验室课题（YN2023JZ17）；广东省中医院朝阳人才科研专项（ZY2022KY06）