Chen Wen-Hua, Guo Bei-Chia, Chen Chia-Hui, Hsu Man-Chen, Wang Chih-Hsien, Lee Tzong-Shyuan
Graduate Institute and Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Cardiovascular Surgery, Department of Surgery, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
J Food Drug Anal. 2023 Aug 31;31(3):519-533. doi: 10.38212/2224-6614.3472.
Statins induce nitric oxide (NO) bioavailability by activating endothelial nitric oxide synthase via kinase- and calcium-dependent pathways in endothelial cells (ECs). However, their effect on the metabolism of L-arginine, the precursor for NO biosynthesis, and regulatory mechanism have not yet been investigated. In this study, we investigated the role of the autophagy-urea cycle-L-arginine pathway in simvastatin-mediated NO bioavailability in ECs. Griess's assay was used to determine the NO bioavailability. Protein expression was assessed using Western blot analysis. Further, immunocytochemistry was performed to observe autophagosome formation, while conventional assay kits were used to quantify the levels of different intermediate substrates of the urea cycle. In ECs, treatment with simvastatin induced the activation of autophagy flux, as evidenced by the increased levels of microtubule-associated protein 1A/1B-light chain 3 II and autophagolysosome formation and decreased levels of p62. Inhibition of autophagy by ATG7 small interfering RNA (siRNA), chloroquine and bafilomycin A1 abolished simvastatin-induced NO bioavailability, EC proliferation, migration, and tube formation. Additionally, simvastatin increased the intermediate substrates levels of the urea cycle, including glutamate, acetyl-CoA, urea, and L-arginine, all of which were abrogated by chloroquine or bafilomycin A1. Genetic knockdown of argininosuccinate lyase using siRNA abrogated simvastatin-induced increase in NO bioavailability and EC-related functions. Moreover, inhibition of AMP-activated protein kinase (AMPK) and transient receptor potential vanilloid 1 (TRPV1) prevented simvastatin-induced activation of the autophagy-urea cycle pathway and NO production. Our findings suggest that simvastatin activates the autophagy-urea cycle pathway via TRPV1-AMPK signaling, which increases L-arginine bioavailability and ultimately promotes NO production in ECs.
他汀类药物通过在内皮细胞（ECs）中经由激酶和钙依赖途径激活内皮型一氧化氮合酶来诱导一氧化氮（NO）的生物利用度。然而，它们对NO生物合成的前体L-精氨酸代谢的影响及其调控机制尚未得到研究。在本研究中，我们调查了自噬-尿素循环-L-精氨酸途径在辛伐他汀介导的ECs中NO生物利用度中的作用。采用格里斯试剂法测定NO生物利用度。使用蛋白质印迹分析评估蛋白质表达。此外，进行免疫细胞化学以观察自噬体形成，并使用传统检测试剂盒定量尿素循环中不同中间底物的水平。在ECs中，辛伐他汀处理诱导了自噬通量的激活，微管相关蛋白1A/1B轻链3 II水平增加、自噬溶酶体形成以及p62水平降低证明了这一点。用ATG7小干扰RNA（siRNA）、氯喹和巴弗洛霉素A1抑制自噬消除了辛伐他汀诱导的NO生物利用度、EC增殖、迁移和管腔形成。此外，辛伐他汀增加了尿素循环的中间底物水平，包括谷氨酸、乙酰辅酶A、尿素和L-精氨酸，所有这些都被氯喹或巴弗洛霉素A1消除。使用siRNA对精氨琥珀酸裂解酶进行基因敲低消除了辛伐他汀诱导的NO生物利用度增加和EC相关功能。此外，抑制AMP激活的蛋白激酶（AMPK）和瞬时受体电位香草酸亚型1（TRPV1）可阻止辛伐他汀诱导的自噬-尿素循环途径激活和NO生成。我们的研究结果表明，辛伐他汀通过TRPV1-AMPK信号传导激活自噬-尿素循环途径，这增加了L-精氨酸的生物利用度，并最终促进了ECs中NO的生成。
