Effects of autophagy inhibitor 3-methyladenine on a diabetic mice model

AIM: To investigate the role of autophagy inhibitor 3-methyladenine (3-MA) on a diabetic mice model (DM) and the potential mechanism. METHODS: Male C57BL/6J mice were randomly divided into a normal control group (NC group) and an DM group. DM were induced by multiple low-dose intraperitoneal injection of streptozotocin (STZ) 60 mg/kg.d for 5 consecutive days. DM mice were randomly subdivided into untreated group (DM group), 3-MA (10 mg/kg. d by gavage) treated group ( DM+3-MA group) and chloroquine (CQ; 50 mg/kg by intraperitoneal injection) treated group (DM+CQ group). The fasting blood glucose (FBG) levels were recorded every week. At the end of experiment, retinal samples were collected. The expression levels of pro-apoptotic proteins cleaved caspase-3, cleaved poly ADP-ribose polymerase 1 (PARP1) and Bax, anti-apoptotic protein Bcl- 2, fibrosis-associated proteins Fibronectin and type 1 collagen a1 chain (COL1A1), vascular endothelial growth factor (VEGF), inflammatory factors interleukin (IL)-1 beta and tumor necrosis factor (TNF)-a, as well as autophagy related proteins LC3, Beclin-1 and P62 were determined by Western blotting. The oxidative stress indicators 8- hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) were detected by commercial kits. RESULTS: Both 3- MA and CQ had shor t- term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1 beta and TNF-alpha in DM mice. 3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA, decreased the expression of fibrosisrelated proteins Fibronectin and COL1A1, pro-apoptotic proteins cleaved caspase- 3, cleaved PARP1, as well as the ratio of Bax/Bcl-2. CQ had no significant impact on the oxidative stress indicators, fibrosis, and apoptosis related proteins. The results of Western blotting for autophagy related proteins showed that the ratio of LC3 II/LC3 I and the expression of Beclin-1 in the retina of DM mice were decreased by 3-MA treatment, and the expression of P62 was further increased by CQ treatment. CONCLUSION: 3-MA has anti-apoptotic and anti-fibrotic effects on the retina of DM mice, and can attenuate retinal oxidative stress, VEGF expression and the production of inflammatory factors in the retina of DM mice. The underlying mechanism of the above effects of 3-MA may be related to its inhibition of early autophagy and hypoglycemic effect.