Department of Orthopedics, The First Hospital of China Medical University, Shenyang, Liaoning 110000, P.R. China.
Science Experiment Center of China Medical University, Shenyang, Liaoning 110122, P.R. China.
Mol Med Rep. 2019 May;19(5):3676-3684. doi: 10.3892/mmr.2019.10040. Epub 2019 Mar 15.
Vitamin K2 likely exerts its protective effects during osteoporosis by promoting osteoblast differentiation and mineralization. However, the precise mechanism remains to be fully elucidated. Autophagy maintains cell homeostasis by breaking down and eliminating damaged proteins and organelles. Increasing evidence in recent years has implicated autophagy in the development of osteoporosis. The aim of the present study was to verify whether vitamin K2 (VK2) can induce autophagy during the differentiation and mineralization of osteoblasts. In the present study, MC3T3‑E1 osteoblasts were treated with various doses of VK2 (10‑8‑10‑3 M) for 1‑5 days. The results revealed no cytotoxicity at concentrations below 10‑5 M, but cell viability was reduced in a dose‑dependent manner at concentrations above 10‑5 M. Furthermore, MC3T3‑E1 osteoblasts were seeded in 6‑well plates in complete medium supplemented with dexamethasone, β‑glycerophosphate and vitamin C (VC) for osteogenic differentiation. MC3T3‑E1 osteoblasts treated with different concentrations (10‑5, 10‑6 and 10‑7 M) of VK2 for 24 h on days 1, 3, 5 and 7 of the differentiation protocol. It was confirmed that VK2 promoted osteoblast differentiation and mineralization by using alkaline phosphatase (ALP) and alizarin red staining. Using western blotting, immunofluorescence, monodansylcadaverine staining and reverse transcription‑quantitative polymerase chain reaction, it was observed that VK2 induced autophagy in osteoblasts. The results revealed that VK2 (1 µM) significantly increased ALP activity and the conversion of microtubule associated protein 1 light chain 3‑α (LC3)II to LC3I in MC3T3‑E1 osteoblasts (P<0.05) at every time point. The number of fluorescent bodies and the intensity increased with VK2, and decreased following treatment with 3‑MA+VK2. There was an increase in the mRNA expression levels of ALP, osteocalcin (OCN) and Runt‑related transcription factor 2 in VK2‑treated cells (P<0.01). The present study further confirmed the association between autophagy and osteoblast differentiation and mineralization through treatment with an autophagy inhibitor [3‑methyladenine (3‑MA)]. Osteoblasts treated with 3‑MA exhibited significant inhibition of ALP activity and osteogenic differentiation (both P<0.05). In addition, ALP activity and osteogenesis in the VK2+3‑MA group was lower compared with VK2‑treated cells (P<0.05 for both). The present study confirmed that VK2 stimulated autophagy in MC3T3 cells to promote differentiation and mineralization, which may be a potential therapeutic target for osteoporosis.
维生素 K2 通过促进成骨细胞分化和矿化，可能发挥其在骨质疏松症中的保护作用。然而，确切的机制仍有待充分阐明。自噬通过分解和消除受损的蛋白质和细胞器来维持细胞内环境稳定。近年来越来越多的证据表明自噬参与了骨质疏松症的发展。本研究旨在验证维生素 K2（VK2）是否可以在成骨细胞的分化和矿化过程中诱导自噬。在本研究中，用不同浓度的 VK2（10-8-10-3 M）处理 MC3T3-E1 成骨细胞 1-5 天。结果显示，浓度低于 10-5 M 时无细胞毒性，但浓度高于 10-5 M 时细胞活力呈剂量依赖性降低。此外，将 MC3T3-E1 成骨细胞接种于 6 孔板中，在完全培养基中加入地塞米松、β-甘油磷酸钠和维生素 C（VC）进行成骨分化。在分化方案的第 1、3、5 和 7 天，用不同浓度（10-5、10-6 和 10-7 M）的 VK2 处理 24 h。结果证实 VK2 通过碱性磷酸酶（ALP）和茜素红染色促进成骨细胞分化和矿化。通过 Western blot、免疫荧光、单丹磺酰尸胺染色和逆转录-定量聚合酶链反应观察到 VK2 诱导成骨细胞自噬。结果表明，VK2（1 μM）在每个时间点均显著增加 MC3T3-E1 成骨细胞中微管相关蛋白 1 轻链 3-α（LC3）Ⅱ向 LC3I 的转化和 ALP 活性（P<0.05）。荧光体的数量和强度随 VK2 增加而增加，随 3-MA+VK2 处理而减少。VK2 处理的细胞中 ALP、骨钙素（OCN）和 runt 相关转录因子 2 的 mRNA 表达水平均增加（P<0.01）。本研究进一步通过用自噬抑制剂[3-甲基腺嘌呤（3-MA）]处理证实了自噬与成骨细胞分化和矿化之间的关联。用 3-MA 处理的成骨细胞中 ALP 活性和成骨分化明显受到抑制（均 P<0.05）。此外，VK2+3-MA 组的 ALP 活性和成骨作用均低于 VK2 处理组（均 P<0.05）。本研究证实 VK2 刺激 MC3T3 细胞中的自噬以促进分化和矿化，这可能是骨质疏松症的潜在治疗靶点。
