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王倩, 陆昊, 任玮, 王青, 范丽桢.地黄苷A通过调控DLL4/Notch信号通路保护缺血性脑卒中大鼠血脑屏障并改善认知障碍[J].湖南中医药大学学报,2026,46(2):222-231[点击复制] |
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| 地黄苷A通过调控DLL4/Notch信号通路保护缺血性脑卒中大鼠血脑屏障并改善认知障碍 |
| 王倩,陆昊,任玮,王青,范丽桢 |
| (南京鼓楼医院/南京大学医学院附属鼓楼医院, 江苏 南京 210000) |
| 摘要: |
| 目的 探究地黄苷A调节Delta样配体4（DLL4）/Notch信号通路对缺血性脑卒中（IS）大鼠血脑屏障和认知障碍的影响。方法 按照随机数字表法将大鼠分为假手术组、模型组、地黄苷A低剂量组、地黄苷A中剂量组、地黄苷A高剂量组、尼莫地平组、地黄苷A中剂量+MK-0752组、MK-0752组，每组12只。除假手术组外，其余各组均采用大脑中动脉闭塞（MCAO）法建立IS模型。地黄苷A低、中、高剂量组分别腹腔注射地黄苷A 30、60、120 mg/kg并灌胃等量生理盐水；尼莫地平组灌胃12 mg/kg尼莫地平并腹腔注射等量生理盐水；地黄苷A中剂量+MK-0752组腹腔注射60 mg/kg地黄苷A并灌胃100 mg/kg MK-0752；MK-0752组灌胃100 mg/kg MK-0752并腹腔注射等量生理盐水；假手术组、模型组大鼠腹腔注射并灌胃等量生理盐水。1次/d，持续14 d。通过Morris水迷宫试验评估认知功能；采用改良神经功能缺损评分（mNSS）评价神经功能缺损程度；干湿重法测定脑含水量；EB渗漏法检测血脑屏障通透性，TTC染色测量脑梗死面积，Nissl染色观察神经元损伤；qPCR检测脑组织DLL4、Notch同源蛋白1（Notch1）、闭锁小带蛋白-1（ZO-1）、闭合蛋白（Occludin）和基质金属蛋白酶-9（MMP-9）的mRNA表达水平；Western blot检测脑组织DLL4、Notch1、ZO-1、Occludin和MMP-9的蛋白表达水平。结果 与假手术组相比，模型组大鼠逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05），目标象限停留时间缩短（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均升高（P<0.05），脑梗死面积增大（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均降低（P<0.05）。与模型组相比，尼莫地平组、地黄苷A低、中、高剂量组及地黄苷A中剂量+MK-0752组大鼠逃避潜伏期均缩短（P<0.05），穿越平台次数均增加（P<0.05），目标象限停留时间均延长（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均降低（P<0.05），脑梗死面积均缩小（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均升高（P<0.05）。与尼莫地平组相比，地黄苷A低、中剂量组及地黄苷A中剂量+MK-0752组大鼠逃避潜伏期均延长（P<0.05），穿越平台次数均减少（P<0.05），目标象限停留时间均缩短（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均升高（P<0.05），脑梗死面积均增大（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均降低（P<0.05）。与地黄苷A低剂量组比较，地黄苷A中、高剂量组大鼠逃避潜伏期均缩短（P<0.05），穿越平台次数均增加（P<0.05），目标象限停留时间均延长（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均降低（P<0.05），脑梗死面积均缩小（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均升高（P<0.05）。与地黄苷A中剂量组比较，地黄苷A高剂量组大鼠逃避潜伏期缩短（P<0.05），穿越平台次数增加（P<0.05），目标象限停留时间延长（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均降低（P<0.05），脑梗死面积缩小（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均升高（P<0.05）；地黄苷A中剂量+MK-0752组逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05），目标象限停留时间缩短（P<0.05），mNSS、脑含水量、脑组织EB含量及MMP-9 mRNA与蛋白表达水平均升高（P<0.05），脑梗死面积增大（P<0.05），DLL4、Notch1、ZO-1及Occludin mRNA与蛋白表达水平均降低（P<0.05）。结论 地黄苷A可能通过激活DLL4/Notch信号通路，降低血脑屏障通透性，改善IS大鼠的认知障碍。 |
| 关键词: 缺血性脑卒中 血脑屏障 地黄苷A DLL4/Notch信号通路 认知障碍 紧密连接蛋白 |
| DOI：10.3969/j.issn.1674-070X.2026.02.002 |
| 投稿时间：2025-11-18 |
| 基金项目:国家自然科学基金项目(82101425)。 |
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| Rehmannioside A protects blood-brain barrier and improres cognitive impairment in rats with ischemic stroke by regulating the DLL4/Notch signaling pathway |
| WANG Qian, LU Hao, REN Wei, WANG Qing, FAN Lizhen |
| (Nanjing Drum Tower Hospital/The Affiliated Drum Tower Hospital of the Medical School of Nanjing University, Nanjing, Jiangsu 210000, China) |
| Abstract: |
| Objectives To explore the effects of Rehmannioside A on regulating the delta-like ligand 4 (DLL4)/Notch signaling pathway on the blood-brain barrier dysfunction and cognitive impairment in rats with ischemic stroke (IS). Methods Rats were randomly assigned to the sham-operated group, model group, low-dose, medium-dose, and high-dose Rehmannioside A groups, nimodipine group, medium-dose Rehmannioside A+MK-0752 group, and MK-0752 group (n=12 per group). Except for the sham group, the middle cerebral artery occlusion (MCAO) method was used to establish the IS model in the other groups. The low-dose, medium-dose, and high-dose groups of Rehmannioside A were intraperitoneally injected with 30, 60 and 120 mg/kg of Rehmannioside A respectively, and intragastric administration of an equal volume of normal saline. The nimodipine group received intragastric administration of nimodipine (12 mg/kg) and intraperitoneal injection of an equal volume of normal saline. The medium-dose Rehmannioside A+MK-0752 group received intraperitoneal injection of Rehmannioside A (60 mg/kg) and intragastric administration of MK-0752 (100 mg/kg). The MK-0752 group received intragastric administration of MK-0752 (100 mg/kg) and intraperitoneal injection of an equal volume of normal saline. The sham and model groups received intraperitoneal injection plus intragastric administration of equal volumes of normal saline. All treatments were given once daily for 14 days. Cognitive function was evaluated using the Morris water maze test. Neurological deficits were assessed by the modified neurological severity score (mNSS). Brain water content was measured by the wet-dry method. Blood-brain barrier permeability was assessed by Evans blue extravasation, cerebral infarct area was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and neuronal damage was examined by Nissl staining. Quantitative PCR (qPCR) was performed to determine mRNA expression levels of DLL4, Notch homolog 1 (Notch1), zonula occludens-1 (ZO-1), Occludin, and matrix metalloproteinase-9 (MMP-9). Western blotting was used to determine protein expression levels of DLL4, Notch1, ZO-1, Occludin, and MMP-9. Results Compared with the sham group, the model group exhibited a prolonged escape latency (P<0.05), reduced platform crossings (P<0.05), and shortened time spent in the target quadrant (P<0.05). The mNSS, brain water content, Evans Blue (EB) content in brain tissue, and MMP-9 mRNA and protein expression levels were higher (P<0.05), accompanied by a larger infarct area (P<0.05). In contrast, the mRNA and protein expression levels of DLL4, Notch1, ZO-1, and occludin were lower (P<0.05). Compared with the model group, the nimodipine group, low-, medium-, and high-dose Rehmannioside A groups, and the medium-dose Rehmannioside A+MK-0752 group showed shortened escape latency (P<0.05), increased platform crossings (P<0.05), and prolonged time spent in the target quadrant (P<0.05). Meanwhile, mNSS scores, brain water content, EB content, and MMP-9 mRNA and protein expression levels decreased (P<0.05), the infarct area was reduced (P<0.05), and the mRNA and protein expression levels of DLL4, Notch1, ZO-1, and Occludin increased (P<0.05). Compared with the nimodipine group, the low- and medium-dose Rehmannioside A groups, and medium-dose Rehmannioside A+MK-0752 group demonstrated prolonged escape latency (P<0.05), reduced platform crossings (P<0.05), and shortened time in the target quadrant (P<0.05). In addition, mNSS scores, brain water content, EB content, and MMP-9 mRNA and protein expression levels increased (P<0.05), the infarct area was enlarged (P<0.05), and the mRNA and protein expression levels of DLL4, Notch1, ZO-1, and Occludin decreased (P<0.05). Compared with the low-dose Rehmannioside A group, the medium- and high-dose Rehmannioside A groups exhibited shortened escape latency (P<0.05), increased platform crossings (P<0.05), and prolonged time in the target quadrant (P<0.05). Moreover, mNSS scores, brain water content, EB content, and MMP-9 mRNA and protein expression levels were lower (P<0.05), the infarct area was reduced (P<0.05), and the mRNA and protein expression levels of DLL4, Notch1, ZO-1, and Occludin were higher (P<0.05). Compared with medium-dose Rehmannioside A group, rats in the high-dose group showed further shortened escape latency (P<0.05), increased platform crossings (P<0.05), and prolonged time in the target quadrant (P<0.05), along with decreased mNSS scores, brain water content, EB content, and MMP-9 mRNA and protein expression levels (P<0.05), a reduced infarct area (P<0.05), and upregulated mRNA and protein expression of DLL4, Notch1, ZO-1, and occludin (P<0.05). In contrast, the medium-dose Rehmannioside A+MK-0752 group displayed prolonged escape latency (P<0.05), reduced platform crossings (P<0.05), and shortened time in the target quadrant (P<0.05), accompanied by increased mNSS scores, brain water content, EB content, and MMP-9 mRNA and protein expression levels (P<0.05), an enlarged infarct area (P<0.05), and decreased mRNA and protein expressions of DLL4, Notch1, ZO-1, and occludin (P<0.05). Conclusion Rehmannioside A may reduce blood-brain barrier permeability and alleviate cognitive impairment in IS rats by activating the DLL4/Notch signaling pathway. |
| Key words: ischemic stroke blood-brain barrier Rehmannioside A DLL4/Notch signaling pathway cognitive impairment tight junction proteins |
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