Effects Of Mitophagy In MC3T3-E1 Osteoblasts Induced By 17β-estradiol Via G Protein-coupled Estrogen Receptor And AMPK/mTOR Signaling Pathway

Objective:Osteoarthritis is a chronic joint disease which is characterized by chondral degenerative change,synovitis and secondary periarticular osteoproliferation.It is the most frequent form of arthritis and a major cause of pain and disability worldwide,especially in middle-age and older adults.With the aged and overweight tendency of population worldwide,the incidence of osteoarthritis and the accompanied disability continue to increase.Therefore,the interest in research into osteoarthritis pathogenesis and its prevention is increasing.A published study has shown that osteoblast dysregulation and dysfunction play a critical role in osteoarthritis pathogenesis,maintaining the normal physiological function of osteoblasts is the key to prevent the progression of osteoarthritis.Estrogen can reduce the apoptosis of osteoblasts and maintain their normal physiological functions through a variety of molecular mechanisms,which may be one of the mechanisms of estrogen in the prevention and treatment of osteoarthritis.Some estrogen receptors such as ERα、ERβ、GPR30 are expressed in osteoblasts.As a kind of membrane estrogen receptor,GPR30 has been proved to be involved in regulating autophagy in a variety of cells.The normal physiological function of osteoblasts also depends on an appropriate level of autophagy.Previous studies have found that AMPK/m TOR signaling pathway can be involved in regulating mitophagy in osteoblasts.At present,it is still unclear whether estrogen can regulate mitophagy in osteoblasts through GPR30 and AMPK/mTOR signaling pathway so as to protect osteoblasts.Therefore,the purpose of this study was to make sure whether estrogen induces mitophagy through GPR30 and AMPK/mTOR signaling pathway in MC3T3-E1 osteoblasts and elucidate the molecular mechanism.Methods:MC3T3-E1 osteoblasts were cultured in vitro and processed with different concentrations of 17β-estradiol,G15（a selective GPR30 antagonist）and compound C（an AMPK inhibitor）were also used to process the cells.RT-PCR technique and Western blot analysis were applied to detect the expression of GPR30 mRNA and protein.Immunofluorescence analysis was used to detect the expression and distribution of GPR30.In addition,Western blot analysis can also be used to detect the expression levels of LC3、Tom20、Hsp60、AMPK and mTOR.And the mitochondrial autophagosomes in MC3T3-E1 osteoblasts were observed by transmission electron microscopy analysis.Results:1.There is endogenous expression of GPR30 in MC3T3-E1 osteoblasts.17β-estradiol can improve the expression levels of GPR30 and the effect is strongest when the estradiol concentration is 10^-7M.G15,a GPR30 antagonist,can block the promoting effect of 17β-estradiol on GPR30 expression.2.17β-estradiol can improve the expression levels of mitophagy related proteins such as LC3、Tom20 and Hsp60 in MC3T3-E1 osteoblasts.G15 can block these promoting effects.3.Transmission electron microscopy analysis shows that more mitochondrial autophagosomes were observed in MC3T3-E1 osteoblasts treated with 17β-estradiol.4.17β-estradiol enhances the phosphorylation level of AMPK and reduces the phosphorylation level of mTOR in MC3T3-E1 osteoblasts.Compound C,an AMPK antagonist,can reduces the phosphorylation level of AMPK which enhanced by17β-estradiol.Conclusion:17β-estradiol can promote the expression of GPR30 in MC3T3-E1osteoblasts,and 17β-estradiol can induce mitophagy in MC3T3-E1 osteoblasts through GPR30 and AMPK/mTOR signaling pathway.