School of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang, China.
School of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Arch Oral Biol. 2019 Dec;108:104525. doi: 10.1016/j.archoralbio.2019.104525. Epub 2019 Aug 14.
This study aims to investigate the role of Cannabinoid receptor 2 (CB2) on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) under hypoxia.
BMSCs were isolated from Sprague-Dawley rats and cultured in the presence of cobalt chloride (CoCl) to induce intracellular hypoxia. Cell proliferation was measured with MTT assay. Quantitative real-time PCR and western blot were applied to evaluate the mRNA and protein expressions of CB2 and osteogenic indicators including osteocalcin, RUNX2, collagen-1 and osterix (SP7). The osteogenic differentiation of BMSCs was further examined by ALP assay and alizarin red S (ARS) staining. Moreover, the activation of MAPKs signaling pathways was analyzed by western blot.
CoCl dose-dependently increased hypoxia inducible factor while higher concentrations (200 and 400 μM) of CoCl markedly inhibited cell proliferation. CoCl induced hypoxia significantly increased the protein and mRNA expressions of osteocalcin, RUNX2, collagen-1 and osterix, along with enhanced ALP and ARS staining. Interestingly, such effects can be inhibited by the addition of CB2 inhibitor AM630. Moreover, AM630 partially inhibited hypoxia-induced p38 and ERK pathways, which may lead to a decrease in the osteogenic transcripts of RUNX2, collagen-1 and osterix.
CoCl induced hypoxia could promote osteogenesis of rat BMSCs possibly through CB2.
本研究旨在探讨大麻素受体 2（CB2）在缺氧条件下对骨髓间充质干细胞（BMSCs）成骨的作用。
从 Sprague-Dawley 大鼠中分离 BMSCs，并在氯化钴（CoCl）存在下培养以诱导细胞内缺氧。用 MTT 法测定细胞增殖。定量实时 PCR 和 Western blot 用于评估 CB2 和成骨指标（包括骨钙素、RUNX2、胶原-1 和 osterix（SP7））的 mRNA 和蛋白表达。通过碱性磷酸酶（ALP）测定和茜素红 S（ARS）染色进一步检测 BMSCs 的成骨分化。此外，通过 Western blot 分析 MAPKs 信号通路的激活。
CoCl 呈剂量依赖性增加缺氧诱导因子，而较高浓度（200 和 400μM）的 CoCl 明显抑制细胞增殖。CoCl 诱导的缺氧显著增加了骨钙素、RUNX2、胶原-1 和 osterix 的蛋白和 mRNA 表达，并增强了 ALP 和 ARS 染色。有趣的是，这种作用可以通过添加 CB2 抑制剂 AM630 来抑制。此外，AM630 部分抑制了缺氧诱导的 p38 和 ERK 通路，这可能导致 RUNX2、胶原-1 和 osterix 的成骨转录物减少。
CoCl 诱导的缺氧可能通过 CB2 促进大鼠 BMSCs 的成骨。
