Integrated Traditional Chinese and Western Medicine Hospital of Wenzhou Affilated Hospital of Zhejiang Chinese Medicine University, Zhe Jiang, China.
Department of Sport Rehabilitation, Shanghai University of Sport, Shanghai, China.
PLoS One. 2018 Jul 10;13(7):e0200367. doi: 10.1371/journal.pone.0200367. eCollection 2018.
Icariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to reveal the mechanism of senile osteoporosis and the therapeutic mechanism of drug activity. However, little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin.
We isolated and cultured osteoblasts from SAMP6 or SAMR1 mice and compared their proliferation, migration, and differentiation by performing the CCK-8 assay, cell counting assay, EdU staining, cell cycle analysis, ALP staining and activity measurement, Alizarin red staining, and RT-qPCR analysis to measure the levels of osteoblast markers, including RUNX2, Colla1 and Oc. To assess the effects of icariin on BMP-2-induced osteoblast differentiation, after BMP-2 treatment, osteoblast markers were analyzed by RT-qPCR and semi-quantitative Western blotting. The effects of icariin on connective tissue growth factor (CTGF) were measured by RT-qPCR. shRNA targeting CTGF mRNA was employed to knockdown its expression level in osteoblasts.
The SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease. Moreover, SAMP6 osteoblasts presented upregulated CTGF compared with SAMR1 osteoblasts. Icariin enhanced BMP-2-induced osteoblast differentiation by downregulating CTGF expression, which tightly regulates osteoblast differentiation. By downregulating CTGF, icariin treatment upregulated phosphate-Smad1/5/8, indicating its activating effects on the BMP signaling pathway.
These results suggest that decreased osteoblast development and function potentially contributes to age-associated disease. Icariin exerts enhancing effects on BMP-2-mediated osteoblast development via downregulating CTGF.
淫羊藿苷是一种主要的黄酮类糖苷，具有抑制成骨细胞生成和促进成骨作用，广泛用于临床治疗骨损伤和重建。快速老化小鼠 SAMP6 被认为是揭示衰老性骨质疏松症机制和药物活性治疗机制的有用鼠模型。然而，关于 SAMP6 成骨细胞的特征及其与淫羊藿苷相关的调节作用知之甚少。
我们从 SAMP6 或 SAMR1 小鼠中分离培养成骨细胞，并通过 CCK-8 检测、细胞计数、EdU 染色、细胞周期分析、碱性磷酸酶（ALP）染色和活性测定、茜素红染色和 RT-qPCR 分析比较它们的增殖、迁移和分化，以测量成骨细胞标志物的水平，包括 runt 相关转录因子 2（RUNX2）、I 型胶原（Colla1）和骨钙素（Oc）。为了评估淫羊藿苷对 BMP-2 诱导的成骨细胞分化的影响，在 BMP-2 处理后，通过 RT-qPCR 和半定量 Western 印迹分析成骨细胞标志物。通过 RT-qPCR 检测淫羊藿苷对结缔组织生长因子（CTGF）的影响。采用 shRNA 靶向 CTGF mRNA 敲低成骨细胞中 CTGF 的表达水平。
与 SAMR1 成骨细胞相比，SAMP6 成骨细胞的发育和分化活性降低，表明其是与年龄相关疾病相关的潜在机制。此外，与 SAMR1 成骨细胞相比，SAMP6 成骨细胞中 CTGF 的表达上调。淫羊藿苷通过下调 CTGF 表达增强 BMP-2 诱导的成骨细胞分化，从而紧密调节成骨细胞分化。通过下调 CTGF，淫羊藿苷处理后磷酸化 Smad1/5/8 上调，表明其对 BMP 信号通路具有激活作用。
这些结果表明，成骨细胞发育和功能的降低可能导致与年龄相关的疾病。淫羊藿苷通过下调 CTGF 对 BMP-2 介导的成骨细胞发育发挥增强作用。
