Objective: To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI).
Methods: Forty Sprague-Dawley male rats (weight 220-250 g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 μL 3-MA (10 mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110 mmHg for 60 min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting.
Results: The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05).
Conclusions: Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.
Keywords: 3-Methyladenine; Autophagy; Retinal ischemia–reperfusion.