Effect And Mechanism Of Estradiol Mediated SIRT1-FOXO3a On Autophagy In Osteoblasts

Objective:The incidence of osteoporosis is increasing rapidly,and its etiology is related to many factors,such as gender and age.Osteoporosis in postmenopausal women is caused by a decrease in estrogen levels in postmenopausal women due to ovarian dysfunction,resulting in an unbalanced interaction between the osteogenesis of osteoblasts and bone resorption by osteoclasts.A metabolic disease in postmenopausal women with reduced bone mass,accelerated bone loss,and reduced bone strength.Therefore,a decrease in estrogen is considered to be one of the key factors that cause osteoporosis in postmenopausal women.Sirtuins is a histone deacetylase with transcriptional silencing function originally found in yeast.It has been widely studied in mammals for its ability to extend lifespan.As an important member of Sirtuin,silencing information regulator 1 is Histone deacetylase,which relies on nicotinamide adenine dinucleotide（NAD+）,has been reported to be involved in the control of many diseases,such as aging,tumor suppression,energy metabolism,anti-osteoporosis,diabetes,and cardiovascular disease.And how SIRT1 regulates bone quality has attracted more attention.Studies have found that estrogen can bind to ligands through receptor alpha（ERα）,adenosine monophosphate-activated protein kinase（AMPK）through phosphorylation,and silencing information regulator 2 related enzyme l（SIRTl）through deacetylation modification They jointly regulate the functions of osteoblasts and osteoclasts,and there are certain upstream and downstream relationships between them.Estrogen activates AMPK through phosphorylation.The interaction between phosphorylated AMPK can participate in the regulation of osteogenesis Cell autophagy or apoptosis,etc.,have all suggested the important role of 17β-E2,AMPK,and Sirtl in the pathogenesis of osteoporosis.Studies have found that overexpression of SIRTl can prevent age-related bone loss in mice.Osteoblast or osteoclast-specific SIRT1 knockout studies in rats can result in reduced bone formation leading to reduced bone volume and absorption.SIRTl has been shown to regulate oxidative stress through the deacetylation of various substrates.Through a forkhead box O（FOXO）-dependent mechanism,modest overexpression of SIRTl（up to 7.5-fold）protects the heart from oxidative stress and increases expression of antioxidants.Studies have found that overexpression of SIRTl increases bone formation in osteoblasts,and through FOXO3a deacetylation and oxidative stress inhibition,reduces osteoblast apoptosis and partially restores bone growth and osteogenesis defects in mice.Therefore,studying whether estrogen can enhance the activity of SIRT 1 and thus affect osteoblasts,and its pathway has become the focus of this study,which is helpful to further explore the mechanism of osteoporosis in postmenopausal women and can guide the disease.Treatment makes better predictions,diagnostics,and prevention measures.Methods:1)Firstly,different concentrations of 17β-E2 were applied to h FOB 1.19After 24 hours,the autophagy of osteoblasts was detected by fluorescence autophagy detection kit（MDC）;（2）the expression of autophagy related protein LC3 was detected by Western blotting;（3）the effect of AMPK,phosphorylated AMPK and SIRT1 on egg white activity was detected by Western blotting;（4）SIRT1 was enhanced or inhibited in17β-E2 environment The changes of cell morphology and key proteins were observed by electron microscopy,confocal laser microscopy and flow cytometry;（5）the effects of17β-E2/SIRT1/AMPK/FOXO3a on autophagy related proteins Bcl-2,BNIP3,m TOR and caspase-3 were detected by immunoblotting and real-time quantitative PCR.Results:（1）17β-E2(10^-8M,10^-6M)increased the autophagy of osteoblasts and the activity of lc3ii protein in osteoblasts;（2）with the increase of 17β-E2 concentration,the expression of SIRT1 protein increased and the activity increased;（3）17β-E2increased the level of phosphorylated AMPK,and AMPK increased the activity of SIRT1in osteoblasts;（4）SRT1720,SIRT1 agonist,and ex527,an inhibitor of SIRT1 It can interfere with the negative regulation of 17β-E2/SIRT1 on the apoptosis of osteoblasts;（5）using laser confocal microscopy and perspective electron microscopy to observe the LC3 protein regulated by 17β-E2/SIRT1 in osteoblasts,SRT1720 was found It can enhance the autophagy mediated by 17β-E2,increase the autophagy bodies in cells,and increase the mitochondrial autophagy bodies with double membrane structure;（6）it can mediate SIRT1 protein,17β-E2 can increase the expression of autophagy related proteins Bcl-2 and BNIP3,reduce the activity of m TOR and increase the activity of FOXO3a.Conclusion:（1）SIRT1 plays a key role in 17β-E2 mediated autophagy of osteoblasts;（2）17β-E2 may mediate autophagy of osteoblasts through AMPK/SIRT1/FOXO3a/mTOR pathway.