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

Abstract

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 α1 chain (COL1A1), vascular endothelial growth factor (VEGF), inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α, 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 short-term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1β and TNF-α in DM mice. 3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA, decreased the expression of fibrosis-related 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.

Keywords: 3-methyladenine; apoptosis; autophagy; diabetic mellitus; fibrosis; mice.

Figure 1. 3-MA had a short-term hypoglycemic effect on FBG in STZ-induced diabetic mice

A: Schematic diagram of animal experimental design; B: Trend graph of weekly FBG; C: Trend chart of 24h water intake per week; D: Serum GSP detected by a commercial biochemical assay kit. All data are represented as mean±SEM, ^aP<0.001 vs the NC group; ^bP<0.05, ^cP<0.001 vs the DM group. FBG: Fasting blood glucose; 3-MA: 3-methyladenine; STZ: Streptozotocin; DM: Diabetes mice model; NC: Normal control; CQ: Chloroquine; SEM: Standard error of mean; GSP: Glycated serum protein.

Figure 2. 3-MA reduced the expression of apoptosis-related proteins and fibrosis-associated proteins in the retina of STZ-induced diabetic mice

A: Retinal tissue lysates were subjected to immunoblot analysis with antibodies to cleaved caspase-3, cleaved PARP1, Bcl-2, Bax, and GAPDH; B: Expression level of cleaved caspase-3 was quantitated by densitometry and normalized with GAPDH; C: Expression level of cleaved PARP1 was quantitated by densitometry and normalized with GAPDH; D: Expression levels of Bcl-2 and Bax were calculated by densitometry and the ratio of Bax/Bcl-2 was determined; E: Retinal tissue lysates were subjected to immunoblot analysis with antibodies to Fibronectin, COL1A1, and GAPDH; F: Expression level of Fibronectin was quantitated by densitometry and normalized with GAPDH; G: Expression level of COL1A1 was quantitated by densitometry and normalized with GAPDH. All data were represented as mean±SEM. ^aP<0.01 and ^bP<0.001 vs the NC group; ^cP<0.05, ^dP<0.01, ^eP<0.001 vs the DM group; ^fP<0.05, ^gP<0.001 vs the DM+3-MA group. PARP1: Poly ADP-ribose polymerase 1; COL1A1: Type 1 collagen α1 chain; 3-MA: 3-methyladenine; STZ: Streptozotocin; DM: Diabetes mice model; NC: Normal control; CQ: Chloroquine; SEM: Standard error of mean; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. 3-MA alleviated oxidative stress, expression of VEGF, and production of inflammatory factors in the retina of STZ-induced diabetic mice

A: Retinal MDA detected by a commercial biochemical assay kit; B: Retinal 8-OHdG detected by a commercial ELISA kit; C: Retina tissue lysates were subjected to immunoblot analysis with antibodies to VEGF, IL-1β, TNF-α, and GAPDH; D: Expression level of VEGF was quantitated by densitometry and normalized with GAPDH; E: Expression levels of IL-1β and TNF-α were quantitated by densitometry and normalized with GAPDH. All data are represented as mean±SEM. ^aP<0.001 vs the NC group; ^bP<0.05, ^cP<0.01, ^dP<0.001 vs the DM group. MDA: Malondialdehyde; 8-OHdG: 8-hydroxydeoxyguanosine; VEGF: Vascular endothelial growth factor; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; 3-MA: 3-methyladenine; STZ: Streptozotocin; DM: Diabetes mice model; NC: Normal control; CQ: Chloroquine; ELISA: Enzyme-linked immunosorbent assay; SEM: Standard error of mean; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 4. 3-MA inhibited the expression of early autophagy related proteins in the retina of STZ-induced diabetic mice

A: Retinal tissue lysates were subjected to immunoblot analysis with antibodies to LC3, Beclin-1, P62, and GAPDH; B: Expression levels of LC3 I and LC3 II were calculated by densitometry and the ratio of LC3 II/LC3 I was determined; C: Expression level of Beclin-1 was quantitated by densitometry and normalized with GAPDH; D: Expression level of P62 was quantitated by densitometry and normalized with GAPDH. All data are represented as mean±SEM. ^aP<0.05, ^bP<0.01, ^cP<0.001 vs the NC group; ^dP<0.05, ^eP<0.01 vs the DM group; ^fP<0.05, ^gP<0.01 vs the DM+3-MA group. LC3: Microtubule-associated protein light chain 3; P62: Sequestosome 1; MDA: Malondialdehyde; 8-OHdG: 8-Hydroxydeoxyguanosine; VEGF: Vascular endothelial growth factor; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; 3-MA: 3-methyladenine; STZ: Streptozotocin; DM: Diabetes mice model; NC: Normal control; CQ: Chloroquine; SEM: Standard error of mean; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.