| Objective:Estrogen is a steroid hormone that plays an important role in cardiovascular,immune,nervous and endocrine systems,among which estradiol（17β-estradiol,E2）is the most effective.Estrogen mediates physiological effects and regulates pathological processes mainly by binding to estrogen receptors to form ligands.G protein coupled estrogen receptor（G protein coupled estrogen receptor,GPER）,also known as GPR30,as a new type of estrogen receptor was found has been more than 20 years,in recent years,a large number of studies have shown that GPER involved physiological processes and life activities,including sugar metabolism,lipid metabolism,bone metabolism and so on.In the heart,blood vessels,brain,liver,breast,pancreatic,nerve and reproductive organs,GPER is widely distributed many organs.Autophagy is a process of self-degradation of abnormal or redundant proteins in cells.Autophagy is another form of cell death besides"type 1 programmed death"--apoptosis,also known as"type 2 programmed death".Autophagy guarantees cell homeostasis through organelle renewal and metabolism,and promotes cell adaptation and survival.Mitophagy refers to the autophagy of mitochondria,which plays an important role in clearing damaged and aged mitochondria and maintaining the structure and function of cells.Type 2 diabetes mellitus（T2DM）is a metabolic disorder syndrome caused by both genetic and environmental factors,with the main manifestation of high blood glucose.Its pathogenesis mainly includes insulin resistance and functional defects of islet beta cells.Previous studies have shown that mitophagy plays an important role in maintaining the structure and function of isletβcells,suggesting that the pathogenesis of T2DM may be related to mitophagy of isletβcells.However,the mechanism of mitophagy in isletβcells,the effect of estrogen and its receptor GPER in it,and the signal transduction pathway have not been fully studiedAfter GPER is activated by estrogen,it exerts biological effects in cells through a variety of signaling pathways,including cAMP/PKA pathway,PI3K/Akt pathway,EGFR/ERK pathway,EGFR/MAPK pathway,etc.Among them,cAMP signaling pathway is closely related to cell proliferation,differentiation and apoptosis in endocrine system,and protein kinase A（PKA）is a major effector molecule of cAMP,which can phosphorylate various proteins in cytoplasm and nucleus.At present,a study has proved that the cAMP/PKA signaling pathway is involved in mediating the autophagy activation of esophageal squamous cell carcinoma,but the effect of the cAMP/PKA pathway on mitophagy in islet beta cells and the specific mechanism are not completely clear.Therefore,mouse islet beta cells（Min6 cells）were selected as the model in this study to explore the mechanism of estrogen binding GPER and regulating mitophagy in islet cells through PKA pathway.Methods:Part one:the expression of GPER in Min6 cellsMin6 cells were cultured in vitro and treated with 17β-E2 of different concentrations(10-9M,10-8M,10-7M,10-6M)for 24h.Western blot was applied to detect the expression of GPER and determine the optimal concentration.GPER expression was detected by immunofluorescence after E2 treatment for 24h.Min6 cells were divided into control group,E2 group,E2+GPER antagonist（G15）group and G15 group.After 24 hours of treatment,western blot was used to detect GPER expression.Part two:the effects of E2 activation of GPER on mitochondrial autophagy in Min6 cellsMin6 cells were cultured in vitro and treated with E2 at an optimal concentration of10-7M for 24h.Mitochondrial autophagosomes were observed by transmission electron microscopy.Min6 cells were divided into control group,10-7M E2 group,10-7M E2+GPER antagonist（G15）group and G15 group.After 24 hours of treatment,western blot was applied to detect the expression levels of mitophagy related proteins HSP60,TOM20 and LC3.The co-localization of mitochondrial outer membrane protein TOM20and lysosomal membrane protein LampII was detected by immunofluorescence.Part three:the effects of E2 activation of GPER on mitochondrial autophagy in Min6 cells via PKA signaling pathwayMin6 cells were cultured in vitro and divided into control group,E2 group,G15group,G15+E2 group,PKA pathway inhibitor（H89）group and H89+E2 group.After 24h of treatment,western blot was applied to detect the phosphorylation level of PKA pathway,total protein level of PKA,HSP60,TOM20 and LC3 expression level.Results:Part one:the expression of GPER in Min6 cells1.GPER was expressed in Min6 cells.2.E2 activated GPER in Min6 cells.3.Different concentrations of E2 affected the expression level of GPER in Min6cells,among which the E2 group with 10-7m concentration had statistical significance.G15 can reduce the expression level of GPER.Part two:the effects of E2 activation of GPER on mitochondrial autophagy in Min6 cells1.E2 reduced the formation of mitochondrial autophagosomes in Min6 cells.2.E2 reduced the expression of TOM20 and LampII co-localization in Min6 cells,and G15 attenuated this effect.3.E2 increased the expression of mitochondrial matrix protein HSP60 and mitochondrial membrane protein TOM20,reduced the expression of autophagy marker protein LC3-Ⅱ/LC3-Ⅰin Min6 cells,and G15 can inhibit this effect.Part three:the effects of E2 activation of GPER on mitochondrial autophagy in Min6 cells via PKA signaling pathway1.E2 up-regulates the phosphorylation level of PKA in Min6 cells（p-PKA/PKA）,and both G15 and H89 can inhibit this effect.2.E2 can increase the expression of HSP60 and TOM20 in Min6 cells,reduce the LC3-Ⅱ/LC3-Ⅰexpression,and H89 can inhibit this effect.Conclusion:1.GPER exists in Min6 cells,and E2 can activate GPER.2.E2 inhibits mitophagy in Min6 cells by activating GPER,thus affecting the function of pancreatic islet cells.3.PKA signaling pathway is involved in the regulation of mitophagy of the above-mentioned E2 in Min6 cells and plays an inhibitory role.Intervention of this pathway may be an effective target to improve the function of islet and treat diabetes. |
