Effects Of Inokosterone On The Proliferation And Differentiation Of Rat Pr Imary Osteoblasts And MC3T3-E1 Cells

ObjectivesTo investigate the effects of inokosterone on the proliferation and the expression of OCN,OPG,OPN,type ? collagen and ALP,which are osteoblast differentiation factors,of primary osteoblasts of rat and MC3T3-E1 cells.Exploration of effects and mechanism of inokosterone on the proliferation and differentiation of osteoblasts provides a theoretical basis for the clinical application of inokosterone in the treatment of osteoporosis.Method The study is divided into seven parts.(1)Rat primary osteoblasts were isolated and cultured by secondary enzyme digestion,and intervented with inokosterone at a concentration of 1ng/?1,5ng/?l,10 ng/?l for 24h and 48h.The proliferative activity of rat osteoblasts was examined by CCK-8 kit.(2)The expression of mRNA of osteocalcin(OCN),osteoprotegerin(OPG),protein(OPN)and collagen ? of rat osteoblasts were detected by real-time quantitative PCR(qPCR)method after the intervention of the above concentrations of inokosterone for 24h and 48h.(3)MC3T3-E1 cells were intervented with inokosterone at a high concentration of 1ng/?l,20ng/?l,50ng/?l,75ng/?l,100ng/?l in vitro for 24h and 48h.The CCK-8 kit was used to detect the proliferative activity of MC3T3-E1 cells.(4)MC3T3-E1 cells were intervented with inokosterone at a low concentration of 10-6ng/?l,10-5ng/?l,10-4ng/?l,10-3ng/?l,10-2ng/?l,10-1ng/?l in vitro for 24h and 48h.The CCK-8 kit was used to detect the proliferative activity of MC3T3-E1 cells.(5)MC3T3-E1 cells were intervented with inokosterone at a low concentration of 10-6g/?l,10-5ng/?l,10-4ng/?l,10-3ng/?l,10-2ng/?l,10-1ng/?l in vitro.Then the qPCR detection was used to detect the expression of mRNA of OCN,OPG,OPN,collagen ? and ALP in MC3T3-E1 cells.(6)MC3T3-E1 cells were intervented with inokosterone at a concentration of 1ng/?l,5ng/?l and 10ng/?l for 7d and 14d.The AKP kit was used to detected the expression of ALP.(7)MC3T3-E1 cells was cultured in an induction solution containing Ing/?l,5ng/?l and 10ng/?l of inokosterone,and the alizarin red was performed on the 7th and 14th day respectively.Result(1)The result of detection of CCK-8 showed that cell viability of primary osteoblasts decreased were intervented at a concentration of inokosterone of 1ng/?l,5ng/?l and 10ng/?l respectively for 48h.(P<0.05).(2)After rat osteoblasts were intervented with inokosterone for 24h,OCNmRNA in 5ng/?l concentration group and mRNA of OPN,OCN,OPG,collagen? in 10ng/?l group increased(P<0.05).After intervented for 48h,the expressions of mRNA of OPN,OCN,OPG in the 1ng/?l group and mRNA of OPN,OCN,OPG,collagen ? in 5ng/?l and 10ng/?l groups were significantly increased,and the expression increases with increasing concentration(P<0.05).(3)The cell viability of MC3T3-E1 cells of the group of 50ng/?l,75ng/?l,100ng/?l after treated with high concentration of inokosterone for 24h and 20ng/?l,50ng/?l,75ng/?l,100ng/?l decreased after 48h(P<0.05).(4)The low concentration of inokosterone was used to interfere with MC3T3-E1 cells for 24h.There was no significant change in cell viability among each group(P>0.05).After intervention of 48h,the cell viability of the group of 10-3ng/?l,10-2ng/?l,10-1ng/?l decreased(P<0.05).(5)After MC3T3-E1 cells cultured with low concentration of inokosterone,except for the expression of ALPmRNA in 10-6ng/?l,10-5ng/?l,10-4ng/?l,10-3ng/?l,10-2ng/?l,10-1ng/?l group and OPNmRNA in 10-5ng/?l,10-4ng/?l group increased(P<0.05),OCNmRNA in 10-6ng/?l,10-3ng/?l and 10-1ng/?l group;OPGmRNA in 10-6ng/?l,10-5ng/?l,10-3ng/?l group;collagen ?mRNAin10-6ng/?l,,10-5ng/?l,10-4ng/?l,10-3ng/?l,10-2ng/?l,10-1ng/?l group decreased(P<0.05).(6)After MC3T3-E1 cells intervented with inokosterone for 7d and 14d,expression of ALP in osteogenic induction group,Ing/?l,5ng/?l,10ng/?l group increased(P<0.05).Compared with the osteogenic induction group,the ALP expression in the group of 5ng/?l and 10ng/?l on the 7th and 14th day also increased(P<0.05).(7)The results of alizarin red staining showed that with intervention of inokosterone for 7d and 14d,the AOD of MC3T3-E1 cells in 1ng/?l,5ng/?l,and 10ng/?ll group increased,the difference was statistically significant(P<0.05).ConclusionInokosterone has a significant inhibitory effect on the proliferation of primary osteoblasts of SD rat and MC3T3-E1 cells.Although under the situation of inhibited proliferation,it can still promote the differentiation of rat primary osteoblasts,which is concentration dependent and timely dependence.Inokosterone can also promote the differentiation and mineralization of MC3T3-E1 cells to promote bone formation.And it may has therapeutic effect on osteoporosis.