Mechanisms Of Icariin Promoting Differentiation Of Mouse Preosteoblasts By Enhancing Autophagy

Objective1.To investigate the effect of icariin on proliferation and differentiation of mouse preosteoblasts.2.To determine whether icariin affects the differentiation of mouse preosteoblasts by regulating autophagy.Methods1.Mouse preosteoblasts MC3T3-E1 cells were divided into control group and treatment group,which were treated with icariin of different concentrations.(1)MTS was used to detect the proliferation activity of preosteoblasts in each group after icariin intervention.(2)Alkaline phosphatase staining and alizarin red staining were used to evaluate the level of osteogenic differentiation.(3)Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the osteoblastic differentiation ability and autophagy activity of preosteoblasts in each group after icariin intervention.(4)Western blot was used to detect the osteoblastic differentiation ability and autophagy activity of preosteoblasts in each group after icariin intervention.2.The optimal concentration of icariin was screened out,and 3-methyladenine(3MA)was added to inhibit autophagy.The experimental groups were as follows:control group,3MA group,ICA group,and 3MA+ICA group.(1)Alizarin red staining was used to evaluate the level of osteogenic differentiation;(2)Cell cycle and apoptosis were detected by flow cytometry.(3)The protein expressions of ALP,Runx2,p62,LC3,NF-κB p65 and p-NF-κB p65 were detected by Western blot.Results1.Icarin can promote the proliferation of mouse preosteoblasts,and the effect was most obvious when the concentration was 10μmol/L(P<0.05).2.Compared with the control group,the formation of alkaline phosphatase chromophore and mineralized nodules were significantly increased in the 10μmol/L icariin group,and the expression levels of ALP,Runx2 gene and protein were significantly increased(P<0.01).3.Compared with the control group,LC3 gene expression was increased and p62 gene expression was decreased in 10μmol/L icariin group(P<0.01).Compared with the control group,the expressions of Beclinl and LC3Ⅱ/LC3 protein were significantly increased in the 10μmol/L icariin group,while the expression of p62 protein was decreased(P<0.01).4.After 3MA inhibition of autophagy,compared with ICA group,the expression of ALP,Runx2,LC3Ⅱ/LC3 protein decreased in 3MA+ICA group,while the expression of p62 protein increased(P<0.05).5.Compared with the control group,the expression of p-p65/p65 decreased in icariin l0μmol/L group(P<0.01).Conclusion1.Icarin promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells.2.Icarin affects osteoblastic differentiation of MC3T3-E1 cells by enhancing autophagy.