Madecassic Acid protects against hypoxia-induced oxidative stress in retinal microvascular endothelial cells via ROS-mediated endoplasmic reticulum stress
Madecassic Acid protects against hypoxia-induced oxidative stress in retinal microvascular endothelial cells via ROS-mediated endoplasmic reticulum stress
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羟基积雪草酸通过 ROS 介导的内质网应激保护视网膜微血管内皮细胞免受缺氧诱导的氧化应激
DOI：
10.1016/j.biopha.2016.10.015
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发表时间：
2016-12-01
影响因子：
7.5
通讯作者：
Liang, Xiaoling
中科院分区：
文献类型：
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作者：
Yang, Boyu;Xu, Yue;Liang, Xiaoling
积雪草酸（Madecassic acid，MA）是积雪草（Centella asiatica（L.）城市。（伞形科），其已被用作伤口愈合剂、抗炎剂和抗癌剂。到目前为止，MA抗氧化应激的作用尚不清楚。本研究探讨了MA对缺氧诱导的人视网膜微血管内皮细胞（human Retinal Microvascular Endothelial Cells，hRMEC）的影响及其机制。用不同浓度的MA（0-50 μ M）预处理hRMEC 30分钟，然后在缺氧条件（37 ℃，5%CO2和95%N-2）下孵育。MTT法和TUNEL法检测细胞凋亡，Western blotting和RT-PCR法检测细胞凋亡和内质网应激相关分子的表达。用DCFH-DA法测定细胞内ROS水平，用相关试剂盒测定细胞内丙二醛（MDA）、脱氢酶（LDH）、谷胱甘肽过氧化物酶（GSH-PX）和超氧化物歧化酶（SOD）活性。采用免疫印迹和免疫荧光染色法检测细胞核转位情况。MA显著降低缺氧诱导的hRMEC中的氧化应激，如通过增加细胞活力、SOD和GSH-PX渗漏、降低TUNEL和ROS阳性细胞比率、LDH和MDA渗漏、caspase-3和-9活性以及Bax/Bcl-2比率所示。此外，MA还减弱了低氧诱导的hRMEC中的ER应激，如通过降低葡萄糖调节蛋白78（GRP 78）、C/EBP同源转录因子（CHOP）的mRNA水平、切割的转录激活因子6（ATF 6）和肌醇需要激酶/核酸内切酶1 α（INK 1 α）的蛋白水平所示。（IRE 1 α）、胰腺ER应激激酶（PERK）和真核起始因子2 α（eIF 2 α）的磷酸化、裂解的半胱天冬酶-12和ATF 4易位至细胞核。本研究表明，MA对氧化应激和内质网应激的调节将是一种很有前途的治疗方法，以扭转缺氧诱导的hRMEC功能障碍的过程和发展。(C)2016 Elsevier Masson SAS。All rights reserved.
Madecassic acid (MA) is an abundant triterpenoid in Centella asiatica (L.) Urban. (Apiaceae) that has been used as a wound-healing, anti-inflammatory and anti-cancer agent. Up to now, the effects of MA against oxidative stress remain unclear. In this study, we investigated the effect of MA and its mechanisms on hypoxia-induced human Retinal Microvascular Endothelial Cells (hRMECs). hRMECs were pre-treated with different concentrations of MA (0-50 mu M) for 30 min before being incubated under hypoxia condition (37 degrees C, 5% CO2 and 95% N-2). Cell apoptosis was evaluated with MTT assay and TUNEL staining, and the expression of apoptosis-and endoplasmic reticulum (ER) stress-related molecules was assessed with western blotting and RT-PCR analysis. Intracellular ROS level was evaluated using DCFH-DA. Intracellular malondialdehyde (MDA), dehydrogenase (LDH), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were evaluated using related Kits. Activating transcription factor 4 (ATF4) nuclear translocation was assessed with western blotting analysis and immunofluorescence staining. MA significantly reduced oxidative stress in hypoxia-induced hRMECs, as shown by increased cell viability, SOD and GSH-PX leakage, decreased TUNEL-and ROS-positive cell ratio, LDH and MDA leakage, caspase-3 and -9 activity, and Bax/Bcl-2 ratio. In addition, MA also attenuated hypoxia-induced ER stress in hRMECs, as shown by reduced mRNA levels of glucose-regulated protein 78 (GRP78), C/EBP homologous transcription factor (CHOP), protein levels of cleaved activating transcription factor 6 (ATF6) and inositolrequiring kinase/endonuclease 1 alpha (IRE1 alpha), phosphorylation of pancreatic ER stress kinase (PERK) and eukaryotic initiation factor 2 alpha (elF2 alpha), cleaved caspase-12 and ATF4 translocation to nucleus. The current study indicated that the regulation of oxidative stress and ER stress by MA would be a promising therapy to reverse the process and development of hypoxia-induced hRMECs dysfunction. (C) 2016 Elsevier Masson SAS. All rights reserved.