Abstract
Purpose :
The unclear pathogenesis of glaucoma limits the progress of treatment . This study aims to explore the role of AMPK/mTOR pathway and the potential molecular mechanisms using experimental glaucoma models, so as to provide a new therapeutic target 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 was used to explore the cell death rate at multiple time points. Western blot, 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 levels of AMPK, phosphorylated AMPK (p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), PERK, ATF4, CHOP, GRP78 and apoptosis-related proteins were detected by WB.<br type="_moz" />
Results :
The level of p-AMPK slightly increased and the p-mTOR level obviously up-regulated in both OGD/R and retinal I/R models. The level of Bcl-2 decreased, the levels of pro-apoptotic proteins cleaved-caspase 3 and Bax were increased.
Metformin and acadesine (AICAR) are the activators of AMPK, both increased the level of p-AMPK, and inhibited the level of p-mTOR. Both up-regulated the level of anti-apoptotic protein Bcl-2, down-regulated the expression levels of Bax and cleaved-caspase 3, and decreased the rate of TUNEL positive cells. AMPK inhibitor Compound C and AMPK siRNA inhibited the activation of p-AMPK, promoted the expression level of p-mTOR, down-regulated the expression level of Bcl-2, up-regulated the expression levels of Bax and cleaved-caspase 3, and increased the rate of TUNEL positive cells.
The activation of AMPK/mTOR pathway inhibited the expression of p-PERK, ATF4, CHOP and GRP78 proteins induced by OGD/R. Compound C reversed the effect on the above proteins that by inhibiting the AMPK/mTOR pathway. 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.
Conclusions :
Activation of AMPK/mTOR pathway can protect against apoptosis by inhibiting PERK/ATF4/CHOP pathway in experimental glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.