摘要
目的探讨微小RNA(miR)-199a-5p保护脑缺血后血-脑屏障(BBB)完整性的机制。方法通过SPF级成年雄性SD大鼠建立永久性大脑中动脉闭塞(MCAO)模型,48只大鼠随机分为假手术组、模型组、MCAO+miR-199a-5p组和MCAO+miR-199a-5p阴性对照组,每组12只。应用Ludmila Belayev 12分评分法评估大鼠的神经行为学表现;采用Evans蓝染色检测脑缺血后BBB的完整性;免疫荧光染色检测脑缺血后细胞凋亡;Western blotting技术检测claudin-5、磷脂酰肌醇-3激酶调节亚基2(PIK3R2)、p-Akt、Akt和血管内皮生长因子(VEGF)-A的蛋白表达;利用Real-time PCR检测脑缺血半暗带、梗死区miR-199a-5p、claudin-5及VEGF-A表达水平。结果MiR-199a-5p mimic干预增强MCAO大鼠本体感觉和运动能力。MiR-199a-5p降低脑缺血后PIK3R2的表达,激活Akt信号通路,并增加缺血半暗带中claudin-5和VEGF-A的表达。此外,miR-199a-5p减轻了脑缺血后的炎症。MiR-199a-5p降低脑缺血后BBB渗透性,减少神经细胞凋亡。结论MiR-199a-5p可降低缺血性脑卒中后PIK3R2的表达,激活Akt信号通路,降低炎性细胞因子的表达,减轻缺血性脑卒中对BBB的破坏。
Objective To investigate whether microRNA(miR)-199a-5p regulates blood-brain barrier(BBB)integrity through PI3K/Akt pathway after cerebral ischemia.Methods A permanent middle cerebral artery occlusion(MCAO)model was established in SPF adult male SD rats.Totally 48 rats were randomly divided into sham group(n=12),model group(n=12),MCAO+miR-199a-5p group(n=12),and MCAO+miR-199a-5p negative control group(n=12).The Ludmila Bellayev 12 point score was used to evaluate the neurobehavioral performance of rats;The integrity of the BBB after ischemia stroke was detected through Evans blue staining;Immunofluorescent staining was used to determine apoptosis after cerebral ischemia;Western blotting technology was used to detect the protein expression of claudin-5,phosphatidylinositol-3 kinase regulatory subunit 2(PIK3R2),p-Akt,Akt,and vascular endothelial growth factor(VEGF)-A;Real-time PCR was used to investigate the expression levels of miR-199a-5p,claudin-5,and VEGF-A in the ischemic penumbra and infarcted area of the brain.Results The result showed that miR-199a-5p mimic intervention improved proprioception and motor ability in MCAO rats.MiR-199a-5p mimic reduced the expression of PIK3R2 following ischemia stroke,activated the Akt signaling pathway,and increased the expression of claudin-5 and VEGF-A in the ischemic penumbra.In addition,miR-199a-5p alleviated inflammation after cerebral ischemia.MiR-199a-5p mimic reduced BBB permeability and reduced neuronal apoptosis after cerebral ischemia.Conclusion MiR-199a-5p can reduce the expression of PIK3R2 following ischemic stroke,activate the Akt signaling pathway,reduce the expression of inflammatory cytokines,and alleviate the damage to the blood-brain barrier.
作者
倪广晓
段春巧
寇璐璐
孟然
王晓青
王璞
NI Guang-xiao;DUAN Chun-qiao;KOU Lu-lu;MENG Ran;WANG Xiao-qing;WANG Pu(Department of Rehabilitation,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;Stomatological Laboratory,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)
出处
《解剖学报》
CAS
CSCD
2024年第4期460-467,共8页
Acta Anatomica Sinica
基金
河北省自然科学基金(H2023206115)
河北省中医药管理局科技计划项目(2022127)。
