Luo Wei, Zhou Jiming, Liang Feng, Chou Xianghui, Peng Zhengliang, Tan Weihua, Yu Ziying, Wan Huan
Department of Cardiology, The First Affiliated Hospital of University of South China, No. 69, Chuanshan Road, Shigu District, Hengyang, 421001, Hunan, China.
Department of Anesthesiology, The First Affiliated Hospital of University of South China, Hengyang, 421001, Hunan, China.
Genes Genomics. 2025 Apr 10. doi: 10.1007/s13258-024-01610-x.
Cell senescence-associated endothelia dysfunction is a vital point in the pathological progression of atherosclerosis (AS). G-protein coupled receptor 4 (GPR4) is a proton-sensing receptor involved in developing endothelial dysfunction.
In this study, we investigated the protective role of NE 52-QQ57, a GPR4 inhibitor in endothelial cell senescence induced using an oxidized low-density lipoprotein (ox-LDL). We also unravel the underlying molecular mechanism of NE 52-QQ57 as a therapeutic agent.
Endothelial cell senescence model was established using human aortic endothelial cells (HAECs) stimulated with ox-LDL. The expression levels of GPR4, p53, p16, and sirtuin1 (SIRT1) were evaluated using real-time PCR and western blot assays. ROS production was determined using dihydroethidium (DHE) staining. Further, interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) secretion and expression were determined using ELISA and real-time PCR analysis, respectively. Finally, β-galactosidase (SA-β-Gal) staining associated with cell senescence, telomerase activity, and cell cycle assay were used to determine the state of cell senescence.
Firstly, GPR4 was found to be upregulated in the ox-LDL-stimulated HAECs. We also identified elevated ROS, IL-6, and MCP-1 levels induced by ox-LDL and significantly abrogated by NE 52-QQ57 treatment. Second, a reversal in SA-β-Gal activity, telomerase activity, and G0/G1 proportion, with an upregulation in p53 and p16 expressions was observed on NE 52-QQ57 treatment in the ox-LDL induced model. Lastly, the decreased expression level of SIRT1 was extremely elevated by NE 52-QQ57. Notably, the inhibitory effect of NE 52-QQ57 against ox-LDL-induced cell senescence was abolished by the SIRT1 inhibitor EX-527.
The GPR4 antagonist NE 52-QQ57 might prevent cellular senescence by promoting the expression of SIRT1.
细胞衰老相关的内皮功能障碍是动脉粥样硬化（AS）病理进展中的一个关键点。G蛋白偶联受体4（GPR4）是一种质子感应受体，参与内皮功能障碍的发生。
在本研究中，我们研究了GPR4抑制剂NE 52-QQ57在氧化型低密度脂蛋白（ox-LDL）诱导的内皮细胞衰老中的保护作用。我们还揭示了NE 52-QQ57作为治疗剂的潜在分子机制。
使用ox-LDL刺激人主动脉内皮细胞（HAECs）建立内皮细胞衰老模型。使用实时PCR和蛋白质印迹分析评估GPR4、p53、p16和沉默调节蛋白1（SIRT1）的表达水平。使用二氢乙锭（DHE）染色测定活性氧（ROS）的产生。此外，分别使用酶联免疫吸附测定（ELISA）和实时PCR分析测定白细胞介素-6（IL-6）和单核细胞趋化蛋白1（MCP-1）的分泌和表达。最后，使用与细胞衰老相关的β-半乳糖苷酶（SA-β-Gal）染色、端粒酶活性和细胞周期分析来确定细胞衰老状态。
首先，发现在ox-LDL刺激的HAECs中GPR4上调。我们还确定ox-LDL诱导的ROS、IL-6和MCP-1水平升高，而NE 52-QQ57处理可显著消除这些升高。其次，在ox-LDL诱导的模型中，用NE 52-QQ57处理后观察到SA-β-Gal活性、端粒酶活性和G0/G1比例的逆转，以及p53和p16表达的上调。最后，NE 52-QQ57使SIRT1的降低表达水平显著升高。值得注意的是，SIRT1抑制剂EX-527消除了NE 52-QQ57对ox-LDL诱导的细胞衰老的抑制作用。
GPR4拮抗剂NE 52-QQ57可能通过促进SIRT1的表达来预防细胞衰老。
