Chen Sheng, Dai Min
Department of Orthopedic, Shaoxing Second Hospital, No.123 Yan'an Road, Shaoxing, 312000, Zhejiang, China.
Department of Orthopedic, The First Affiliated Hospital of Nanchang University, No.17 Yongwai Zheng Street, Donghu District, Nanchang, 330006, Jiangxi, China.
Hum Cell. 2023 Nov;36(6):1965-1977. doi: 10.1007/s13577-023-00929-z. Epub 2023 Jul 24.
Melatonin has been shown to exert an inhibitory effect on osteoporosis. This study investigates the function of the miR-224-5p/SIRT3/AMPK/mTOR axis in melatonin-mediated effects against osteoporosis. Human bone marrow mesenchymal stem cells (hBMSCs) were treated with glucocorticoid dexamethasone to induce an in vitro osteoporosis model. After melatonin treatment, miR-224-5p and SIRT3 levels were measured by RT‒PCR. Transmission electron microscopy and immunofluorescence were conducted for evaluating autophagy. Western blotting was carried out to determine the expression of osteogenesis-related proteins (Runx2, OSX, OPN, and OCN), SIRT3-AMPK-mTOR axis, and autophagy-related markers (LC3 and p62). Alizarin red staining was used to measure matrix mineralization. The data showed that melatonin inhibited dexamethasone-induced osteoporosis in vitro, and enhanced autophagic levels (as indicated by increased LC3 puncta, LC3II/I ratio, and autophagic vacuoles). In terms of the mechanisms, melatonin decreased miR-224-5p expression and increased SIRT3. SRIT3 was shown to be a direct target of miR-224-5p. miR-224-5p upregulation or SIRT3 downregulation reversed the effects of melatonin on osteoporosis and suppressed autophagy. Additionally, miR-224-5p inhibited SIRT3 expression and AMPK pathway activation. In summary, we discovered that melatonin suppressed glucocorticoid-induced osteoporosis and autophagy inhibition via the miR-224-5p/SIRT3/AMPK/mTOR axis.
褪黑素已被证明对骨质疏松症具有抑制作用。本研究探讨了miR-224-5p/SIRT3/AMPK/mTOR轴在褪黑素介导的抗骨质疏松作用中的功能。用人骨髓间充质干细胞（hBMSCs）与糖皮质激素地塞米松处理以诱导体外骨质疏松模型。褪黑素处理后，通过RT-PCR检测miR-224-5p和SIRT3水平。进行透射电子显微镜和免疫荧光以评估自噬。进行蛋白质印迹法以确定成骨相关蛋白（Runx2、OSX、OPN和OCN）、SIRT3-AMPK-mTOR轴和自噬相关标志物（LC3和p62）的表达。茜素红染色用于测量基质矿化。数据表明，褪黑素在体外抑制地塞米松诱导的骨质疏松症，并提高自噬水平（表现为LC3斑点增加、LC3II/I比率增加和自噬泡增加）。在机制方面，褪黑素降低miR-224-5p表达并增加SIRT3。SRIT3被证明是miR-224-5p的直接靶点。miR-224-5p上调或SIRT3下调逆转了褪黑素对骨质疏松症的影响并抑制自噬。此外，miR-224-5p抑制SIRT3表达和AMPK途径激活。总之，我们发现褪黑素通过miR-224-5p/SIRT3/AMPK/mTOR轴抑制糖皮质激素诱导的骨质疏松症和自噬抑制。
