Department of Orthopaedics, The First Hospital of China Medical University, Shenyang 110001, China.
Department of Orthopaedics, The First Hospital of China Medical University, Shenyang 110001, China.
Life Sci. 2020 Sep 15;257:118044. doi: 10.1016/j.lfs.2020.118044. Epub 2020 Jul 2.
High-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown.
An examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting.
In the present study, we found that 100 μM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 μM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells.
These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.
大剂量糖皮质激素（GC）的使用会导致骨质疏松。本研究小组和其他研究小组的许多先前研究表明，褪黑素参与了成骨细胞增殖和分化的调节，尤其是低浓度的褪黑素可以增强成骨细胞的成骨作用。然而，褪黑素在糖皮质激素诱导的成骨细胞分化中的作用尚不清楚。
通过检测碱性磷酸酶染色和碱性磷酸酶酶活性测定来评估成骨细胞分化和矿化，以检测地塞米松（Dex）和褪黑素对 MC3T3-E1 细胞分化的影响，同时还检测了成骨细胞分化标志物（ALP、OCN、COLL-1）的表达。我们使用免疫荧光染色来检测褪黑素处理的 MC3T3-E1 细胞中 Runx2 的表达。通过 qRT-PCR 确定 mRNA 的表达，通过 Western blot 测定蛋白水平。
在本研究中，我们发现 100μM Dex 显著降低了 MC3T3-E1 细胞中的成骨细胞分化和矿化，而 1μM 褪黑素则减弱了这些抑制作用。我们发现，只有抑制 PI3K/AKT（MK2206）和 BMP/Smad（LDN193189）信号通路才能消除褪黑素诱导的分化和矿化。同时，MK2206 降低了 P-AKT 和 P-Smad1/5/9 的表达，LDN193189 降低了 P-Smad1/5/9 的表达，但对褪黑素处理和 Dex 诱导的 MC3T3-E1 细胞中的 P-AKT 表达没有明显影响。
这些发现表明，褪黑素通过 PI3K/AKT 和 BMP/Smad 信号通路挽救了 Dex 诱导的 MC3T3-E1 细胞成骨细胞分化抑制，PI3K/AKT 信号通路可能是 BMP/Smad 信号通路的上游信号。
