A major finding of the present study was that ghrelin may provide retinal neuronal protection by inhibiting neuronal autophagy and apoptosis in COH retinas, which is supported by the following evidence. First, ghrelin treatment rescued the IOP elevation-induced increase in protein levels of the autophagy-related proteins LC3-II/I and beclin1 (
Fig. 5). During physiological conditions, autophagy plays an important role in maintaining cellular homeostasis. However, impaired autophagy could contribute to neurodegenerative disorders.
42–45 Usually, beclin1 and LC3-II are considered biomarkers of ongoing autophagy; they are involved in the nucleation and assembly of the isolation membrane and/or phagophore elongation step during autophagosome formation.
46 LC3-II, a lipidated form of LC3, is generated from the modification of LC3-I (a cytosolic form of LC3) and is the only protein marker that is reliably associated with completed autophagosomes.
47 Additionally, we found that ghrelin treatment rescued the decreased activity of the Akt/mTOR signaling pathway in COH retinas, further confirming the involvement of autophagy inhibition in the effects of ghrelin (
Fig. 6). Second, ghrelin treatment significantly reduced the IOP elevation-induced increase in the protein levels of the cleaved caspase3, and the number of TUNEL-positive cells in COH retinas (
Fig. 5). Apoptosis is the culmination of a complex series of molecular events, including activation of caspases in injured cells.
48 Caspase3 plays a central role in the execution phase of cell apoptosis, which is activated by both intrinsic and extrinsic apoptotic pathways. Caspase3 zymogen has no activity until it is cleaved into 17-kDa and 12-kDa subunits, which are considered to be markers of apoptosis.
49 The anti-apoptotic activity of ghrelin has been reported in numerous of pathophysiological conditions, such as ischemia, inflammation, and nutrient deprivation.
50–53 In an experimental glaucoma model, ghrelin treatment reduces the TUNEL positivity in COH retinas by inhibiting the levels of oxidation products in anterior chamber fluid, suggesting that the anti-apoptotic effect of ghrelin is mediated by acting on the anterior segment of the eye.
22 It should be noted that ghrelin treatment did not completely rescue the IOP elevation-induced increase in protein levels of the cleaved caspase3, suggesting that neuronal apoptosis in COH retinas involves multiple paths. Our results also showed that ghrelin treatment induced an increase in protein levels of GHSR-1a (
Fig. 6). Because GHSR-1a is localized to RGCs, it is possible that the elevated expression of GHSR-1a may provide a direct protective role for RGCs against apoptosis during ghrelin treatment. Consistent with this possibility, we observed that ghrelin treatment increased the survival of RGCs in COH retinas (
Fig. 5).