Metformin Protects Against Cell Apoptosis By Activating AMPK/mTOR Pathway In Experimental Glaucoma

Purpose:Glaucoma is the leading cause of irreversible blindness worldwide,characterized by the progressive loss of the visual field and optic nerve damage.At present,there is no effective therapy for treating and stopping the progression of glaucoma.Metformin,as a classic hypoglycemic drug,more attention has been focused on its effects other than hypoglycemic.This study aims to explore the role of metformin in experimental glaucoma models and the potential molecular mechanisms,so as to provide a new therapeutic strategy for the treatment of glaucoma.Methods:The Oxygen-glucose deprivation/recovery(OGD/R)model on the R28 cell line and the ischemia/reperfusion(I/R)model on the retina were established to simulate the glaucomatous injury.Propidium iodide(PI)staining and Cell Counting Kit-8(CCK-8)were used to explore the effects of metformin on cell death and retinal damage induced by the experimental glaucoma models at multiple time points and concentrations.Western blot(WB),TUNEL detect kit and flow cytometry were used to detect apoptosis related indicators.The retinal apoptosis was detected by the co-staining of immunofluorescence and TUNEL.By using agonists,inhibitors,and gene silencing interventions,the expression levels of AMP-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(m TOR),phosphorylated m TOR(p-m TOR)and apoptosis-related proteins were detected by WB.Agonists and inhibitors interventions were used to explore the relationship between AMPK/m TOR pathway and PERK/ATF4/CHOP pathway,and the relationship between the latter and apoptosis.WB was used to detect the levels of AMPK,p-AMPK,m TOR,p-m TOR,pancreatic kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP),glucose-regulated protein 78(GRP78)and apoptosis-related proteins.TUNEL staining was used to detect apoptosis.Results:1.Metformin inhibited the cell apoptosis and relieved the retinal damage induced by the OGD/R and retinal I/R model.It increased the expression level of anti-apoptotic protein,decreased the expression levels of pro-apoptotic proteins and the cell apoptosis rate.Metformin improved the thickness of retinas,increased the number of retinal ganglion cells(RGCs)and decreased the TUNEL positive RGCs.2.Both metformin and acadesine(AICAR)increased the expression level of p-AMPK induced by OGD/R and retinal I/R,and inhibited the expression of p-m TOR.AICAR up-regulated the level of anti-apoptotic protein Bcl-2,down-regulated the expression levels of pro-apoptotic proteins Bax and cleaved-caspase 3,and decreased the rate of TUNEL positive cells.AMPK inhibitor Compound C and AMPK si RNA reversed the metformin’s effect,inhibited the activation of p-AMPK,promoted the expression level of p-m TOR,down-regulated the expression level of anti-apoptotic protein Bcl-2,up-regulated the expression levels of pro-apoptotic proteins Bax and cleaved-caspase 3,and increased the rate of TUNEL positive cells.3.Metformin inhibited the expression of p-PERK,ATF4,CHOP and GRP78 proteins induced by OGD/R.Compound C reversed the effect of metformin on the above proteins.PERK activator CCT020312 increased the expression levels of p-PERK,ATF4,CHOP,GRP78,Bax and activated caspase 3 proteins,inhibited the expression of Bcl-2,and increased the rate of TUNEL positive cells.However,the effect of metformin on the expression of p-AMPK,AMPK,p-m TOR and m TOR protein was not affected by using CCT020312.Conclusion:Metformin can protect against apoptosis by inhibiting PERK/ATF4/CHOP pathway via activating the AMPK/m TOR pathway in experimental glaucoma.