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

Abstract

<i>Achyranthes bidentata</i> is a herbal plant commonly used in the treatment of osteoporosis and bone nonunion with traditional Chinese medicine. <i>Achyranthes bidentata</i> alcohol is a major component extracted from <i>Achyranthes bidentata</i>, 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 <i>Achyranthes bidentata</i> alcohol on the proliferation of osteoblasts was detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of <i>Achyranthes bidentata</i> alcohol on the alkaline phosphatase (ALP) activity in osteoblasts was analyzed via ALP assay. The effect of <i>Achyranthes bidentata</i> 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 <i>Achyranthes bidentata</i> alcohol was analyzed using western blotting. <i>Achyranthes bidentata</i> 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 <i>Achyranthes bidentata</i> 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, <i>Achyranthes bidentata</i> 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.