CoCl2 induced hypoxia enhances osteogenesis of rat bone marrow mesenchymal stem cells through cannabinoid receptor 2.

OBJECTIVES:

This study aims to investigate the role of Cannabinoid receptor 2 (CB2) on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) under hypoxia.

MATERIALS AND METHODS:

BMSCs were isolated from Sprague-Dawley rats and cultured in the presence of cobalt chloride (CoCl₂) 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.

RESULTS:

CoCl₂ dose-dependently increased hypoxia inducible factor while higher concentrations (200 and 400 μM) of CoCl₂ markedly inhibited cell proliferation. CoCl₂ 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.

CONCLUSIONS:

CoCl₂ induced hypoxia could promote osteogenesis of rat BMSCs possibly through CB2.

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