Promotion Of MC3T3-E1 Preosteoblast Differentiation By Icariin Through The Hedgehog Signaling Pathway

Purpose:To study the effect of icariin on the proliferation and differentiation of osteogenic precursor cells MC3T3-E1 and its molecular mechanism,and to provide a theoretical basis for screening drugs for clinical treatment of bone loss diseases.Methods:Immunofluorescence was used to detect the expression of Ki67 in the control and icariin-added groups;Western blot was used to detect the expression of cyclinD1 and cyclinD2 proliferation-related genes in the control and icariin groups;osteogenic differentiation and the formation of calcified nodules was detected by alizarin red stain;the detection of ALP activity;RT-PCR and Western blot methods were used to detect the expression of osteogenic genes(collagen ??OPN?Runx2?OCN);To clarify the molecular mechanism of icariin on osteogenic differentiation of osteogenic precursor MC3T3-E1 cells,Western blot was used to detect gene expression related to Hedgehog signaling pathway(shh?Gli1?Gli3?PTCH2).After adding Hedgehog signaling pathway inhibitor cyclopamine,the effect of icariin on osteoblast differentiation and molecular mechanism were further verified by alizarin red staining and Western blot.Results:1.We set up the control and icariin-added groups.The results of the study showed that Ki67 immunofluorescence was significantly enhanced after the addition of icariin,so that the protein expression of cyclinD1 and cyclinD2 increased significantly.2.The alizarin red staining in the icariin-treated group was significantly enhanced compared with the control group.The ALP activity test was increased.The RNA levels and protein expression of osteogenic related genes increased.3.Compared with the control group,the protein expression of Hedgehog signaling pathway related genes(shh,Gli1)in the icariin group was significantly increased,and the expression levels of Gli3 and PTCH2 protein were greatly reduced.4.Compared with the icariin group,the addition of the inhibitor cyclopamine reduced the expression of genes(shh,Gli2)related to the Hedgehog signaling pathway.Conclusions:1.Icariin promotes the proliferation of osteogenic precursor MC3T3-E1 cells.2.Icariin promotes the differentiation and mineralization of MC3T3-E1 cells.3.Icariin promotes the differentiation of MC3T3-E1 cells through the Hedgehog signaling pathway.4.The addition of the inhibitor cyclopamine inhibits the effects of icariin on promoting osteogenic differentiation and the expression of genes related to osteogenesis,further indicating that icariin promotes osteogenic differentiation at least in part through the Hedgehog signaling pathway.