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BACKGROUND: Studies have found that icariin can promote the proliferation and differentiation of mouse preosteoblasts. Nuclear factor-κB signaling pathways and autophagy play an important role in the regulation of osteoblast differentiation. Whether they are involved in icariin promoting the proliferation and differentiation of mouse preosteoblasts remains unclear. OBJECTIVE: To investigate the effect of icariin on the proliferation and differentiation of mouse preosteoblasts and the role of nuclear factor-κB signaling and autophagy. METHODS: (1) Screening out the best intervention concentration of icariin: Mouse preosteoblasts MC3T3-E1 cells were divided into control group and various icariin groups (0.01, 0.1, 1, 10, 100 μmol/L). MTS was used to detect the proliferation activity of mouse preosteoblasts from different groups after icariin intervention. Osteogenic differentiation was assessed by Alizarin red staining. The osteoblastic differentiation ability and autophagy activity in mouse preosteoblasts from different groups were evaluated by western blot assay after icariin intervention. (2) 3-Methyladenine was added to inhibit autophagy. Four groups were set up: control group, 3-methyladenine group, icariin group, and 3-methyladenine + icariin group. Alizarin red staining was used to observe the mineralized nodules. Western blot assay was utilized to determine the expression levels of alkaline phosphatase, Runx2, p62, LC3, NF-κB p65 and p-NF-κB p65 protein. RESULTS AND CONCLUSION: (1) Icariin could promote the proliferation of mouse preosteoblasts and the effect was the most obvious at 10 μmol/L (P < 0.05). The formation of mineralized nodules in the 10 μmol/L icariin group was significantly more than that in the control group and the expression levels of alkaline phosphatase and Runx2 protein were significantly increased (P < 0.05). Compared with the control group, the expression levels of Beclin1, LC3 II/LC3 I increased significantly and the expression of p62 decreased in the 10 μmol/L icariin group (P < 0.05). (2) After 3-methyladenine inhibited autophagy, compared with the icariin group, the expression of alkaline phosphatase, Runx2, LC3 II/LC3 I decreased and the expression of p62 increased in the 3-methyladenine + icariin group (P < 0.05). (3) Compared with the control group, the expression of p-p65/p65 decreased in the 10 μmol/L icariin group (P < 0.05). (4) These results indicate that icariin may promote osteogenic differentiation of MC3T3-E1 cells by down-regulation of the nuclear factor-κB signaling and enhancing autophagy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Icariin is an effective active component of traditional Chinese medicine which is used to prevent osteoporosis, but the specific mechanism remains unclear. Existing studies have indicated that autophagy can participate in regulating bone metabolism; however, whether icariin can prevent osteoporosis by changing autophagy is unknown. OBJECTIVE: To investigate whether icariin can prevent osteoporosis by regulating autophagy and promoting osteoblast differentiation. METHODS: Mouse preosteoblast cell lines MC3T3-E1 in logarithmic phase were selected and treated by different concentrations of icariin (10-6 to 10-3 mmol/L). Cell viability, apoptosis rate, alkaline phosphatase activity, the number of mineralized nodules and the expression level of osteogenic differentiation markers were observed in order to determine the optimal concentration of icariin. Then cells were treated by the optimal concentration of icariin and divided into three groups: control group, icariin group, icariin+3-methyladenine group. The number of autophagosomes, the expression level of osteogenic differentiation markers and autophagy related genes, the expression level of autophagy related proteins were evaluated by MDC staining, RTqPCR and western blot assay, respectively. In an in vivo test, an ovariectomized rat model was established and all the rats were divided into four groups: sham group, ovariectomized group, icariin group, estradiol group. Micro-CT was used to detect the bone micro parameters, and western blot was used to detect the expression level of autophagy related proteins. RESULTS AND CONCLUSION: Icariin could promote the proliferation and differentiation of osteoblasts, and at the same time inhibit apoptosis of the cells. The maximum stimulatory effect of icariin was observed at the concentration of 10-5 mmol/L (P < 0.05). In addition, icariin could increase the number of autophagosomes and upregulate the expression of osteogenic differentiation markers and autophagy-related genes or proteins (P < 0.05), which were antagonized by 3-methyladenine (P < 0.05). Icariin could also improve the parameters of bone microstructure in ovariectomized rats and upregulate the expression of autophagy-related proteins (P < 0.05). All these findings indicate that icariin can prevent osteoporosis by activating autophagy and promoting osteoblast differentiation. [ABSTRACT FROM AUTHOR]

BACKGROUND: It is an effective strategy for the treatment of osteoporosis to promote the proliferation and differentiation of osteoblasts, but the influence of inokosterone onosteoblast proliferation and differentiation is rarely reported.OBJECTIVE: To investigate the effects of inokosterone on theproliferation 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 genessuch 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. [ABSTRACT FROM AUTHOR]

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