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The Effects And Mechanisms Of 17?-estradiol Interferes In Mitophagy Via SIRT1/AMPK Signaling Pathway In ATDC5 Chondrocytes

Posted on:2020-12-22Degree:MasterType:Thesis
Country:ChinaCandidate:R H MeiFull Text:PDF
GTID:2404330596495910Subject:Surgery
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Background Osteoarthritis?OA?is a very common chronic disease that affects all joint tissues throughout the body,causing progressive irreversible damage and ultimately loss of joint function.Among the various pathophysiological mechanisms involved in OA,those mechanisms associated with sex hormone control have attracted much attention,particularly estrogen.Many studies have shown that estrogen appears to have potential protective effects on chondrocytes.However,the mechanism of action of estrogen in osteoarthritis remains controversial.Mitochondria are one of the major metabolic sites in the cell.Mitochondrial autophagy is a selective autophagy that is capable of degrading dysfunctional mitochondria through the autophagic lysosomal system.Autophagy-associated proteins play an important role in the regulation of autophagy,such as microtubule-associated protein-1 light chain-3?LC3?,a marker located on the autophagosomes membrane,which is an important marker of autophagy in cells.In addition,translocase of the outer membrane 20?TOM 20?and heat shock protein 60?HSP60?are proteins involved in mitochondrial autophagySIRT1?silent information regulator 2 related enzyme 1?is a member of the Sirtuins family,which induces expression of target genes involved in stress protection,as well as inhibition of cell cycle arrest and apoptotic genes.SIRT1 is involved in many physiological functions of humans such as DNA repair,senescence,gene expression and apoptosis.AMPK?adenylate-activated protein kinase?is considered to be the primary energy metabolism sensor that is activated to cope with the reduction of cellular energy charge and further regulate energy metabolism homeostasis.Studies have shown that the SIRT1/AMPK pathway has protective effects in ischemic stroke and cardiovascular dysfunction in postmenopausal women with metabolic syndrome,but the effects of estrogen on OA are still rarely studied.Objective Estrogen,especially 17?-estradiol,has been shown to protect chondrocytes from apoptosis.However,whether mitochondrial autophagy is involved in the protective effect of 17?-estradiol on chondrocytes,or whether 17?-estradiol regulates mitochondrial autophagy through its specific nuclear receptors SIRT1,AMPK and its signaling pathway remains unknown.The aim of this study was to elucidate the mechanism by which 17?-estradiol interferes with mitochondrial autophagy in ATDC5chondrocytes via the SIRT1/AMPK signaling pathway.Methods In vitro cultured mouse chondrocyte cell line ATDC5,chondrocytes were treated with increasing concentrations of 17?-estradiol(0M,10-9M,10-8M,10-7M)with or without NAM?SIRT1 antagonist,15?M?,AMPK inhibitor?Compound C,20?M?.The expression levels of SIRT1 and AMPK in ATDC5 cells were detected by Western blot and real-time quantitative PCR with the concentration of 17?-estradiol.The expression and distribution of SIRT1 were determined by immunofluorescence staining.The 17?was observed by transmission electron microscopy?TEM?.In ATDC5chondrocytes,the transmission electron microscopy has used to detect mitochondrial autophagosomes and autophagy treated with or without NAM?SIRT1 antagonist?,Compound C?AMPK inhibitor?.In addition,Western blot can also be used to detect the expression levels of p-AMPK,LC-3,TOM20 and Hsp60 in ATDC5 chondrocytes.Results 1.We found the presence of SIRT1 expression in ATDC5 chondrocytes.17?-estradiol increased the expression levels of SIRT1 mRNA and protein in a dose-and time-dependent manner and reached the highest level at 10-7 M,but NAM was able to inhibit the expression of SIRT1 and was not reversed by 17?-estradiol.IF staining showed that an increase in the expression level of SIRT1 protein in the membrane and nucleus of ATDC5 chondrocytes.2.It was observed by transmission electron microscopy?TEM?that fewer mitochondrial autophagosomes were observed in ATDC5 chondrocytes treated with 10-7M 17?-estradiol for 24 hours.3.10-7M 17?-estradiol can decrease the expression of LC3-II in ATDC5 cells and increase the expression levels of TOM20 and Hsp60 proteins,but add specific inhibitor of SIRT1?NAM??15?M?and AMPK-specific inhibitor Compound C?20?M?can block the above effects of 17?-estradiol.4.Phosphorylation-AMPK protein levels were increased in 17?-estradiol-treated ATDC5 cells,but the opposite results were observed when cells were pretreated with NAM or AMPK inhibitors.Conclusion The expression of SIRT1 mRNA and protein was observed in mouse ATDC5 chondrocytes cultured in vitro,and 17?-estradiol?10-7M?activates the AMPK signaling pathway by enhancing the activity of SIRT1,inhibiting mitochondrial autophagy in ATDC5 chondrocytes.
Keywords/Search Tags:17?-estradiol, SIRT1, AMPK, autophagy, chondrocytes
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