Resveratrol Induces Proliferation And Autophagy In Pre-Osteoblast Cells MC3T3-E1 Via Activating GATA-1

Objectives:Multiple studies had confirmed that resveratrol can promote osteogenesis in vivo and in vitro,but there still needed more research to illuminate the inner mechanism of this function.GATA-1 was an important transcription factor in the differentiation of megakaryocytes and erythrocytes,and directly activated autophagy in erythrocytes as well.Newly evidences showed that GATA-1 could impact on bone metabolism,but its specific mechanisms were still unknow.Therefore,this study intended to observe and study the role of GATA-1 and autophagy in a resveratrol-induced MC3T3-E1 proliferation model and to study connection between autophagy and GATA-1 in the process to find more mechanism during proliferation regulation in osteoblasts.Methods:Osteoblast precursor cell strain MC3T3-E1 were cultured in vitro,CCK-8 was used to detect cell viability,crystal violet was used to detect colony formation,and western-blotting was used to detect the expression of Cyclin-D1.Thus,the effect of resveratrol on MC3T3-E1 proliferation was determined.The changes of autophagy levels during resveratrol-promoted proliferation were observed by the expression levels of LC3II and ATG7,and labeled autophagosomes and lysosomes.By using autophagy inhibitor 3-MA and autophagy flux inhibitor bafilomycin,the change in proliferation under the reduced autophagy level was observed,and whether adding resveratrol can improve it was tested.The expression levels of GATA-1 and AMPKa were detected by western-blotting and the position of GATA-1 was detected by immunofluorescence.To observe whether resveratrol and AMPKa agonist AICAR affected the expression changes and nuclear translocation of GATA-1,a small interfering RNA Construct to interfere with the expression of GATA-1 in MC3T3-E1,and observe its effect on autophagy and proliferation,and whether adding resveratrol can change this effectResults:1.0.1～10μM resveratrol is a safe application range for the osteoblast precursor MC3T3-E1 strain cultured in vitro;Among them,1 μM resveratrol can significantly promote the osteoblast precursor cell strain MC3T3-E1 Proliferation(p<0.005),and 10 μM resveratrol up-regulated the expression of cyclin D1(p<0.01).2.10 μM resveratrol can increase the expression levels of LC3II and ATG7 at the same time(p<0.01),and promote the formation of autophagosomes and autophagolysosomes.The use of autophagy inhibitors can downregulate the expression of cyclin D1,but the addition of resveratrol can improve the effect on proliferation;the use of serum starvation to induce autophagy within 12 h can increase the expression of cyclin D1.3.The effect of 10 μM resveratrol for 12 h can increase the expression of GATA-1 and promote its nuclear translocation.After using siRNA to interfere with the expression of GATA-1,the expression of LC3II and cyclin D1 both decreased.This indicates that GATA-1 plays an important role in promoting the proliferation of osteoblast precursor MC3T3-E1 and inducing its autophagy.4.10 μM resveratrol can promote AMPKa phosphorylation after 12 h.The AMPKa agonist AICAR can also up-regulate the expression of GATA-1 and promote its nuclear translocation,but the effect is not as good as resveratrol,indicating that AMPKa may be one of the upstream regulators of GATA-1.Conclusions:These results indicate that RSV induces proliferation in MC3T3-E1 by increasing GATA-1 expression which thence activates autophagy;and of note,AMPKα is one of the upstream regulators of GATA-1.