Effect And Molecular Mechanism Of Mangiferin On Bone Formation Of Osteoblasts Induced By H₂O₂

Aim:In this study,the oxidative stress model of osteoblasts was established by H₂O₂.Mangiferin was used to interfere with osteoblasts induced by H₂O₂.To investigate the effects of mangiferin on proliferation,differentiation and bone formation of osteoblasts under oxidative stress,and to study the relationship between mangiferin-mediated H₂O₂-induced osteoblastic bone formation and autophagy apoptosis.In addition,after blocking ERK1/2 signal by PD98059 and interfering with FoxO1 transcription factor by FoxO1 siRNA,compared upstream and downstream protein expression to explore the molecular mechanism of mangiferin’s protective effect on osteoblasts induced by H₂O₂.Methods:1.Establishment of oxidative stress model and intervention of mangiferin:taking osteoblasts as the research object,using mangiferin intervention,using H₂O₂ treatment,combined with MTT method to screen the optimal concentration of H₂O₂ to induce oxidative stress model,selecting the appropriate concentration of mangiferin as the concentration of administration;MTT method to detect the effect of Mangiferin on the proliferation of osteoblasts induced by H₂O₂;To detect the effect of Mangiferin on the activity and content of ALP in osteoblasts induced by H₂O₂.2.Effects of mangiferin on bone formation of osteoblasts under oxidative stress:Osteoblast mineralization by alizarin red staining;OC expression of osteoblasts by immunocytochemistry;The expression of BMP-2,OPG and COL I in osteoblasts was detected by Western Blot.The expression ofβ-catenin,Runx2,OPG and BMP-2 mRNA in osteoblasts was detected by Real-time PCR.3.Mangiferin regulates the relationship between H₂O₂-induced osteoblast reduced bone formation and autophagy apoptosis:Flow cytometry was used to detect the apoptotic rate of osteoblasts;DCFH-DA probe and Fluo-3AM probe were used to detect ROS and calcium ions in osteoblasts respectively;the expression of autophagy-related proteins Beclin 1,p62,LC3 II/I and apoptosis-related proteins Bax and Bcl-2 in osteoblasts was detected by Western Blot.4.Mangiferin regulates the signal pathway of osteoblast osteogenesis induced by H₂O₂:The effect of Mangiferin on osteoblasts bone formation induced by H₂O₂ was observed after blocking ERK1/2 signaling.MTT assay was used to detect the toxicity of PD98059 to osteoblasts.Osteoblasts were divided into normal control group（Control）,model group（Model）,PD98059 group（PD98059,25μM）,mangiferin group（Mangiferin,80μM）,mangiferin+PD98059 group（Mangiferin+PD98059）;The expression of ERK1/2 and p-ERK1/2 protein in osteoblasts was detected by Western Blot.After determining the blocking efficiency,the expression of BMP-2,OPG and COLI and the expressions of PTEN,p-PTEN,FoxO1 and p-FoxO1 in osteoblasts were detected by Western Blot.The expression ofβ-catenin,Runx2,OPG and BMP-2 mRNA in osteoblasts was detected by Real-time PCR.The effect of Mangiferin on osteoblasts bone formation induced by H₂O₂ was detected after blocking FoxO1 signal.Western Blot was used to detect the transfection efficiency of Lip2000transfected FoxO1 siRNA;The osteoblasts were divided into normal control group（Control）,model group（Model）,FoxO1 siRNA group（si FoxO1,10μL）,mangiferin group（Mangiferin,80μM）,mangiferin+Fox O1 siRNA group（Mangiferin+si FoxO1）;the expression of COLI and FoxO1,p-FoxO1,ERK1/2,p-ERK1/2,PTEN,p-PTEN,AKT and p-AKT was detected by Western Blot.The mRNA expression ofβ-catenin,Runx2,OPG and BMP-2 in osteoblasts was detected by Real-time PCR.Results:1.The results showed that the survival rate of cells induced by 400μM H₂O₂ for 3h was about 70-80%,which can be used as a modeling condition for oxidative stress in subsequent experimental.The concentration of mangiferin at 20,40 and 80μM was selected as the low,medium and high intervention concentration of mangiferin in this study.Mangiferin improved the proliferation of osteoblasts induced by H₂O₂.Mangiferin increased the ALP staining positive results and increased the ALP content in osteoblasts under oxidative stress.The effect of mangiferin in 40μM and 80μM groups was the most significant.2.Mangiferin can increase the mineralization area and OC expression of osteoblasts under oxidative stress,up-regulate the expression BMP-2,OPG and COL I protein induced by H₂O₂,and increase the mRNA expression of BMP-2,OPG,β-catenin and Runx2 in different degrees.3.Mangiferin significantly decreased the apoptosis rate of osteoblast induced by H₂O₂,decreased ROS and calcium ion content in osteoblasts induced by H₂O₂,and mangiferin significantly up-regulated LC3II/I ratio,Beclin 1 expression and significantly down-regulated P62 expression.Mangiferin significantly increased the expression of Bcl-2 and the expression of Bax was significantly decreased in osteoblasts under oxidative stress.4.PD98059 blocked mangiferin up-regulation the protein expression of BMP-2,OPG,COLⅠin osteoblasts under oxidative stress,and PD98059 inhibited mangiferin to increase the mRNA expression of BMP-2,OPG,β-catenin,Runx2 in osteoblasts induced by H₂O₂.The results of signal protein assay showed that H₂O₂ significantly decreased the expression of ERK1/2,p-ERK1/2 and p-AKT in osteoblasts compared with the control group,while the expression of p-PTEN,FoxO1 and p-FoxO1 was up-regulated.PD98059 blocks the expression of p-ERK1/2 protein,and PD98059 inhibits mangiferin up-regulation of p-ERK1/2 expression in osteoblasts.PD98059 can enhance the down-regulation of PTEN and p-PTEN by mangiferin but has no significant effect on FoxO1 and p-FoxO1 proteins.5.The si FoxO1 group inhibited mangiferin up-regulation of COLI protein and the mRNA expression ofβ-catenin,Runx2,OPG in osteoblasts induced by H₂O₂,but significantly increased BMP-2 mRNA expression.It is indicated that the protection of mangiferin on the bone formation of osteoblasts after inhibition of FoxO1 signal is not a simple reversal effect.When the signal protein expression was detected,it was found that compared with the model group,FoxO1 was down-regulated after osteoblasts were directly interfered with FoxO1 siRNA,but p-FoxO1 was up-regulated,AKT was increased,p-AKT was down-regulated,PTEN was down-regulated,and p-PTEN was up-regulated.In addition,the si FoxO1 group had no effect on ERK1/2 protein expression,but increased p-ERK1/2 protein expression.When mangiferin was combined with FoxO1 siRNA,osteoblasts significantly decreased the expression of FoxO1 and p-FoxO1 and increased the expression of PTEN,p-PTEN,ERK1/2 and AKT,but it plays an inhibitory role on the expression of phosphorylated proteins of ERK1/2 and AKT.Conclusion:1.Mangiferin promotes the proliferation and differentiation of osteoblasts induced by H₂O₂.2.Mangiferin protects H₂O₂-induced osteoblast bone formation by increasing osteoblast mineralization,bone formation-related protein expression and bone formation-related gene expression.3.One of the mechanisms of mangiferin protection against H₂O₂-induced osteoblasts is to is to reduce ROS and calcium content in osteoblasts induced by H₂O₂.4.Mangiferin increases the autophagy of osteoblasts under oxidative stress and reduces osteoblast apoptosis to exert its protective effect on osteoblast formation induced by H₂O₂..5.Mangiferin may enhance osteoblast bone formation induced by H₂O₂ through regulating PTEN/AKT/ERK/FoxO1 signaling pathway.