Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

IF 4.4 1区 医学 Q1 CLINICAL NEUROLOGY Stroke and Vascular Neurology Pub Date : 2024-05-02 DOI:10.1136/svn-2023-003080
Zhongfang Deng, Xiaoyu Chen, Ran Zhang, Lingchao Kong, Yang Fang, Jizheng Guo, Bing Shen, Lesha Zhang
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Abstract

Background Local brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by acute I/R injury in brain microvascular endothelial cells (BMECs). Objective This study aims to explore the mechanism by which DADLE enhances the level of mitophagy in BMECs by upregulating the expression of transient receptor potential vanilloid subtype 4 (TRPV4). Methods BMECs were extracted and made to undergo oxygen-glucose deprivation/reoxygenation (OGD/R) accompanied by DADLE. RNA-seq analysis revealed that DADLE induced increased TRPV4 expression. The CCK-8 method was used to assess the cellular viability; quantitative PCR (qPCR) was used to determine the mRNA expression of Drp1 ; western blot was used to determine the expression of TRPV4 and autophagy-related proteins; and calcium imaging was used to detect the calcium influx. Autophagosomes in in the cells’ mitochondria were observed by using transmission electron microscopy. ELISA was used to measure ATP content, and a JC-1 fluorescent probe was used to detect mitochondrial membrane potential. Results When compared with the OGD/R group, OGD/R+DADLE group showed significantly enhanced cellular viability; increased expression of TRPV4, Beclin-1, LC3-II/I, PINK1 and Parkin; decreased p62 expression; a marked rise in calcium influx; further increases in mitophagy, an increase in ATP synthesis and an elevation of mitochondrial membrane potential. These protective effects of DADLE can be blocked by a TRPV4 inhibitor HC067047 or RNAi of TRPV4. Conclusion DADLE can promote mitophagy in BMECs through TRPV4, improving mitochondrial function and relieving I/R injury. Data are available upon reasonable request. The data from western blot strips have been uploaded as supplementary material, other data are available upon reasonable request.
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δ类阿片肽[D-ala2, D-leu5]-脑啡肽通过 TRPV4 增强有丝分裂能力,缓解脑微血管内皮细胞的缺血再灌注损伤
背景局部脑组织会受到缺血再灌注(I/R)损伤,从而导致血管内皮损伤。多肽δ阿片受体(δOR)激动剂[D-ala2, D-leu5]-脑啡肽(DADLE)可减少急性I/R损伤引起的脑微血管内皮细胞(BMECs)凋亡。目的 本研究旨在探讨 DADLE 通过上调瞬时受体电位香草素亚型 4(TRPV4)的表达来提高脑微血管内皮细胞有丝分裂水平的机制。方法 提取 BMECs 并使其在 DADLE 的作用下进行氧-葡萄糖剥夺/复氧(OGD/R)。RNA-seq分析显示,DADLE诱导TRPV4表达增加。CCK-8方法用于评估细胞活力;定量PCR(qPCR)用于测定Drp1的mRNA表达;Western印迹用于测定TRPV4和自噬相关蛋白的表达;钙成像用于检测钙离子流入。透射电子显微镜观察了细胞线粒体中的自噬体。用酶联免疫吸附法测定 ATP 含量,用 JC-1 荧光探针检测线粒体膜电位。结果 与 OGD/R 组相比,OGD/R+DADLE 组的细胞活力明显增强;TRPV4、Beclin-1、LC3-II/I、PINK1 和 Parkin 表达增加;p62 表达减少;钙离子流入明显增加;有丝分裂进一步增加;ATP 合成增加;线粒体膜电位升高。TRPV4 抑制剂 HC067047 或 TRPV4 的 RNAi 可阻断 DADLE 的这些保护作用。结论 DADLE 可通过 TRPV4 促进 BMECs 的有丝分裂,改善线粒体功能并缓解 I/R 损伤。如有合理要求,可提供相关数据。Western 印迹条的数据已作为补充材料上传,其他数据可应合理要求提供。
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来源期刊
Stroke and Vascular Neurology
Stroke and Vascular Neurology Medicine-Cardiology and Cardiovascular Medicine
CiteScore
11.20
自引率
1.70%
发文量
63
审稿时长
15 weeks
期刊介绍: Stroke and Vascular Neurology (SVN) is the official journal of the Chinese Stroke Association. Supported by a team of renowned Editors, and fully Open Access, the journal encourages debate on controversial techniques, issues on health policy and social medicine.
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