高压氧修复缺血再灌注损伤大鼠血脑屏障的自噬机制

doi:10.3969/j.issn.1006-9771.2022.04.007

《中国康复理论与实践》 ›› 2022, Vol. 28 ›› Issue (4): 415-420.doi: 10.3969/j.issn.1006-9771.2022.04.007

高压氧修复缺血再灌注损伤大鼠血脑屏障的自噬机制

黄菲菲¹,王万松²,董晓阳²,冯珍²()

1.皖南医学院弋矶山医院康复医学科,安徽芜湖市 241000
2.南昌大学第一附属医院康复医学科,江西南昌市 330006

收稿日期:2022-01-11 修回日期:2022-03-04 出版日期:2022-04-25 发布日期:2022-05-05
通讯作者: 冯珍 E-mail:fengzhenly@sina.com
作者简介:黄菲菲(1991-),女,汉族,安徽怀宁县人,硕士,住院医师,主要研究方向：神经系统疾病的康复。
基金资助:
国家自然科学基金项目(81260295);江西省自然科学基金青年项目(20202BAB216018)

Repair of blood-brain barrier by hyperbaric oxygen via autophagy in rats with cerebral ischemia-reperfusion injury

HUANG Feifei¹,WANG Wansong²,DONG Xiaoyang²,FENG Zhen²()

1. Department of Rehabilitation Medicine, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui 241000, China
2. Department of Rehabilitation Medicine, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China

Received:2022-01-11 Revised:2022-03-04 Published:2022-04-25 Online:2022-05-05
Contact: FENG Zhen E-mail:fengzhenly@sina.com
Supported by:
National Natural Science Foundation of China(81260295);Jiangxi Natural Science Foundation(Youth)(20202BAB216018)

摘要/Abstract

摘要：

目的观察高压氧舱治疗对缺血再灌注损伤大鼠脑血管内皮细胞微管相关蛋白1轻链3 (LC3)和Beclin-1表达的影响,探究高压氧修复缺血再灌注损伤大鼠血脑屏障的机制。方法 54只成年雌性Sprague-Dawley大鼠随机分为假手术组(n=12)、缺血再灌注损伤模型组(n=18)、高压氧组(n=12)和抑制剂组(n=12)。模型组、高压氧组和抑制剂组采用线栓法建立大鼠大脑中动脉缺血2 h再灌注损伤模型。高压氧组和抑制剂组建模后予高压氧舱治疗;抑制剂组治疗前侧脑室注射3-甲基腺嘌呤。损伤后72 h,各组进行伊文思蓝染色,观察梗死区伊文思蓝含量;模型组采用免疫荧光双标法观察LC3在CD₃₁⁺血管内皮细胞的表达;采用Western blotting检测各组梗死区皮质微血管段LC3-Ⅱ和Beclin-1的表达水平。结果与假手术组相比,模型组梗死区伊文思蓝含量明显升高(P < 0.01);与模型组相比,高压氧组梗死区伊文思蓝含量明显降低(P < 0.01);与高压氧组相比,抑制剂组梗死区伊文思蓝含量增高(P<0.05)。模型组损伤区CD₃₁⁺血管内皮细胞可见LC3表达;模型组梗死区微血管段Beclin-1和LC3-Ⅱ的表达水平均明显高于假手术组(P < 0.01);高压氧组的LC3-Ⅱ和Beclin-1蛋白表达水平较模型组均升高(P<0.05);抑制剂组较高压氧组和模型组均明显下降(P<0.01)。结论缺血再灌注损伤大鼠损伤区的血管内皮细胞存在自噬现象,高压氧舱治疗能够上调梗死区血管内皮细胞自噬蛋白LC3-Ⅱ和Beclin-1的表达,从而促进血脑屏障修复。

关键词: 高压氧, 缺血再灌注损伤, 自噬, 脑血管内皮细胞, 血脑屏障

Abstract:

Objective To observe the effect of hyperbaric oxygen chamber treatment on the expression of autophagy protein microtubule associated protein 1 light chain 3 (LC3) and Beclin-1 in cerebral vascular endothelial cells of rats with ischemia-reperfusion injury, and to explore the mechanism of hyperbaric oxygen in repairing blood-brain barrier in rats with ischemia-reperfusion injury.Methods A total of 54 Sprague-Dawley rats were randomly divided into sham operated group (n=12), cerebral ischemia-reperfusion injury (CIRI) model group (n=18), hyperbaric oxygen group (n=12) and inhibitor group (n=12). The CIRI model was made by a suture method in the model group, hyperbaric oxygen group and inhibitor group. The hyperbaric oxygen group and inhibitor group accepted hyperbaric oxygen, and the inhibitor group injected 3-methyladenine in lateral cerebral ventricle before treatment. They were stained with Evans blue and Evans blue content in the infarct area was detected 72 hours after CIRI. Double immunofluorescent staining was used to observe the expression of LC3 in vascular endothelial cells marked with CD₃₁ in the model group, and LC3-II and Beclin-1 in infarcted cortical microvascular were examined by Western blotting in all the groups.Results Compared with the sham operation group, Evans blue content in the infarct area was significantly higher in the model group (P<0.01); compared with the model group, the content of Evans blue in the infarct area decreased significantly in hyperbaric oxygen group (P<0.01); compared with the hyperbaric oxygen group, the content of Evans blue in the infarct area was significantly higher in the inhibitor group (P < 0.05). There was obvious expression of LC3 in CD₃₁⁺ cells. The expression levels of Beclin-1 and LC3-Ⅱ in the infarcted area were significantly higher in the model groupt than in the sham operation group (P<0.01); and it was significantly higher in the hyperbaric oxygen group than in the model group (P<0.05) and was lower in the inhibitor group than in the hyperbaric oxygen group and model group (P < 0.01).Conclusion Autophagy exists in vascular endothelial cells in the injured area of rats with CIRI. Hyperbaric oxygen can upregulate the expression of autophagy proteins LC3-Ⅱ and Beclin-1 in vascular endothelial cells in the infarcted area, to promote repairing blood-brain barrier.

Key words: hyperbaric oxygen, ischemia-reperfusion injury, autophagy, brain endothelial cell, blood-brain barrier

黄菲菲,王万松,董晓阳,冯珍. 高压氧修复缺血再灌注损伤大鼠血脑屏障的自噬机制[J]. 《中国康复理论与实践》, 2022, 28(4): 415-420.

HUANG Feifei,WANG Wansong,DONG Xiaoyang,FENG Zhen. Repair of blood-brain barrier by hyperbaric oxygen via autophagy in rats with cerebral ischemia-reperfusion injury[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2022, 28(4): 415-420.

图/表 4

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组别	n	EB含量
假手术照组	6	0.887±0.191
模型组	6	2.423±0.223^a
高压氧组	6	1.989±0.163^b
抑制剂组	6	2.249±0.174^c
F值		79.726
P值		< 0.001

组别	n	LC3-Ⅱ/LC3-Ⅰ	Beclin-1/β-actin
假手术组	6	0.255±0.041	0.873±0.174
模型组	6	0.385±0.064^a	1.235±0.090^a
高压氧组	6	0.487±0.066^b	1.549±0.660^b
抑制剂组	6	0.268±0.082^c,d	0.934±0.048^c,d
F值		16.927	51.700
P值		< 0.001	< 0.001

高压氧修复缺血再灌注损伤大鼠血脑屏障的自噬机制

Repair of blood-brain barrier by hyperbaric oxygen via autophagy in rats with cerebral ischemia-reperfusion injury

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