Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: The GPR4 antagonist NE 52-QQ57 might prevent cellular senescence by promoting the expression of SIRT1.

Keywords: Atherosclerosis; Cell senescence; Endothelial dysfunction; GPR4; SIRT1.

© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.

背景： 细胞衰老相关的内皮功能障碍是动脉粥样硬化（AS）病理进展中的关键点。G蛋白偶联受体4 (GPR4) 是一种参与发展内皮功能障碍的质子感应受体。

目的： 在这项研究中，我们调查了NE 52-QQ57（一种GPR4抑制剂）在氧化低密度脂蛋白 (ox-LDL) 引起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。

方法： 使用ox-LDL刺激的人主动脉内皮细胞（HAECs）建立了内皮细胞衰老模型。通过实时定量PCR和Western blot测定GPR4、p53、p16 和 sirtuin1 (SIRT1) 的表达水平。ROS的产生用二氢乙锭 (DHE) 染色法测定。进一步，使用ELISA和实时定量 PCR 分析分别确定白细胞介素-6 (IL-6) 和单核趋化蛋白 1 (MCP-1) 的分泌和表达。最后，通过β-半乳糖苷酶 (SA-β-Gal) 染色、端粒酶活性测定及细胞周期分析来判断细胞衰老状态。

结果： 首先，在ox-LDL刺激的HAECs中发现GPR4上调。我们还确定了ox-LDL诱导ROS水平升高，以及IL-6和MCP-1水平上升，并且这些升高的指标在使用NE 52-QQ57处理后显著下降。其次，在ox-LDL引起的模型中用NE 52-QQ57治疗观察到SA-β-Gal活性、端粒酶活性及G0/G1比例逆转，同时p53和p16表达上调。最后，SIRT1的降低表达水平在使用NE 52-QQ57后显著升高。值得注意的是，用SIRT1抑制剂EX-527处理后，NE 52-QQ57对抗ox-LDL诱导的细胞衰老的作用被消除。

结论： GPR4拮抗剂NE 52-QQ57可能通过促进SIRT1的表达来预防细胞衰老。

关键词： 动脉粥样硬化；细胞衰老；内皮功能障碍；GPR4；SIRT1.