基于磷脂酰肌醇-3激酶/蛋白激酶B/核因子κB通路探讨高原缺氧对大鼠蛛网膜下腔出血后血脑屏障的作用

doi:10.16098/j.issn.0529-1356.2023.02.005

摘要/Abstract

摘要：

目的探讨高原缺氧对大鼠蛛网膜下腔出血（SAH）后血脑屏障的作用及其机制。方法 SPF级健康成年雄性SD大鼠（n=78），随机分为4组：假手术组（sham）、蛛网膜下腔出血模型组（SAH）、高原缺氧假手术组（Hp sham）和高原缺氧蛛网膜下腔出血模型组（Hp SAH）。通过低压模拟舱（海拔5000 m）饲养4 d建立高原缺氧大鼠动物模型，采用颈内动脉刺破法建立SAH大鼠动物模型。SAH后24 h，各组大鼠进行神经功能学评分和出血严重程度评估，Nissl染色和TUNEL染色分别观察大鼠海马组织CA1区神经元形态变化和神经细胞凋亡，Western blotting检测大鼠海马组织磷酸化PI3K（p-PI3K）、PI3K、磷酸化Akt（p-Akt）、Akt 、磷酸化核因子κB（p-NF-κB）、 NF-κB及基质金属蛋白酶9（MMP-9）、闭锁蛋白（occludin）、紧密连接蛋白-5（claudin-5）的表达，免疫荧光染色观察大鼠海马组织CA1区occludin、claudin5蛋白表达。结果 SAH后24 h，SAH组与Sham组、Hp SAH组与Hp Sham组比较，大鼠神经功能评分明显下降，颅底出血量明显增加（P<0.05），Hp SAH组与SAH组比较，神经功能评分明显下降（P<0.05）；SAH组与sham组比较，大鼠海马组织CA1区神经元形态萎缩变形，神经元细胞排列紊乱，神经元数量明显下降，神经细胞凋亡明显增加（P<0.05），高原缺氧后上述神经元形态损伤和神经细胞凋亡进一步加重；SAH组与sham组、Hp SAH组与Hp sham组比较，p-PI3K/PI3K、p-Akt/Akt、occludin及claudin5蛋白的表达明显下降，p-NF-κB/NF-κB和MMP-9蛋白表达明显升高（P<0.05），Hp SAH组与SAH组比较，p-PI3K/PI3K和MMP-9蛋白表达明显升高，Hp sham组与sham组比较，claudin-5蛋白表达明显升高，差异均具有统计学意义（P<0.05）；免疫荧光染色发现，SAH组大鼠海马组织CA1区occludin、claudin-5蛋白的表达下降，高原缺氧后occludin、claudin-5蛋白的表达进一步降低。结论高原缺氧通过抑制PI3K/Akt/NF-κB通路加重蛛网膜下腔出血大鼠模型后血脑屏障破坏。

关键词: 高原缺氧, 蛛网膜下腔出血, 血脑屏障, 磷脂酰肌醇-3激酶/蛋白激酶B/核因子κB通路, 免疫印迹法, 免疫荧光, 大鼠

Abstract:

Objective To investigate the effect of plateau hypoxia on the blood-brain barrier after subarachnoid hemorrhage (SAH) in rats. Methods Adult male SD rats (n=78) were randomly divided into 4 groups: sham group (sham), SAH model group (SAH), plateau hypoxia sham group (Hp sham) and plateau hypoxia SAH model group (Hp SAH). The rat model of plateau hypoxia was established through low-pressure simulation chamber (altitude 5000 m), and the SAH model was established by endovascular perforation method. At 24 hours after SAH, neurobehavior score and SAH grade were assessed. The morphological changes of neurons and apoptosis of nerve cells in the CA1 region of hippocampal were observed by the staining of Nissl and TUNEL. The expression of phosphorylated PI3K (p-PI3K), PI3K, phosphorylated Akt (p-Akt), Akt, phosphorylated nuclear factor κB(p-NF-κB), NF-κB, matrix metalloproteinase-9 (MMP-9), occludin and claudin-5 in hippocampal were detected by the method of Western blotting. The expression of occludin and claudin-5 proteins in the CA1 region of hippocampal were observed by immunofluorescent staining. Results At 24 hours after SAH, the neurobehavior score decreased significantly and SAH grade increased significantly in the SAH and Hp SAH group (P <0.05). Neurobehavior score decreased significantly in the Hp SAH group compared with the SAH group (P< 0.05). In the SAH group, neurons in the CA1 region of hippocampus were atrophied and deformed, the arrangement were disordered, the number of neurons decreased significantly, and the apoptosis of nerve cells increased significantly(P< 0.05). Plateau hypoxia could aggravate the morphological damage of neurons and apoptosis of nerve cells. The expression of p-PI3K/PI3K, p-Akt/Akt, occludin and claudin-5 proteins decreased significantly, while the expression of p-NF-κB/NF-κB and MMP-9 proteins increased significantly in the SAH and Hp SAH group (P< 0.05). The expression of p-PI3K/PI3K and MMP-9 proteins increased significantly in Hp SAH group compared with the SAH group. The expression of claudin-5 protein increased significantly in Hp sham group compared with the sham group (P<0.05). Immunofluorescent staining showed that the expression of occludin and claudin-5 proteins in the CA1 region of hippocampus decreased in the SAH group. Plateau hypoxia could further decreased the expression of occludin and claudin-5 proteins. Conclusion Plateau hypoxia aggravates blood-brain barrier disruption after subarachnoid hemorrhage in rats through inhibiting PI3K/Akt/NF-κB pathway.

Key words: Plateau hypoxia, Subarachnoid hemorrhage, Blood-brain barrier, Phosphatidylinositol 3-kinase/protein kinase B/nuclear factor κBpathway, Westernblotting, Immunofluorescence, Rat

中图分类号:

R743.3

魏艳娜王锋存马香莲索田莎陈胜王兰桂孙娟赵秀丽. 基于磷脂酰肌醇-3激酶/蛋白激酶B/核因子κB通路探讨高原缺氧对大鼠蛛网膜下腔出血后血脑屏障的作用[J]. 解剖学报, 2023, 54(2): 156-164.

WEI Yan-na WANG Feng-cun MA Xiang-lian SUO Tian-sha CHEN Sheng WANG Lan-gui SUN Juan ZHAO Xiu-li .

Effect of altitude hypoxia on blood-brain barrier after subarachnoid hemorrhage based on phosphatidylinositol 3-kinase/protein kinase B/nuclear factor κBpathwayin rats

[J]. Acta Anatomica Sinica, 2023, 54(2): 156-164.

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