The State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, University Town, Guian New District, Guizhou, China; Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, China; The Key Laboratory of Optimal Utilization of Natural Medicine Resources, School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, China.
The State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, University Town, Guian New District, Guizhou, China; Department of Pharmacology of Materia Medica (The High Educational Key Laboratory of Guizhou Province for Natural Medicinal Pharmacology and Druggability, The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province), Guizhou Medical University, University Town, Guian New District, Guizhou, China; The Key Laboratory of Optimal Utilization of Natural Medicine Resources, School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, China.
Pharmacol Res. 2021 Aug;170:105629. doi: 10.1016/j.phrs.2021.105629. Epub 2021 Jun 3.
Endoplasmic reticulum (ER) stress-mediated phenotypic switching of vascular smooth muscle cells (VSMCs) is key to vascular calcification (VC) in patients with chronic kidney disease (CKD). Studies have shown that activation/upregulation of SIRT1 has a protective effect on CKD-VC. Meanwhile, although terpinen-4-ol has been shown to exert a protective effect against cardiovascular disease, its role and underlying mechanism in VC remain unclear. Herein, we explored whether terpinen-4-ol alleviates ER stress-mediated VC through sirtuin 1 (SIRT1) and elucidated its mechanism to provide evidence for its application in the clinical prevention and treatment of VC. To this end, a CKD-related VC animal model and β-glycerophosphate (β-GP)-induced VSMC calcification model were established to investigate the role of terpinen-4-ol in ER stress-induced VC, in vitro and in vivo. Additionally, to evaluate the involvement of SIRT1, mouse and VSMC Sirt1-knockdown models were established. Results show that terpinen-4-ol inhibits calcium deposition, phenotypic switching, and ER stress in VSMCs in vitro and in vivo. Furthermore, pre-incubation of VSMCs with terpinen-4-ol or a SIRT1 agonist, decreased β-GP-induced calcium salt deposition, increased SIRT1 protein level, and inhibited PERK-eIF2α-ATF4 pathway activation, thus, alleviating VC. Similar results were observed in VSMCs induced to overexpress SIRT1 via lentivirus transcription. Meanwhile, the opposite results were obtained in SIRT1-knockdown models. Further, results suggest that SIRT1 physically interacts with, and deacetylates PERK. Specifically, mass spectrometry analysis identified lysine K889 as the acetylation site of SIRT1, which regulates PERK. Finally, inhibition of SIRT1 reduced the effect of terpinen-4-ol on the deacetylation of PERK in vitro and in vivo and weakened the inhibitory effect of terpinen-4-ol against ER stress-mediated VC. Cumulatively, terpinen-4-ol was found to inhibit post-translational modification of PERK at the K889 acetylation site by upregulating SIRT1 expression, thereby ameliorating VC by regulating ER stress. This study provides insights into the underlying molecular mechanism of terpinen-4-ol, supporting its development as a promising therapeutic agent for CKD-VC.
内质网（ER）应激介导的血管平滑肌细胞（VSMCs）表型转换是慢性肾脏病（CKD）患者血管钙化（VC）的关键。研究表明，SIRT1 的激活/上调对 CKD-VC 具有保护作用。同时，尽管萜品-4-醇已被证明对心血管疾病具有保护作用，但它在 VC 中的作用和潜在机制尚不清楚。在此，我们探讨了萜品-4-醇是否通过 Sirtuin 1（SIRT1）减轻 ER 应激介导的 VC，并阐明了其机制，为其在 VC 的临床预防和治疗中的应用提供了依据。为此，建立了 CKD 相关 VC 动物模型和β-甘油磷酸（β-GP）诱导的 VSMC 钙化模型，以研究萜品-4-醇在 ER 应激诱导的 VC 中的作用，在体外和体内。此外，为了评估 SIRT1 的参与，建立了小鼠和 VSMC Sirt1 敲低模型。结果表明，萜品-4-醇抑制体外和体内 VSMCs 中的钙沉积、表型转换和 ER 应激。此外，用萜品-4-醇或 SIRT1 激动剂预孵育 VSMCs 可减少 β-GP 诱导的钙盐沉积，增加 SIRT1 蛋白水平，并抑制 PERK-eIF2α-ATF4 通路激活，从而减轻 VC。通过慢病毒转录使 SIRT1 过表达诱导的 VSMCs 也观察到类似结果。同时，在 SIRT1 敲低模型中得到了相反的结果。此外，结果表明 SIRT1 与 PERK 相互作用并使其去乙酰化。具体而言，质谱分析鉴定出 SIRT1 的赖氨酸 K889 是 PERK 的乙酰化位点，调节 PERK。最后，SIRT1 的抑制减少了萜品-4-醇在体外和体内对 PERK 去乙酰化的影响，并削弱了萜品-4-醇对 ER 应激介导的 VC 的抑制作用。总之，发现萜品-4-醇通过上调 SIRT1 表达抑制 PERK 在 K889 乙酰化位点的翻译后修饰，从而通过调节 ER 应激改善 VC。这项研究为萜品-4-醇的潜在分子机制提供了新的见解，支持其作为 CKD-VC 有前途的治疗药物的发展。
