The combined effect of oleonuezhenide and wedelolactone on proliferation and osteoblastogenesis of bone marrow mesenchymal stem cells

Background: Regulation of the survival and differentiation of bone marrow mesenchymal stem cells is an essential consideration in the development of targeted drugs for treatment of osteoporosis.

Purpose: The present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide, two compounds from Chinese formula Er-Zhi-Wan, on osteoblastogenesis and the underlying molecular mechanisms.

Methods: MTT assay was taken to evaluate cell proliferation. The Alkaline Phosphatase (ALP) activity assay was used to determine the activity of ALP. Alizarin red S (ARS) staining was taken to indicate the intensity of the calcium deposits. Quantitative Real-Time PCR and Western blot were performed to the levels of Runx2, Osteocalcin, and Osterix expression in mouse bone marrow mesenchymal stem cells (BMSCs). Ovariectomized mouse model and bone histomorphometric analysis were also used to research the effects of wedelolactone and oleonuezhenide on bone loss caused by ovariectomy.

Results: Wedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization. Osteoblastogenesis-related marker genes including osteocalcin, Runx2, and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide. At the molecular level, oleonuezhenide did not affect GSK-3β phosphorylation induced by wedelolactone, but elevated Casein Kinase 2-alpha (CK2α) expression, resulting in β-catenin and Runx2 nuclear translocation. In addition, 30 µM wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 µM oleonuezhenide. These cells were protected by oleonuezhenide and maintained osteoblastic activity. Oleonuezhenide increased Wnt5a and CK2α expression. Wedelolactone-reduced extracellular signal-regulated kinase (ERK) phosphorylation was reversed by oleonuezhenide. In ovariectomized mice, administration of wedelolactone and oleonuezhenide prevented ovariectomy-induced bone loss by enhancing osteoblastic activity.

Conclusion: These results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis. These two compounds could be developed as a combined therapeutic agent for osteoporosis.