Effects Of Ipriflavone On The Proliferation And Osteogenesis Of MC3T3-E1 Cells Based On GPER1-mediated P38MAPK Pathway

Ipriflavone is a kind of isoflavone in phytoestrogens,which is similar to estradiol in chemical structure and plays an important role in promoting formation of bone tissue,inhibiting resorption of bone tissue,and resulting in estrogen-like anti-bone effect.The role of osteoporosis is currently widely used in clinical and for the treatment of osteoporosis in postmenopausal women.It improves primary OP and other bone metabolic diseases because its metabolic process is similar to the physiological process in vivo.And it does not produce side effects caused by estrogen application,so it has good application prospects.G coupled-protein estrogen receptor 1(GPER1)is a functional membrane estrogen receptor mainly located in organelle membranes.Related studies have shown that estrogen could bind to it and activate a variety of downstream intracellular responses,including kinase activation,ion channels,and activation of multiple signaling pathways.G15 is a cell-permeable non-steroidal antagonist of GPER1,which specifically inhibits GPER1 activation.The p38MAPK signaling pathway is one of the most important components of the MAPKs family.Sun Xianchang and other studies have shown that ginsenoside Rgl can inhibit the activation of NF-?B and MAPKs(ERK1/2,JNK,p38)signaling pathways through the GPER1 receptor.SB 203580 is a selective,ATP-competitive inhibitor of the p38MAPK signaling pathway.It is widely used in experimental studies and can provide strong evidence to prove that the p38MAPK signaling pathway is involved in biological regulation.Objectives:This study investigated whether ipriflavone had effects on the proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro,and explored the role and mechanism of GPER1-mediated p38MAPK signaling pathway in the process of promoting osteogenic differentiation with the optimal concentration,which provided a theoretical basis for the feasibility of ipriflavone in the clinical application of bone defect treatment.Methods:1.Culture of MC3T3-E1 cells in vitro:including cell culture,cell resuscitation,and cell cryopreservation.2.Here,we used cell-counting kit-8(CCK-8)and Alkaline phosphatase(ALP)activity kits to verify the effects of ipriflavone on MC3T3-E1 cells proliferation and osteogenic differentiation.According to the data acquired,screen the optimal concentration of IP that promotes its proliferation and osteogenic differentiation.3.Alizarin red staining and NBT/BCIP alkaline phosphatase coloration kit were further used to verify the effect of the obtained optimal drug concentration on promoting the osteogenic differentiation of MC3T3-E1.4.Westem Blot and qRT-PCR were used to verify the protein and mRNA expression levels of GPER1,ALP,and Runx2.The expression levels of p38 and p-p38 were detected by Western Blot,which may have changed,to investigate whether p38MAPK signaling pathway play a part in this process.5.The p38MAPK signaling pathway inhibitor SB 203580 and GPER1 specific inhibitor G15 were applied to further verify the possible role of GPER1-mediated p38MAPK pathway during this process.6.Statistical analysis:The data obtained in this experiment was analyzed by using Graphpad Prism 6.The t test was used to compare the two sets of experimental data.The paired t test was used to compare the two sets of experimental data at different time points.The comparison between multiple groups of data was performed by one-way analysis of variance.The experimental group and the control group were considered statistically significant with P<0.05.Results:1×10-7 mol/L ipriflavone was proved to be the optimal concentration to promote MC3T3-E1 proliferation,ALP activity,and mineral deposition in vitro(P<0.05).The gene expression levels of GPER1,ALP,Runx2 and p-p38 were all up-regulated compared with the control group(P<0.05).Application of GPER1 specific inhibitor G15 could inhibit the activation of p38MAPK pathway to a certain extent;application of SB203580 could block the promotion of ALP and Runx2 expression by IP to a certain extent.Conclusion:1.The proliferation and osteogenic differentiation of MC3T3-E1 cells could be promoted significantly by ipriflavone,and the optimal concentration for promotion is 1×10-7mol/L in vitro.2.GPER1-based p38MAPK signaling pathway may play an important role in the process of ipriflavone promoting MC3T3-E1 cells proliferation and osteogenic differentiation.