定志小丸对血管性痴呆小鼠PINK1/PARKIN/MEF2D线粒体自噬通路的影响  

Effect of Dingzhi Xiaowan Decoction on PINK1/PARKIN/MEF2D Mitophagy Pathway in Vascular Dementia Mice

作　　者：邓敏贞 王宇[1,3] 宁振求 胡达峰 王凯 王成毅[3] 孙景波 程骁 DENG Minzhen;WANG Yu;NING Zhenqiu;HU Dafeng;WANG Kai;WANG Chengyi;SUN Jingbo;CHENG Xiao(State Key Laboratory of Traditional Chinese Medicine Syndrome/Department of Neurology,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510120 Guangdong,China;Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Emergency Research,Guangzhou 510120 Guangdong,China;Guangzhou University of Chinese Medicine,Guangzhou 510006 Guangdong,China)

机构地区：[1]中医证候全国重点实验室/广州中医药大学第二附属医院,广东广州510120 [2]广东省中医急症研究重点实验室,广东广州510120 [3]广州中医药大学,广东广州510006

出　　处：《中药新药与临床药理》2025年第3期328-337,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：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)。

摘　　要：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、S1Objective 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 significantl

关 键 词：定志小丸 血管性痴呆 PINK1/PARKIN/MEF2D通路 线粒体自噬 氧化应激 炎症反应 小鼠 

分 类 号：R285.5[医药卫生—中药学]