Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Clinical Center for Coronary Heart Disease, Capital Medical University, Beijing, China; Department of Cardiology, Nanlou Division, Chinese PLA General Hospital, National Clinical Research Center for Geriatric Diseases, Beijing, China.
Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Clinical Center for Coronary Heart Disease, Capital Medical University, Beijing, China.
Exp Cell Res. 2020 Apr 1;389(1):111883. doi: 10.1016/j.yexcr.2020.111883. Epub 2020 Feb 1.
Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. Autophagy may affect vascular calcification by inhibiting apoptosis and the transdifferentiation process. This study sought to explore whether melatonin attenuates vascular calcification by regulating autophagy via the AMP-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2, an osteogenic transcription factor), light chain 3 (LC3) II/I, and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were downregulated, whereas LC3 II/I was increased in response to melatonin, and was accompanied by decreased apoptosis. An immunofluorescence assay revealed that melatonin treatment markedly decreased Runx2 expression and upregulated LC3 expression. Treatment with the autophagy inhibitor 3-methyladenine reversed this phenomenon. Melatonin significantly increased expression of p-AMPK and p-ULK1, and decreased mTOR expression. Treatment with compound C (an inhibitor of AMPK) or MHY1485 (an agonist of mTOR) ablated the observed benefits of melatonin treatment. Melatonin protects VSMCs against calcification by activating autophagy via the AMPK/mTOR/ULK1 pathway.
褪黑素已被证明可预防环孢素肾毒性中的钙化。自噬可能通过抑制细胞凋亡和转分化过程来影响血管钙化。本研究旨在探讨褪黑素是否通过调节自噬来减轻血管钙化，其作用机制是通过 AMP 激活的蛋白激酶/哺乳动物雷帕霉素靶蛋白/UNC-51 样激酶 1（AMPK/mTOR/ULK1）信号通路。在血管平滑肌细胞（VSMCs）中研究了褪黑素对血管钙化的影响。通过茜素红染色来观察钙沉积，而钙含量和碱性磷酸酶（ALP）活性则用于评估成骨分化。通过 Western blot 来测量 runt 相关转录因子 2（Runx2，成骨转录因子）、LC3 Ⅱ/Ⅰ和裂解的 caspase 3 的表达。褪黑素明显减少钙沉积和 ALP 活性。Runx2 和裂解的 caspase 3 下调，而 LC3 Ⅱ/Ⅰ则随着褪黑素的增加而上调，并且伴随着凋亡减少。免疫荧光测定表明褪黑素处理明显降低了 Runx2 表达并上调了 LC3 表达。自噬抑制剂 3-甲基腺嘌呤处理逆转了这种现象。褪黑素显著增加了 p-AMPK 和 p-ULK1 的表达，并降低了 mTOR 的表达。用 AMPK 抑制剂（compound C）或 mTOR 激动剂（MHY1485）处理则消除了褪黑素处理的观察到的效果。褪黑素通过 AMPK/mTOR/ULK1 通路激活自噬来保护 VSMCs 免受钙化。
