Asiatic acid stabilizes cytoskeletal proteins and prevents TNF-α-induced disorganization of cell-cell junctions in human aortic endothelial cells
Asiatic acid stabilizes cytoskeletal proteins and prevents TNF-α-induced disorganization of cell-cell junctions in human aortic endothelial cells
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DOI：
10.1016/j.vph.2018.08.005
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发表时间：
2019-06-01
影响因子：
4
通讯作者：
Ahmad, Zuraini
中科院分区：
文献类型：
--
作者：
Fong, Lai Yen;Ng, Chin Theng;Ahmad, Zuraini
内皮细胞通透性增高是早期动脉粥样硬化的起始步骤，通常是内皮屏障功能障碍的结果。积雪草酸是从积雪草（Centella asiatica（L.）Urban先前已被证明可防止肿瘤坏死因子（TNF）-α诱导的内皮屏障功能障碍。本研究旨在探讨积雪草酸对人主动脉内皮细胞屏障保护作用的机制。用免疫细胞化学技术和共聚焦显微镜研究了F-actin、二磷酸化肌球蛋白轻链（diphosphoylated myosin light chain，diphospho-MLC）、粘附连接（adherens junction，AJs）和紧密连接（tight junction，TJs）的定位。Western blot分析其总蛋白表达。使用体外血管渗透性测定试剂盒评估内皮渗透性。此外，使用亚细胞分离试剂盒评价连接蛋白的细胞内再分布。我们发现，积雪草酸稳定的F-肌动蛋白和二磷酸-MLC在细胞周边，并防止其重排刺激TNF-α。然而，积雪草酸未能减弱细胞松弛素D诱导的通透性增加。此外，积雪草酸废除TNF-α诱导的结构重组的血管内皮（VE）-钙粘蛋白和β-连环蛋白通过保留其网状结构在细胞-细胞接触区域。此外，积雪草酸还抑制TNF-α诱导的不同亚细胞组分中occludin和occludens（ZO）-1的再分布。总之，积雪草酸的屏障稳定作用可能与保护AJs和防止TNF-α引起的TJ再分布有关。本研究为积雪草酸在预防由内皮屏障功能障碍引起的早期动脉粥样硬化中的潜在应用提供了证据。
Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-alpha-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-alpha. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-alpha-induced structural reorganization of vascular endothelial (VE)-cadherin and beta-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-alpha-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-alpha. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction.