摘要
目的探讨丹蒌片对血管性痴呆(VD)大鼠认知功能的影响及作用机制。方法随机选择10只SD大鼠作为假手术组,另外50只作为造模组,采用双侧颈总动脉结扎(BCCAO)法复制VD大鼠模型。将模型复制成功的VD大鼠随机分为模型组、丹蒌片低剂量组(0.4 g·kg^(-1))、丹蒌片高剂量组(0.8 g·kg^(-1))及盐酸多奈哌齐组(0.45 mg·kg^(-1)),每组10只。灌胃给药,灌胃体积均为10 mL·kg^(-1),每日1次,持续8周。通过行为学测试(Morris水迷宫实验、Y迷宫实验)检测大鼠的学习记忆及空间探索记忆能力;HE染色法观察海马区神经元结构变化;普鲁士蓝染色法观察海马、皮层区铁沉积情况;透射电镜观察海马区神经元、神经突触及线粒体超微结构变化;微量法检测脑组织中Fe^(2+)、Fe^(3+)、谷胱甘肽(GSH)、丙二醛(MDA)含量;Western Blot法检测海马组织中TfR1、FPN、FTH1、GPX4蛋白表达水平;免疫组化法检测海马区FTH1蛋白表达情况。结果与假手术组比较,模型组大鼠第3~5天的逃避潜伏期显著延长(P<0.01),目标象限停留时间占比及平台穿越次数显著减少(P<0.01);总进臂次数无明显变化(P>0.05),自发交替准确率显著降低(P<0.01);神经元细胞排列紊乱,层次模糊,数量减少,神经元之间间隙增宽,核固缩、碎裂和溶解现象增多,间质明显水肿,部分区域存在“断裂带”“空泡化”改变;病灶脑组织中铁沉积量明显升高,可见黄棕色或棕褐色的阳性细胞数量显著增多;电镜观察可见,神经元细胞内的细胞器数量稀疏,胞核异染色质边集,多聚集在周围,膜结构模糊,前膜内囊泡数量减少,突触间隙显著增宽(P<0.01),线粒体结构损伤明显,数量显著减少,局部基质空化,部分线粒体肿胀破裂,膜密度增加,线粒体嵴模糊甚至消失;脑组织中的Fe^(2+)、Fe^(3+)、MDA含量显著增加(P<0.01),而GSH含量显著降低(P<0.01);海马组织中TfR1蛋白表达量明显升高(P<0.05),而FPN1、GPX4、FTH1蛋白表达量显著降低(P<0.01);海马区FTH1蛋白表达下调,着色较浅,平均光密度值显著降低(P<0.01)。与模型组比较,各给药组大鼠第3~5天的逃避潜伏期显著缩短(P<0.05,P<0.01),目标象限停留时间占比及平台穿越次数明显增多(P<0.05);总进臂次数无明显变化(P>0.05),而自发交替准确率明显升高(P<0.05);神经元细胞排列相对规则紧密,数量丢失显著降低,神经元细胞形态基本恢复,胞浆水肿现象得到改善;病灶脑组织中铁沉积量明显降低,黄棕色或棕褐色的阳性细胞数量显著减少;电镜观察可见,神经元细胞膜清晰可见,细胞器数量相对增加,胞核分布中央,体积较大,染色质边缘化现象显著减轻,神经突触结构改善,突触间隙显著变窄(P<0.01),线粒体膜清晰可见,损伤程度减轻,线粒体嵴断裂现象显著改善;脑组织中的Fe^(2+)、Fe^(3+)、MDA含量显著降低(P<0.01),而GSH含量显著升高(P<0.01);海马组织中TfR1蛋白表达量显著降低(P<0.01),而FPN1、GPX4、FTH1蛋白表达量显著升高(P<0.05,P<0.01);海马区FTH1蛋白表达显著上调,平均光密度值显著升高(P<0.01)。结论丹蒌片可以改善VD大鼠的认知功能障碍,减轻海马组织神经元病理改变,可能与其抑制铁依赖的氧化应激反应,进而抑制神经元细胞发生铁死亡有关。
Objective To investigate the effects of Danlou Tablets on cognitive function in vascular dementia(VD)rats and its underlying mechanisms.Methods Ten SD rats were randomly selected as the sham-operated group,while 50 rats underwent bilateral common carotid artery occlusion(BCCAO)to establish the VD model.Successfully modeled rats were randomly divided into the model group,low-dose Danlou Tablets group(0.4 g·kg^(-1)),high-dose Danlou Tablets group(0.8 g·kg^(-1)),and Donepezil Hydrochloride group(0.45 mg·kg^(-1)),with 10 rats per group.All groups received intragastric administration(10 mL·kg^(-1))once daily for 8 weeks.Behavioral tests(Morris water maze,Y-maze)were conducted to assess learning,memory,and spatial exploration abilities.Hippocampal neuronal structure was examined via hematoxylin-eosin(HE)staining;iron deposition in the hippocampus and cortex was evaluated using Prussian blue staining;ultrastructural changes in hippocampal neurons,synapses,and mitochondria were observed via transmission electron microscopy(TEM).Brain tissue levels of Fe^(2+),Fe^(3+),glutathione(GSH),and malondialdehyde(MDA)were quantified.Western Blot measured hippocampal protein expression of transferrin receptor 1(TfR1),ferroportin 1(FPN1),ferritin heavy chain 1(FTH1),and glutathione peroxidase 4(GPX4).Immunohistochemistry assessed FTH1 expression in the hippocampus.Results Compared with the sham group,the model group exhibited significantly prolonged escape latency(days 3-5,P<0.01),reduced target quadrant dwell time and platform crossings(P<0.01),unchanged total arm entries(P>0.05),and decreased spontaneous alternation accuracy(P<0.01).Neurons were disorganized,with widened intercellular gaps,nuclear pyknosis,fragmentation,and vacuolation;interstitial edema and“fracture zones”were observed.Iron deposition(brown/yellow-positive cells)increased markedly.TEM revealed sparse organelles,marginated heterochromatin,blurred membranes,reduced synaptic vesicles,widened synaptic clefts(P<0.01),and damaged mitochondria(swelling,cristae disruption).Fe^(2+),Fe^(3+),and MDA levels rose(P<0.01),while GSH declined(P<0.01).Hippocampal TfR1 protein expression increased(P<0.05),whereas FPN1,GPX4,and FTH1 protein expression decreased(P<0.01).FTH1 immunoreactivity weakened(P<0.01).Compared with the model group,all treatment groups showed shortened escape latency(P<0.05,P<0.01),improved target quadrant dwell time and platform crossings(P<0.05),unchanged total arm entries(P>0.05),and elevated alternation accuracy(P<0.05).Neuronal arrangement normalized,with reduced edema and iron deposition(P<0.01).TEM demonstrated restored organelles,centralized nuclei,narrower synaptic clefts(P<0.01),and intact mitochondrial cristae.Fe^(2+),Fe^(3+),and MDA levels declined(P<0.01),while GSH rose(P<0.01).TfR1 protein expression decreased(P<0.01),whereas protein expression of FPN1,GPX4,and FTH1 increased(P<0.05,P<0.01).FTH1 protein expression was significantly upregulated in the hippocampus,and the average optical density value was significantly increased(P<0.01).Conclusion Danlou Tablets ameliorates cognitive dysfunction and hippocampal neuronal damage in VD rats,potentially by inhibiting iron-dependent oxidative stress and subsequent ferroptosis.
作者
张雅涵
高爱社
田浩
任金玲
曹珊
陈芳
宋军营
张妍
ZHANG Yahan;GAO Aishe;TIAN Hao;REN Jinling;CAO Shan;CHEN Fang;SONG Junying;ZHANG Yan(School of Medicine,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China)
出处
《中药新药与临床药理》
北大核心
2025年第9期1413-1422,共10页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
中国博士后科学基金项目(2023M741089)
河南省科技攻关项目(232102310434)
河南省自然科学基金面上项目(242300421295)
河南省高等学校重点科研项目(24A310004)。
关键词
丹蒌片
血管性痴呆
认知功能障碍
铁死亡
氧化应激
大鼠
Danlou Tablets
vascular dementia
cognitive dysfunction
ferroptosis
oxidative stress
rats
