Effect Of TRPM3 On Proliferation,Differentiation And Mineralization Of Osteoblasts Induced By Puerarin

Objective:To investigate the effect of puerarin on MC3T3-El osteoblastic cells proliferation,differentiation and mineralization in vitro and the role of TRPM3 in this process.Methods:1.The activity of MC3T3-E1 cells in blank control group,puerarin group and positive control E2 group were detected by CCK-8 assay after treatment with 0.1,1,and 10?M puerarin or 0.01?M E2 for 24,48 and 72h.2.Flow cytometry was used to detect the cell cycle of osteoblasts after treatment with 0.1,1,and 10?M puerarin or 0.01?M E2 for 48h.3.The differentiation of osteoblasts was examined by Alkaline phosphatase(ALP)activity assay after treatment with 0.1 ?M puerarin or 0.01 ?M E2 for 48h.4.Alizarin red-calcium nodule staining method was used to investigate the mineralization of osteoblasts after treatment with 0.1 ?M puerarin or 0.01?M E2 for 14,21 and 28d.5.Following treatment with 0.1?M puerarin or 0.01?M E2 for 48h,the expression of TRPM3 mRNA and protein was detected using Q-PCR and Western Blotting.6.Flow cytometry and calcium detection kits were used to detect intracellular calcium concentration([Ca2+]i),and extracellular calcium concentration([Ca2+]0),respectively,after treatment with 0.1?M puerarin for 2,24 and 48h.7.After transfection of TRPM3-siRNA,and treatment with TRPM3 agonist pregnenolone sulfate(PS),and TRPM3 inhibitor 2-aminoethoxy-diphenyl borate(2-APB)on MC3T3-E1 cells,the expression level of Runx2 and osteoblasts proliferation,differentiation,and mineralization were investigated.Results:1.0.1,1,and 10?M puerarin significantly promoted the proliferation of MC3T3-E1 cells,and reduced the proportion of cells in G1 phase,increased the proportion of G2+S phase,among which 0.1 pM concentration had the best effect.Compared with the blank control group,0.1?M puerarin significantly enhanced the ALP activity of osteoblasts and promoted the formation of mineralized nodules.2.After treatment with 0.1?M puerarin for 48h,the expression level of TRPM3 decreased and the expression level of Runx2 increased.3.TRPM3-siRNA and 2-APB increased the expression of Runx2,which then promoted the proliferation and differentiation of osteoblasts,while PS inhibited this effects.4.0.5,5,10,and 20 mM calcium ions were added to the cell culture medium for 24,36,and 48 hours,respectively.Compared with the blank control group,the cell activity and ALP activity of the calcium ion group were significantly enhanced.5.After puerarin treated cells for 2h,compared with the blank control group,the[Ca2+]i in the puerarin group increased,and[Ca2+]0 decreased.For 24h,there was no significant difference in[Ca2+]i and[Ca2+]0 between the puerarin group and the control group.When the action time was prolonged to 48 h,[Ca2+]i decreased and[Ca2+]0 increased in puerarin group.Conclusions:Puerarin promotes MC3T3-E1 cells proliferation,differentiation and mineralization,which may be due to two reasons.On the one hand,puerarin increases the expression of Runx2 by down-regulating the expression of TRPM3,which promotes the proliferation and differentiation of osteoblasts.On the other hand,puerarin can increase[Ca2+]i within a short time(2h)and promote cell proliferation and differentiation.After a long time(48h),it will increase[Ca2+]0 and promote mineralization by increasing the binding to extracellular matrix proteins.After treatment with puerarin for a long time(48h),the expression of TRPM3 in osteoblasts decreased,and Ca2+ permeated into the cells decreased,which resultes in the increase of[Ca2+]0 and promotes mineralization by increasing the binding to extracellular matrix proteins.