| Objective:Autophagy is a process in which cells undergo self-digestion of excessive or abnormal components in the hunger and stress to obtain nutrients to maintain homeostasis.It is widely found in eukaryotic cells.Growth and physiological and pathological processes.To a certain extent,autophagy facilitates the recycling of proteins and organelles by cells,and plays an important role in maintaining the normal function and survival of cells;however,if autophagy exceeds a certain limit,cells can also be self-digested and important organelles.Excessive degradation and death.At present,molecular biologists and cell biologists are still in the initial stage of autophagy and autophagic programmed cell death.There are few studies on autophagy in China,and they are mainly limited to autophagy in tumors and neurodegeneration.There are no reports of autophagy on osteoblast research in the study of the role of lesions and cardiovascular diseasesIn the microenvironment of bone metabolism,hormonal regulation,growth factor regulation and the nervous system together form a neuro-endocrine-immuno-local growth factor regulatory network,involved in the entire regulation of bone growth and bone resorption.Among them,17?-E2 has an important effect in maintaining normal bone mass and regulating bone metabolism.Many studies have shown that estrogen deficiency can lead to abnormalities in osteoblastogenesis and function.Estrogen exerts different physiological and pathological effects in various tissues and organs of the human body.The biological effects of estrogen are mainly mediated by the estrogen receptors ERa(estrogen receptora)and ER?(estrogen receptor?),which are located in the nucleus and act as transcription factors in combination with estrogen response elements located in the promoter region of the target gene.In recent years,a novel estrogen receptor-G protein-coupled estrogen receptor(GPER)has been discovered by molecular cloning.GPER is a seven-transmembrane G-protein coupled receptor that mediates the rapid non-genomic effects of estrogen,indirectly regulates gene expression,and exerts various biological functions.GPER is widely distributed in tissue cells of multiple systems,such as reproductive system,circulatory system,nervous system,immune system,urinary system and so on.Other studies have confirmed that GPER is overexpressed in a variety of cancer tissues,including breast cancer,endometrial cancer,ovarian cancer,prostate cancer and lung cancer.At present,there is no conclusion about the subcellular localization of GPER.Some studies suggest that GPER is localized on the cell membrane;others also believe that GPER is localized in the cytoplasmic endoplasmic reticulum and Golgi;and studies on tumor-associated fibroblasts.,the presence of GPER was detected on the nucleus of CAFs).Activation of GPER activates phosphatidylinositol 3 kinases/phosphorylated protein kinase B(PI3K/AKT)signaling pathway,causing intracellular calcium mobilization and phosphatidylinositol triphosphate in the nucleus.The synthesis of(PIP3)activates AKT kinase and promotes cell proliferation.Estrogen regulates gene expression and mechanical tone response of osteoblasts and prevents bone loss through antioxidant action,while its lack of upregulation of osteoclasts.The PI3K-AKT-mTOR signaling pathway is dephosphorylated and inactivated in this process.The inhibition of mitogen-activated protein kinase(MAPK)and PI3K-AKT-mTOR signaling pathway is beneficial to autophagy.Therefore,it can promote the osteogenic differentiation of human mesenchymal stem cells(hMSCs)PI3K/AKT is a ubiquitous and important signal transduction pathway in cells,which has close relation to cell proliferation,differentiation,apoptosis and autophagy.In the pathway,PI3K,AKT,mammalian target of rapamycin(mTOR)and phosphatase and tensin homolog(PTEN)are important acting molecules.PI3K is divided into type ?,type? and type ?,in which type ? PI3K activation can inhibit autophagy;type ? PI3K activation can promote autophagy,while type ? PI3K has little relationship with autophagy.Current research suggests that stress conditions such as starvation,hypoxia,and stress all cause a decrease in AKT activation,induce autophagy,and thereby inhibit cell proliferation until cell death.Studies have found that X-ray radiation may promote autophagy in breast cancer cells by inhibiting the activity of PI3K/AKT signaling pathway.The study also found that during the process of caffeine-induced apoptosis,a large number of autophagosomes appeared,the expression of autophagy-related protein LC3? increased,and the expression of activated AKT and p70S6K was significantly reduced.This suggests that caffeine promotes autophagy by inhibiting the expression of proteins involved in the PI3K/AKT signaling pathway,leading to apoptosis,autophagic programmed cell death.Type ? PI3K also plays an important role in the process of autophagy,which can directly act on mTOR to activate autophagy.mTOR is a key enzyme regulating autophagy and is divided into two forms,mTORC1 and mTORC2 mTORC1 is blocked by sirolimus and regulates autophagy;mTORC2 does not directly regulate autophagy.The homologous gene of mTOR in human is FRAP1(FK506 binding protein rapamycin associated protein 1),which can accept various signals such as Class ?,PI3K,IGF-1/2,MAPK,and the regulation of mTOR signaling pathway and autophagy Extracellular nutritional status is closely related to changes in nutrition and energy Based on the research status at home and abroad and the results of previous studies,we believe that estrogen can maintain osteoblast survival and inhibit apoptosis by regulating mitochondrial autophagy in osteoblasts.At the same time,this effect may be achieved by GPER,which regulates the PI3K/AKT pathway.At present,there is no report on whether estrogen can regulate osteoblast mitochondrial autophagy through GPER intervention in PI3K/AKT pathway.Therefore,this study will explore the mechanism by which 17?-estrogen(17?-E2)inhibits autophagy of osteoblasts and inhibits apoptosis,and whether it is mediated by GPER.At the same time,it also explores the regulation of 17?-E2 by GPER on PI3K/AKT pathway,and clarifies 17?-E2 through GPER Regulation of PI3K/AKT pathway inhibits autophagy of osteoblasts and inhibits osteoblast apoptosis,thereby increasing the survival rate of osteoblasts and providing a more reliable theoretical basis for clinical application of 17?-E2METHODS:Part ?:Intervention of 17?-E2 and GPER on mitochondrial autophagy in MC3T3-E1 osteoblasts1.Effect of 17?-E2 on the expression of GPER mRNA in osteoblasts of MC3T3-E1 mice MC3T3-E1 mouse osteoblasts were treated with increasing concentrations of 17?-E2,such as(0,10-9 M,10-8M,10-7 M,10-6 M).Real-time quantitative PCR was used to detect the expression of GPER mRNA in osteoblasts of MC3T3-E1 mice.2.Effect of 17?-E2 on the expression of GPER protein in MC3T3-E1 cells Mouse osteoblast MC3T3-E1 was treated with different concentrations of 17?-E2(0,10-9 M,10-8M,10-7 M)and/or GPER inhibitor G15(15?M)respectively.Western blot was used to detect the relationship between GPER protein expression and estrogen concentration changes3.Expression of GPER protein in MC3T3-E1 cells was detected by immunofluorescence staining4.The effect of 17?-E2 on autophagosomes in mitochondria of MC3T3-E1 cells was observed by transmission electron microscopyThe osteoblasts of MC3T3-E1 mice were treated with 17?-E2 or GPER inhibitor G15(15?M).The effect of 17?-E2 on the morphological changes of autophagy in MC3T3-E1 cells was studied by transmission electron microscopy5.The effect of 17?-E2 on the number of autophagic lysosomes in MC3T3-E1 cells was observed by immunofluorescence double stainingPart ?:17?-E2 affects the activity of autophagy-associated protein in MC3T3-E1 osteoblasts by GPERMC3T3-E1 mouse osteoblasts were treated with 17?-E2(0,10-7 M),and the activities of autophagy-related proteins of LC3-?,TOM20 and Hsp60 were detected by Western blot.In addition,in the mouse osteoblast cell MC3T3-E1,17?-E2(0,10-7 M)and/or GPER specific inhibitor(G15,15 ?M)and PI3K specific inhibitor LY294002(20 ?M)were added respectively.Western blot was used to detect the activity of autophagy-related proteins of LC3-?,TOM20 and Hsp60.Part ?:Molecular mechanism of 17?-E2 regulating mitochondrial autophagy in MC3T3-E1 osteoblasts1.Western blot analysis of the relationship between 17?-E2 and Akt and mTOR protein activity in MC3T3-E1 cellsDetection of the effect of 17?-E2 on the activation of Akt and mTOR in MC3T3-E1 osteoblastsMC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7 M)for 24 h.The protein activation of Akt and mTOR were detected by western blot.Furthermore,MC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7 M)and/or the GPER inhibitor(G15,15?M)and the PI3K inhibitor(LY294002,20?M)and the mTOR inhibitor(0.1 ?M).The protein activation of Akt and mTOR were detected by western blot.2.Western blot analysis of the relationship between 17?-E2 and p38 protein activity in MC3T3-E1 cellsDetection of the effect of 17?-E2 on the activation of p38 in MC3T3-E1 osteoblasts MC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7 M)for 24 h.The protein activation of p38 was detected by western blot.Furthermore,MC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7 M)and/or the GPER inhibitor(G15,15?M)and the p38 inhibitor(10?M).The protein activation of p38 was detected by western blot.3.Western blot analysis of the relationship between 17?-E2 and JNK protein activity in MC3T3-E1 cellsDetection of the effect of 17?-E2 on the activation of JNK in MC3T3-E1 osteoblasts MC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7M)for 24 h.The protein activation of JNK was detected by western blot.Furthermore,MC3T3-E1 osteoblasts were treated with 17?-E2(0,10-7 M)and/or the GPER inhibitor(G15,15?M)and the JNK inhibitor(10?M).The protein activation of JNK was detected by western blot.Results:1.Effects of 17?-E2 and GPER on mitophagy in MC3T3-E1 osteoblasts Real-time PCR assays demonstrated that GPER mRNA was expressed in MC3T3-E1 osteoblasts.17?-E2 induced a significant increase of GPER mRNA levels and GPER protein levels in a dose-dependent manner.10-8M and 10-7M 17?-E2 induced a significant increase of GPER mRNA levels,with the highest level at 10-7 M.17?-E2(10-7M)induced a significant increase of GPER protein levels.The GPER inhibitor(G15,15?M)can inhibit these effects of estrogen.Compared with the control group,the 17?-E2 could increase the number of GPER labeled red fluorescent particles in MC3T3-E1 cytoplasm and increase the number ofGPER protein in the cytoplasm.Compared with the control group,the 17?-E2(10-7 M)significantly reduced the numberof autophagosomes in the cytoplasm of MC3T3-E1.Compared with the control group,the 17?-E2 could reduce the number of fluorescent particles double stained with TOM20 cytoplasmic red fluorescence and LAMP2 green fluorescence in MC3T3-E1 cytoplasm,and reduce the number of mitochondria autophagic lysosomes in the cytoplasm2.Effects of 17?-E2 on the activation of autophagy-related protein in MC3T3-E1 osteoblasts via GPERWe further examined the activation of LC3-?,TOM20 and Hsp60 with western blot analysis.When the cells were treated with 17?-E2(10-7 M),the activation of LC3-? was significantly decreased,while the activation of TOM20 and Hsp60 were significantly increased.Furthermore,the GPER inhibitor(G15,15?M)and the PI3K inhibitor(LY294002,20?M)can inhibit these effects of 17?-E2.3.The mechanism of 17?-E2 and GPER on mitophagy in MC3T3-E1 osteoblasts When MC3T3-E1 osteoblasts were treated with 17?-E2(10-7 M),the activation of Akt was up-regulated.The GPER inhibitor(G15,15 ?M)and the PI3K inhibitor(LY294002,20?M)can inhibit these effects of 17?-E2.When MC3T3-E1 osteoblasts were treated with 17?-E2(10-7 M),the activation of mTOR was up-regulated.The GPER inhibitor(G15,15?M)and the mTOR inhibitor(0.1 ?M)can inhibit these effects of 17?-E2.These results indicate that 17?-E2 could inhibit mitophagy in MC3T3-E1 osteoblasts through the PI3K-Akt-mTOR signaling pathway.When MC3T3-E1 osteoblasts were treated with 17?-E2(10-7 M),the activation of p38 and JNK were not significantly changed,demonstrating that 17?-E2 could not inhibit mitophagy in MC3T3-E1 osteoblasts through the p38 or the JNK signaling pathways.Conclusions:1.GPER mRNA and protein exist in MC3T3-E1 osteoblasts.17?-E2 improved the mRNA levels of GPER,and increased the expression level of GPER protein,and the role is related to 17?-E2 dose.2.17?-E2 and the GPER receptor decreased the formation of mitophagosomes in the cytoplasm of MC3T3-E1.Therefore,17?-E2 could inhibit mitophagy of MC3T3-E1 osteoblasts through GPER.3.When MC3T3-E1 osteoblasts were treated with 17?-E2,the activation of LC3-? was significantly decreased,while the activation of TOM20 and Hsp60 were significantly increased.Furthermore,the GPER inhibitor and the PI3K inhibitor can inhibit these effects of 17?-E2.Therefore,17?-E2 could inhibit mitophagy of MC3T3-E1 osteoblasts through GPR30 and PI3K signaling pathway4.17?-E2 could up-regulated the activation of Akt and mTOR,while have no significant effects on the activation of p38 and JNK,demonstrating that 17?-E2 may inhibit mitophagy of MC3T3-E1 osteoblasts through GPER via the PI3K-Akt-mTOR signaling pathway. |
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