Effects of Achyranthes bidentata alcohol on proliferation capacity of osteoblasts and miRNA in Runx2

Achyranthes bidentata is a herbal plant commonly used in the treatment of osteoporosis and bone nonunion with traditional Chinese medicine. Achyranthes bidentata alcohol is a major component extracted from Achyranthes bidentata, which has been proved to be able to exert a variety of pharmacological effects, such as anti-inflammation, antipyresis, anti-rheumatism, diuresis and anti-osteoporosis. Thirty male Sprague-Dawley rats aged 4 weeks were used in the experiment. All primary rat osteoblasts were cultured and amplified for further experiments. The osteoblasts were divided into six groups (5 rats in each group): the culture medium control group, the 25 µg/ml achyranthol group, the 50 µg/ml achyranthol group, the 100 µg/ml achyranthol group, 200 µg/ml achyranthol group, and the 25 µM PD98059+200 µg/ml achyranthol group. In this study, the effect of Achyranthes bidentata alcohol on the proliferation of osteoblasts was detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of Achyranthes bidentata alcohol on the alkaline phosphatase (ALP) activity in osteoblasts was analyzed via ALP assay. The effect of Achyranthes bidentata alcohol on the expression of osteoblast marker gene, Runt-related transcription factor 2 (Runx2), was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Moreover, the phosphorylation or activation of extracellular signal-regulated kinase (ERK) in osteoblasts induced by Achyranthes bidentata alcohol was analyzed using western blotting. Achyranthes bidentata alcohol increased cell proliferation in a dose-dependent manner, increased the micro ribonucleic acid (miRNA) level in Runx2, enhanced the ALP activity in osteoblasts, and stimulated the activation of ERK (P<0.05). The expression of Runx2 with the inhibitor PD98059 was decreased significantly compared with that in the Achyranthes bidentata alcohol group (P<0.01). Immunohistochemical results manifested that the percentage of Runx2 positive cells in treated tissues was obviously higher than that in untreated tissues (P<0.01). Therefore, Achyranthes bidentata alcohol promotes the proliferation capacity of osteoblasts in a dose-dependent manner, enhances the expression of miRNA in Runx2, and stimulates the osteogenic differentiation of osteoblasts through activating the ERK signal transduction pathway.