Osteogenic Differentiation Of MC3T3-E1 Regulated By BMSCs-derived Exosomes After Isopsoralen Intervention

ObjectiveTo investigate the effects of exosomes secreted by bone marrow mesenchymal stem cells（BMSCs）stimulated by isopsoralen on the proliferation and differentiation of osteoblast precursor cells（MC3T3-E1）and the mechanism of regulating bone metabolism.Methods（1）BMSCs cells were cultured in large quantities by adherent and subcultured methods.BMSCs were stimulated by different concentrations of isopsoralen（IP）.BMSCs were divided into three groups:NC,10-8mol/ml IP and 10-10 mol/ml IP;（2）BMSCs supernatants were collected and exo,somes（NC-exo,10-8 mol/ml IP-exo,10-10 mol/ml IP-exo）were extracted by ultrafast differential centrifugation.The morphology of exosomes was observed by transmission electron microscopy.The specific markers CD63,CD44,CD29 were detected by Western blotting.The protein content of exosomes was quantitatively detected by BCA,and the particle size of exosomes was analyzed by nano Sight（NTA）;（3）Combining the results of high-throughput sequencing of pre-microRNAs and circRNA chip,the significantly differentially expressed circ₈604 with mm9 conserved sequence and miRNAs related to bone metabolism were screened;（4）BMSCs-derived exoLsomes were co-cultured with MC3T3-E1 cells,CCK-8 assay was used to detect the proliferation activity of MC3T3-E1 cells,qRT-PCR was used to detect the co-culture of exosomes with MC3T3-E1 and the expression of ALP,Runx2,OPG,BMP2 and circ₈604 during osteogenic differentiation;（5）Bioinformatics analysis predicted target miRNAs of circ₈604 and target genes of target miRNA.QRT-PCR preliminarily verified the expression of target miRNAs and corresponding target genes during osteogenic differentiation of exosomes and MC3T3-E1 after co-culture.ResultsThe typical exosome morphology of cup vesicle could be observed under transmission electron microscope in exocrine from BMSCs.It espressed exosome surface markers CD63 and contained exosome specific surface markers CD44 and CD29 derived from BMSCs.NTA results also showed that the diameter of the extracted exosome was within 200 nm,which accorded with the diameter of the exosome covered by the study.Compared with MC3T3-E1 group,exosomes of high-concentration isopsoralen group could inhibit the proliferation of MC3T3-E1 and the expression of osteogenic differentiation related genes OPG,ALP,Runx2,BMP2 mRNA was up-regulated after exosome stimulation.It was more obvious after exosome stimulation in high-concentration isopsoralen group.During the co-culture of BMSCs exosomes with MC3T3-E1 cells and induction of osteogenic differentiation,the osteogenic phenotypes OPG,ALP,Runx2,and BMP2 showed an up-regulation trend in the exosomal stimulation group.Moreover,the expression of circ₈604 was up-regulated after exocrine stimulation of MC3T3-E1 in high concentration isopsoralen group,while the expression of target miR-26b-3p was down-regulated,and the expression of target genes ER a and GDF11,TGFBR1 was up-regulated.The deposition of red calcified nodules was found by alizarin red staining in the high concentration isopsoralen group.ConclusionBMSCs-derived exosomes could promote the expression of osteogenic differentiation phenotype ALP,Runx2,OPG and BMP2 in MC3T3-E1 during osteogenic differentiation,and it was more obviously upregulated after exocrine stimulation in high concentration isopsoralen group.Circ₈604 was also up-regulated in high-concentration isopsoralen group after stimulation of MC3T3-E1,while the expression of target miR-26b-3p was down-regulated,while the expressions of target genes ER a,GDF11 and TGFBR1 was up-regulated.BMSCs-derived exosomes regulated the proliferation and osteogenic differentiation of MC3T3-E1.We speculate that BMSCs-derived exosomes stimulated by isopsoralen at an appropriate concentration（10-8mol/ml）may affect the osteogenic differentiation of MC3T3-E1 through the circ₈604/miR-26b-3p/ER a regulatory axis,which regulates bone metabolism and affects the development,prevention and treatment of osteoporosis.