定志小丸对卒中后认知障碍模型小鼠PI3K/Akt/mTOR/HIF-1α通路的影响  

Effect of Dingzhi Xiaowan on PI3K/Akt/mTOR/HIF-1αPathway in Post-stroke Cognitive Impairment Model Mice

作　　者：张晗 王宇 钟晓琴 宁振求 胡达峰 邓敏贞 ZHANG Han;WANG Yu;ZHONG Xiaoqin;NING Zhenqiu;HU Dafeng;DENG Minzhen(State Key Laboratory of Traditional Chinese Medicine Syndrome(TCM),The Second Affiliated Hospital of Guangzhou University of Chinese Medicine/Guangdong Provincial Hospital of Chinese Medicine/Guangdong Branch of Chinese Academy of Chinese Medical Sciences,Guangzhou 510120,China;Guangdong Provincial Key Laboratory of Research on Emergency in TCM,Guangzhou 510120,China;Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Shenzhen Bao'an TCM Hospital/Bao'an TCM Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518100,China;The First Affiliated Hospital of Guangzhou Medical University,Guangzhou 510210,China)

机构地区：[1]广州中医药大学第二附属医院/广东省中医院/广东省中医药科学院,中医证候全国重点实验室,广州510120 [2]广东省中医急症研究重点实验室,广州510120 [3]广州中医药大学,广州510006 [4]深圳市宝安区中医院/广州中医药大学附属宝安中医院,广东深圳518100 [5]广州医科大学附属第一医院,广州510120

出　　处：《中国实验方剂学杂志》2025年第5期1-11,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：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);国家自然科学基金项目(81904104);广州市科学技术局项目(2023A03J0744);广州中医药大学校院联合科技创新基金项目(GZYZS2024G14,GZYZS2024U20,GZYFT2024Y12)。

摘　　要：目的:研究定志小丸(DZXW)对卒中后认知障碍(PSCI)模型小鼠的作用机制。方法:小鼠脑缺血再灌注损伤模型建立采用大脑中动脉闭塞法。将40只C57BL/6雄性小鼠随机分为假手术组、模型组、DZXW低剂量组(1.43 g·kg^(-1))和DZXW高剂量组(2.56 g·kg^(-1)),每组10只。假手术组和模型组都进行等量的生理盐水灌胃处理,上述4组小鼠连续30 d灌胃,每天1次。采用Morris水迷宫实验评价小鼠学习记忆能力;酶联免疫吸附测定法(ELISA)检测血清中淀粉样前体蛋白(APP)、淀粉样蛋白42(Aβ_(42))、乙酰胆碱酯酶(AChE)和超氧化物歧化酶(SOD)水平;原位末端标记法(TUNEL)检测小鼠海马神经元的细胞凋亡程度;蛋白免疫印迹法(Western blot)检测海马组织中磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、缺氧诱导因子-1α亚单位(HIF-1α)、自噬效应蛋白1(Beclin1)、螯合体1(p62)、微管相关蛋白1轻链3(LC3)、B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达;普鲁士蓝染色检测海马组织的铁沉积情况;透射电镜观察小鼠海马神经元超微结构。结果:与假手术组比较,模型组的潜伏期、APP、Aβ_(42)、AChE、TUNEL阳性率、铁离子沉积、HIF-1α、Beclin1、Bax和LC3Ⅱ/Ⅰ显著增加(P<0.01),而穿越平台次数、SOD、p-PI3K、p-Akt、p-mTOR、p62和Bcl-2显著减少(P<0.01)。与模型组比较,DZXW各剂量组的潜伏期、APP、Aβ_(42)、AChE、TUNEL阳性率、铁离子沉积、HIF-1α、Beclin1、Bax和LC3Ⅱ/Ⅰ明显减少(P<0.05),而穿越平台次数、SOD、p-PI3K、p-Akt、p-mTOR、p62和Bcl-2明显增加(P<0.05)。结论:DZXW可以通过调节PI3K/Akt/mTOR/HIF-1α自噬信号通路来减缓PSCI模型小鼠因氧化应激加重海马神经元损伤诱导的认知障碍。Objective:To investigate the effect of Dingzhi Xiaowan(DZXW)in post-stroke cognitive impairment(PSCI)model mice.Methods:The cerebral ischemia-reperfusion injury model of mice was established by using the middle cerebral artery occlusion method.Forty C57BL/6 male mice were randomly divided into the sham operation group,model group,low-dose DZXW group(1.43 g·kg^(-1)),and high-dose DZXW group(2.56 g·kg^(-1)),with 10 mice in each group.Both the sham operation group and the model group were treated with equal amounts of normal saline by gavage,and the above four groups of mice were gavaged once a day for 30 consecutive days.Morris water maze test was used to evaluate the learning memory ability of mice.Serum levels of amyloid precursor protein(APP),amyloid 42(Aβ_(42)),acetylcholinesterase(AChE),and superoxide dismutase(SOD)were measured by enzyme-linked immunosorbent assay(ELISA).Deoxyribonucleotide end transferase-mediated nick end labelling(TUNEL)assay was applied to detect the degree of apoptosis in the mouse's hippocampal neurons.Western blot was used to detect the protein expression of phosphoinositol-3 kinase(PI3K),protein kinase B(Akt),mammalian target of rapamycin(mTOR),hypoxia-inducible factor 1-alpha(HIF-1α),B-cell lymphoma 2(Bcl-2)homologous structural domain protein(Beclin1),sequestosome 1(p62),microtubule-associated protein light chain 3(LC3),Bcl-2,and Bcl-2-associated X protein(Bax)in hippocampal tissue.Prussian blue staining was used to detect iron deposition in hippocampal tissue.Transmission electron microscopy was taken to observe the ultrastructure of the mouse's hippocampal neurons.Results:Compared with the sham operation group,the latency,APP,Aβ_(42),AChE,TUNEL positivity,ferric ion deposition,HIF-1α,Beclin1,Bax,and LC3Ⅱ/Ⅰwere significantly increased in the model group(P<0.01),while the number of crossing platforms,SOD,p-PI3K,p-Akt,p-mTOR,p62,and Bcl-2 were significantly decreased(P<0.01).Compared with the model group,the latency,APP,Aβ_(42),AChE,TUNEL positivity rate,ferric ion deposit

关 键 词：定志小丸 痴呆 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)/缺氧诱导因子-1α亚单位(HIF-1α)通路 缺血性中风 缺血再灌注 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R259