姜黄色素衍生的三烯生育酚通过抑制 RhoA 激活和 HMG-CoA 还原酶基因表达增强 BMP-2 蛋白表达促进 MC3T3-E1 细胞矿化。
Annatto-Derived Tocotrienol Promotes Mineralization of MC3T3-E1 Cells by Enhancing BMP-2 Protein Expression via Inhibiting RhoA Activation and HMG-CoA Reductase Gene Expression.
机构信息
Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, UKM Medical Centre (UKMMC), Kuala Lumpur 56000, Malaysia.
Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, UKM Medical Centre (UKMMC), Kuala Lumpur 56000, Malaysia.
出版信息
Drug Des Devel Ther. 2020 Mar 3;14:969-976. doi: 10.2147/DDDT.S224941. eCollection 2020.
PURPOSE
Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. However, the mechanism of action of AnTT in achieving these effects is unclear. This study aims to investigate the mechanism of action of AnTT on MC3T3-E1 pre-osteoblasts via the mevalonate pathway.
METHODS
Murine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 10 cells/mL and treated with 4 concentrations of AnTT (0.001-1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.
RESULTS
The results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.
CONCLUSION
AnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.
目的
姜黄色素衍生的生育三烯酚（AnTT）已被证明可改善骨质疏松症动物模型中的骨形成，并促进前成骨细胞的分化。然而，AnTT 实现这些效果的作用机制尚不清楚。本研究旨在通过甲羟戊酸途径研究 AnTT 对 MC3T3-E1 前成骨细胞的作用机制。
方法
将鼠前成骨细胞 MC3T3-E1 以 1×10^5 个/mL 的密度培养，并以 4 种不同浓度的 AnTT（0.001-1μg/mL）处理。用 AnTT 处理 21 天后，通过 qPCR 检测 HMG-CoA 还原酶（HMGR）基因的表达。用免疫测定法检测 RhoA 激活和骨形态发生蛋白-2（BMP-2）在 AnTT 处理 9 和 15 天后的表达。洛伐他汀作为阳性对照。用 Von Kossa 染色法检测 AnTT 处理 21 天后矿化结节的形成。
结果
结果显示，与对照组相比，洛伐他汀组在第 9 天和第 21 天 HMGR 表达上调。洛伐他汀还抑制 RhoA 激活（第 9 天和第 15 天）并增加 BMP-2 蛋白（第 15 天）。另一方面，与对照组相比，AnTT 在 0.001μg/mL（第 3 天）和 0.1μg/mL（第 21 天）时显著下调 HMGR 基因表达。与第 15 天相比，第 21 天所有组的 HMGR 基因表达均显著降低。与对照组相比，AnTT 在 0.1μg/mL 时在第 9 天显著抑制 RhoA 激活。与对照组相比，AnTT 在 1μg/mL 时在第 15 天显著增加 BMP-2 蛋白（P<0.05）。与对照组相比，第 21 天 AnTT 处理组矿化钙结节更丰富。
结论
AnTT 通过下调 HMGR 基因表达和抑制 RhoA 激活抑制甲羟戊酸途径，导致 MC3T3-E1 细胞中 BMP-2 蛋白增加。这解释了 AnTT 对成骨细胞矿化的刺激作用。
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