17?-estrogen Induces Mitophagy In M3T3 Mouse Osteoblast Cells Through The PI3K/AKT Signaling Pathway

Objective:Osteoporosis is the most common bone disease affecting millions of people worldwide,which results in high medical expenditures and substantial morbidity with a decrease in quality of life.The process of osteoporosis is influenced by a sort of factors,but the most obvious factor is age-related bone loss which is contributed by estrogen fall.Estrogen can also block bone resorption by interacting with tissue-specific receptors,estrogen receptor ?(ER?)and estrogen receptor ?(ER?),to increase osteoclast apoptosis However,the certain mechanism of estrogen deficiency influences osteoporosis and osteoblast cells' motility which is completely unknown.This observation has drawn attention to mitophagy.Now mitophagy has been a key role in multiple domains of mitochondrial health and quality control.In this study,we designed to make sure whether estrogen induces mitophagy through GPER/PI3K/AKT signal pathway in M3T3 cells and to illuminate the molecular mechanism involved.Methods: Mouse osteoblast cells cultured in vitro and the expression of m RNA GPER in mouse osteoblast cells was detected by real-time quantitative PCR.The phosphorylation level of GPER protein in mouse osteoblast cells was detected by Western-blot technique.The concentration gradient of 17?-E2 and / or GPER specific inhibitor G15 and / or PI3 K specific antagonist(LY294002)were added respectively to intervene in the concentration gradient.Using western-blot technology to detect the expression of AKT protein and GPER protein and phosphorylated protein level and the activity changes of mitochondrial autophagy associated protein which were also detected by immunofluorescence microscopy(mitochondrial matrix protein Hsp60?TOM20).Results:1?17?-E2 enhances GPER gene expression and protein activity in M3T3 cells.Real time quantitative PCR and Western-blot technology detect that 17?-E2(10-7M,10-8M,10-9M)can increase the level of m RNA GPER and phosphorylated protein,And 10-7M group has statistical significance(P<0.05).When pretreated with GPER protein specific inhibitor G15,the activity of GPER was inhibited,and the expression of GPERm RNA was inhibited,with statistical significance(P<0.05).2?17?-E2 enhances mitochondrial autophagy related protein Hsp60?TOM20?LC3-II activity in M3T3 cells.By Western-blotdetection,Hsp60?TOM20?LC3-II protein activation levels were suppressed in 10-7 M 17?-E2 treatment group,And there was statistical significance(P<0.05).3?17?-E2 affect mitochondrial autophagy related protein Hsp60?TOM20?LC3-II protein activity through the GPER/PI3K/AKT pathway in the M3T3 cell.By Western-blot detection,AKT protein activation level increased in 10-7 M 17?-E2 treatment group,And there was statistical significance(P<0.05).By Western-blot detection,Hsp60?TOM20?LC3-II protein activity had been inhibited in G15?LY294002 and/or 10-7 M 17?-E2 combined treatment group Hsp60?TOM20?LC3-II(P>0.05).Conclusion: Estrogen increased the expression of mitochondrial autophagy-related protein through GPER /P13K/AKT signaling pathway,through above molecular mechanism influences the process of osteoporosis.