微小RNA-199a-5p保护大鼠脑缺血后血-脑屏障完整性

doi:10.16098/j.issn.0529-1356.2024.04.012

摘要/Abstract

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。

关键词: 缺血性脑卒中, 血-脑屏障, 微小RNA-199a-5p, Claudin-5, Akt信号通路, 免疫印迹法, 实时定量聚合酶链反应, 大鼠

Abstract:

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.

Key words: Ischemic stroke, Blood-brain barrier, MicroRNA-199a-5p, Claudin-5, Akt signaling pathway, Western blotting, Real-time PCR, Rat

中图分类号:

R741.02

倪广晓段春巧寇璐璐孟然王晓青王璞. 微小RNA-199a-5p保护大鼠脑缺血后血-脑屏障完整性[J]. 解剖学报, 2024, 55(4): 460-467.

NI Guang-xiao DUAN Chun-qiao KOU Lu-lu MENG Ran WANG Xiao-qing WANG Pu. MicroRNA-199a-5p reducing blood-brain barrier disruption following ischemic stroke in rats[J]. Acta Anatomica Sinica, 2024, 55(4): 460-467.

参考文献

［1］Tu WJ, Chao BH, Wang LD. Prevalence of stroke in China: overestimated［J］. Lancet Public Health, 2022, 7(5): e404.

［2］Ma QF, Li R, Wang LJ, et al. Temporal trend and attributable risk factors of stroke burden in China, 1990-2019: an analysis for the Global Burden of Disease Study 2019［J］. Lancet Public Health, 2021,6 (12): e897-e906.

［3］Zhang GT, Pan YS, Zhang RH, et al. Prevalence and prognostic significance of malnutrition risk in patients with acute ischemic stroke: results from the Third China National Stroke Registry［J］. Stroke, 2022, 53(1):111-119.

［4］Gao SH, Zhang XL, Yang YCh, et al. Polyinosinic- polycytidylic acid affecting the expressions of Bcl-2 and Bax after cerebral ischemia-reperfusion in hyperlipidemic rats and its neuroprotective effect ［J］. Acta Anatomica Sinca, 2023, 54(2): 175-180. (in Chinese)

高赛红, 张小良, 杨迎春, 等.聚肌苷酸聚胞苷酸对高血脂大鼠脑缺血/再灌注后Bcl-2和Bax表达的影响及神经保护作用［J］. 解剖学报, 2023, 54(2): 175-180.

［5］Dong HP, Xie HY, Ma XX, et al. Mechanism of blood-brain barrier damage caused by the inhibition of Wnt7/β-catenin pathway induced by endoplasmic reticulum stress in cerebrovascular endothelial cells after stroke［J］. Journal of Shanghai Jiao Tong University (Medical Science),2023,43(7):829-838. (in Chinese)

董海平,谢海怡,马晓晓,等.脑卒中后脑血管内皮细胞内质网应激抑制Wnt7/β-catenin通路导致血脑屏障损伤的机制研究［J］.上海交通大学学报(医学版),2023,43(7):829-838.

［6］Hu D, Mo XC, Luo JH, et al. 17-DMAG ameliorates neuro inflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats［J］. Int Immunopharmacol, 2023:110698.

［7］Zhang Y, Nozohouri S, Abbruscato TJ. In vivo evaluation of BBB integrity in the post stroke brain［J］. Methods Mol Biol, 2023,2616:191-203.

［8］Wei YN, Wang FC, Ma XL, et al. Effect of altitude hypoxia on blood-brain barrier after subarachnoid hemorrhage based on phosphatidylinositol 3-kinase/protein kinase B/nuclear factor KB pathway in rats［J］. Acta Anatomica Sinca, 2023, 54(2): 156-164. (in Chinese)

魏艳娜, 王锋存, 马香莲, 等. 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.

［9］Xiong Y, Fu YQ, Li ZhL, et al. Laquinimod inhibits microglial activation, astrogliosis, BBB damage, and infarction and improves neurological damage after ischemic stroke［J］. ACS Chem Neurosci, 2023,14(11):1992-2007.

［10］Hou WZ, Yao JP, Liu JJ, et al. USP14 inhibition promotes recovery by protecting BBB integrity and attenuating neuroinflammation in MCAO mice［J］. CNS Neurosci Ther, 2023, 29(11): 3612-3623.

［11］Matsuo K, Nshihara H. Rebuilding insight into the pathophysiology of Alzheimer’s disease through new blood-brain barrier models［J］. Neural Regen Res, 2024,19(9):1954-1960.

［12］Li WR, Zheng Y. MicroRNAs in extracellular vesicles of Alzheimer’s disease［J］. Cells, 2023, 12(10):1378.

［13］Walgrave H, Penning A, Tosoni G, et al. MicroRNA-132 regulates gene expression programs involved in microglial homeostasis［J］. iScience, 2023, 26(6):106829.

［14］Sun P, Ma FF, Xu Y, et al. Genetic deletion of endothelial microRNA-15a/16-1 promotes cerebral angiogenesis and neurological recovery in ischemic stroke through Src signaling pathway［J］. J Cereb Blood Flow Metab, 2021, 41(10):2725-2742.

［15］Gong CC, Gu ZhY, Zhang XK, et al. HMSCs exosomederived miR-199a-5p attenuates sulfur mustard-associated oxidative stress via the CAV1/NRF2 signaling pathway［J］. J Cell Mol Med, 2023,27(15):2165-2182.

［16］Jin HQ, Jiang WF, Zheng XT, et al. MiR-199a-5p enhances neuronal differentiation of neural stem cells and promotes neurogenesis by targeting Cav-1 after cerebral ischemia［J］. CNS Neurosci Ther, 2023,29(12):1.

［17］Zhong W, Li YC, Huang QY, et al. lncRNA ANRIL ameliorates oxygen and glucose deprivation (OGD) induced injury in neuron cells via miR-199a-5p/CAV-1 axis［J］. Neurochem Res, 2020, 45(4):772-782.

［18］Zhang XH, Zhou GE. MiR-199a-5p inhibition protects cognitive function of ischemic stroke rats by AKT signaling pathway［J］. Am J Transl Res, 2020,12(10): 6549-6558.

［19］Dobyns WB, Mirzaa GM. Megalencephaly syndromes associated with mutations of core components of the PI3K-AKT-MTOR pathway: PIK3CA, PIK3R2, AKT3, and MTOR［J］. Am J Med Genet C Semin Med Genet, 2019, 181(4):582-590.

［20］Shanbhag NC, Henning RH, Schilling L. Long-term survival in permanent middle cerebral artery occlusion: a model of malignant stroke in rats［J］. Sci Rep,2016,6:28401.

［21］Belayev L, Alonso OF, Busto R, et al. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model［J］.Stroke,1996,27(9):1616-1622.

［22］Asadi-Yousefabad SL, Nammian P, Tabei SMB, et al. Angiogenesis in diabetic mouse model with critical limb ischemia; cell and gene therapy［J］. Microvasc Res, 2022, 141:104339.

［23］Jia R, Solé-Guardia G, Kiliaan AJ. Blood-brain barrier pathology in cerebral small vessel disease［J］.Neural Regen Res, 2024,19(6)：1233-1240.

［24］Ren S, Han SS, Wang LK, et al. Minimally invasive surgery for ICH evacuation combined with deferoxamine treatment increased perihematomal Claudin-5 and ZO-1 expression levels and decreased BBB permeability in rabbits［J］. Front Neurol, 2022, (13):835494.

［25］Li Y, Liu B, Zhao T, et al. Comparative study of extracellular vesicles derived from mesenchymal stem cells and brain endothelial cells attenuating blood-brain barrier permeability via regulating Caveolin-1-dependent ZO-1 and Claudin-5 endocytosis in acute ischemic stroke［J］. J Nanobiotechnol, 2023,21(1):70.

［26］Cheng D, Talib J, Stanley CP, et al. Inhibition of MPO (myeloperoxidase) attenuates endothelial dysfunction in mouse models of vascular inflammation and atherosclerosis［J］. Arterioscler Thromb Vasc Biol, 2019,39 (7):1448-1457.

［27］Bushueva O, Barysheva E, Markov A, et al. DNA hypo methylation of them po gene in peripheral blood leukocytes is associated with cerebral stroke in the acute phase［J］. J Mol Neurosci, 2017,71 (9):1914-1932.

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