Effect Of Autophagy On Proliferation,Differentiation And Mineralization Of Osteoblasts Induced By Puerarin

Objective:To investigate the effect and mechanism of puerarin on the development of MC3T3-E1 osteoblasts,and to provide theoretical guidance for the clinical treatment of osteoporosis with puerarin.Methods:1.Cell viability assay:After cultured with different concentrations of puerarin(0.1,1,10?M)for 24h,48h and 72 h,the cell viability of the blank control group and puerarin group was detected by CCK-8 method.2.Measurement of cell differentiation level:Alkaline phosphatase activity(ALP)activity was measured after puerarin(1?M)was applied to osteoblasts for 72 h.3.Measurement of cell mineralization level:After treated with puerarin(1?M)for 7,14 and 21 days,the area of cell mineralized nodules was determined by alizarin red S staining.4.Measurement of protein expression level:The protein levels of Beclinl and LC3B were determined by western blotting method Oh,48h and 72h after treated with puerarin(1?M),and 72h after treated with RAP(100?M,autophagy agonist)and 3-MA(75?M,autophagy inhibitor).5.Observation of cell ultrastructure:puerarin(1?M)and 3-MA(100?M)were incubated with osteoblasts for 72h,and the ultrastructure of cells was observed under electron microscope and photographed.6.Effect of autophagy on osteoblasts:MC3T3-E1 cells were treated with 3-MA(100?M)to detect changes in the proliferation,differentiation and mineralization of osteoblasts.7.The effect of puerarin on miR-204 expression:The expression of miR-204 after puerarin treatment was detected by RT-qPCR.8.Prediction of the target of miR-204:TargetScan prediction sof-tware was used to analyze the possible binding sites of miR-204 with mouse LC3B gene.9.Effect of miR-204 on autophagy level and ultrastructure of osteoblasts:After transfecting osteoblasts with miR-204-siRNA,the expression level of LC3B protein was detected and the ultrastructure of cells was observed under electron microscope.Results:1.Puerarin(0.1,1,10 ?M)could significantly promote the proliferation of mouse MC3T3-E1 osteoblasts,and the optimum concentration of puerarin was 1?M.Compared with the blank control group,puerarin treatment of 1?M for 72 hours significantly increased ALP activity and promoted the formation of mineralized nodules.2.Compared with the blank control group,Beclinl and LC3B protein expression levels of autophagy markers were significantly increased after the action of puerarin at 1?M concentration,with the best effect after 72h.The LC3B protein expression in puerarin group was higher than those in autophagy agonist group after 72h treatment.In addition,the specific bilayer membrane structure of autophagy was observed under electron microscopy in puerarin group.3.Autophagy inhibitor(3-MA)significantly inhibited the proliferation and differentiation of osteoblasts after 72 hours,but no significant effect on the formation of mineralized nodules was detected.4.MiR-204 expression was significantly down-regulated after 72h treatment with puerarin(1?M).5.Bioinformatics analysis showed that the binding site of microRNA-204 in LC3B was a widely conserved element between 44 and 50 BP of LC3B 3'UTR.6.Compared with the blank control group,the protein expression of LC3B was significantly increased after transfection of miR-204-siRNA.Moreover,under the electron microscope,the cells showed the special double-layer membrane structure of autophagy.Conclusions:(1)Puerarin was capable of promoting the proliferation,differentiation and mineralization of osteoblasts.(2)The effect of puerarin on osteoblasts could induce cell autophagy.(3)The autophagy induced by puerarin on osteoblasts could promote the proliferation and differentiation of osteoblasts,but had no significant effect on mineralization.(4)Down-regulation of miR-204 expression induced by puerarin leads to enhanced expression of LC3B,this might be involved in the mechanism of osteoblast autophagy.