Inokosterone effects on proliferation and differentiation of osteoblasts from neonatal sprague-dawley rats / 中国组织工程研究

ABSTRACT

BACKGROUND: It is an effective strategy for the treatment of osteoporosis to promote the proliferation and differentiation of osteoblasts, but the influence of inokosterone on osteoblast proliferation and differentiation is rarely reported. OBJECTIVE: To investigate the effects of inokosterone on the proliferation and differentiation of primary osteoblasts and the underlying molecular mechanism. METHODS: Primary osteoblasts from neonatal Sprague-Dawley rats were isolated via the second enzyme digestion, and the cells were then cultured in osteogenic induction medium and identified. Cell counting kit 8 assay was performed to evaluate the effect of different concentrations of inokosterone (1, 5, 10 mg/L) on cell viability of primary osteoblasts. Early differentiation ability of osteoblasts was evaluated by alkaline phosphatase staining and alkaline phosphatase activity assay. To evaluate the mineralization ability of osteoblasts, alizarin red staining was performed to observe the number of calcium nodules. The expression level of osteogenic genes was detected by RT-qPCR at different time points. Furthermore, MDC staining was also used to observe the number of autophagosomes. RESULTS AND CONCLUSION: Compared with the control group, inokosterone could inhibit the cell viability of primary osteoblast to some degree (P < 0.05) while significantly promoting alkaline phosphatase activity and mineralized nodules formation (P < 0.05). In addition, inokosterone upregulated the expression of osteogenic genes such as Collagen I, osteoprotegerin, osteopontin, osteocalcin and increased the number of autophagosomes. To conclude, inokosterone can promote osteogenic differentiation by upregulating osteogenic genes expression and activating autophagy of primary osteoblasts.