[关键词]
[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 Fe2+ 、Fe3+ 、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 Fe2+ 、Fe3+ 、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 Fe2+ 、Fe3+ 、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。
[Key word]
[Abstract]
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 Fe2+ ,Fe3+ ,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) . Fe2+ , Fe3+ , 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. Fe2+ ,Fe3+ ,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.
[中图分类号]
R285.5
[基金项目]
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) 。