Sulodexide protects endothelial cells against 4-hydroxynonenal-induced oxidative stress and glutathione-dependent redox imbalance by modulation of sestrin2/nuclear factor erythroid 2-related factor 2 pathway.

IF 2 4区 医学 Q3 PHYSIOLOGY
Journal of Physiology and Pharmacology Pub Date : 2024-08-01 Epub Date: 2024-10-10 DOI:10.26402/jpp.2024.4.03
K Bontor, B Gabryel
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引用次数: 0

Abstract

The lipid peroxidation product 4-hydroxynonenal (HNE) may be involved in vascular endothelial cell damage by induction of oxidative stress, apoptosis, and loss of redox homeostasis. There is evidence that stimulation of endothelial cells with 4-HNE induces the activation of the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap-1) pathway. Sestrin2 protein (SESN2) is one of the key regulators of Nrf2 and is involved in the cellular response to oxidative stress. However, the function of SESN2 in HNE-induced endothelial injury is not yet understood. Sulodexide (SDX) is a mixture of glycosaminoglycans used in clinical practice in the treatment of chronic venous and arterial diseases. While SDX has well-documented endothelial protective properties, little is known about its antioxidant effects. The aim of this study was to elucidate the molecular mechanisms activated by SDX in human umbilical endothelial cells (HUVECs) under HNE-induced oxidative stress. In this experimental model, we decided to evaluate the anti-apoptotic and antioxidant potential of SDX and its effect on the SESN2/Nrf2/GSH pathway. HUVECs were treated with 25 _M HNE or HNE combined with 0.5 LRU/mL SDX for 4 hours. Cell viability, apoptosis and intracellular reactive oxygen species (ROS) production were assessed by MTT assay and fluorescence microscopy. The expressions of Bax, cleaved caspase-3, Keap-1 and Nrf2 were determined by Western blot analysis. The intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by colorimetric assay. SESN2, glutamate-cysteine ligase catalytic subunit (GCLc) and glutathione synthase (GSS) were assessed using ELISA. RT-qPCR was performed to detect Nrf2, GCLc and GSS mRNA levels. Transient Nrf2 silencing was obtained by short interfering RNA (siRNA). We have demonstrated that SDX can reduce the negative impact of HNE on HUVECs. SDX significantly protected HNE-treated HUVECs from apoptosis (p<0.001) and oxidative stress (p<0.001). SDX treatment significantly reduced Bax (p<0.05) and cleaved caspase-3 (p<0.01) expression. Co-administration of HNE and SDX increased GSH content (p<0.001) and GSH:GSSG ratio (p<0.001) as well as decreased SESN2 concentration (p<0.001) and Nrf2 (p<0.01), GCLc (p<0.05) and GSS (p<0.01) gene expression compared with the HNE group. Moreover, we revealed a negative correlation between SESN2 levels and GSH concentrations (p<0.001). Nrf2 silencing significantly decreased the effect of HNE and SDX on the induction of GCLc and GSS genes. SDX also significantly ameliorated the increase of nuclear Nrf2 in response to HNE (p<0.05). The results confirmed that SDX may protect against HNE-induced endothelial damage through its antioxidant effect and modulation of the SESN2/Nrf2/GSH signaling pathway.

舒洛地特通过调节雌三醇2/核因子红细胞2相关因子2通路,保护内皮细胞免受4-羟基壬烯醛诱导的氧化应激和谷胱甘肽依赖性氧化还原失衡的影响。
脂质过氧化产物 4-羟基壬烯醛(HNE)可能通过诱导氧化应激、细胞凋亡和氧化还原平衡的丧失而参与血管内皮细胞的损伤。有证据表明,4-HNE 对血管内皮细胞的刺激可诱导激活核因子红细胞 2 相关因子 2/Kelch 样 ECH 相关蛋白 1(Nrf2/Keap-1)通路。Sestrin2 蛋白(SESN2)是 Nrf2 的关键调节因子之一,参与细胞对氧化应激的反应。然而,SESN2 在 HNE 诱导的内皮损伤中的功能尚不清楚。舒洛地塞(SDX)是一种糖胺聚糖混合物,临床上用于治疗慢性静脉和动脉疾病。虽然 SDX 对内皮的保护作用已得到充分证实,但对其抗氧化作用却知之甚少。本研究旨在阐明在 HNE 诱导的氧化应激下,SDX 在人脐带内皮细胞(HUVECs)中激活的分子机制。在这一实验模型中,我们决定评估 SDX 的抗凋亡和抗氧化潜力及其对 SESN2/Nrf2/GSH 通路的影响。用 25 _M HNE 或 HNE 联合 0.5 LRU/mL SDX 处理 HUVEC 4 小时。细胞活力、细胞凋亡和细胞内活性氧(ROS)的产生通过 MTT 试验和荧光显微镜进行评估。通过 Western 印迹分析测定了 Bax、caspase-3、Keap-1 和 Nrf2 的表达。细胞内还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的浓度通过比色法测定。SESN2、谷氨酸-半胱氨酸连接酶催化亚基(GCLc)和谷胱甘肽合成酶(GSS)采用酶联免疫吸附法进行评估。采用 RT-qPCR 检测 Nrf2、GCLc 和 GSS mRNA 水平。通过短干扰 RNA(siRNA)获得瞬时 Nrf2 沉默。我们已经证明,SDX 可以减轻 HNE 对 HUVEC 的负面影响。SDX 能明显保护 HNE 处理的 HUVEC 免受细胞凋亡(p
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来源期刊
CiteScore
4.00
自引率
22.70%
发文量
0
审稿时长
6-12 weeks
期刊介绍: Journal of Physiology and Pharmacology publishes papers which fall within the range of basic and applied physiology, pathophysiology and pharmacology. The papers should illustrate new physiological or pharmacological mechanisms at the level of the cell membrane, single cells, tissues or organs. Clinical studies, that are of fundamental importance and have a direct bearing on the pathophysiology will also be considered. Letters related to articles published in The Journal with topics of general professional interest are welcome.
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