Abstract: Objective To investigate the effects of 3-methyladenine (3-MA) on mouse mesangial cell line MES-13 cultured in high glucose, and on the kidney of streptozotocin (STZ) - induced mouse model of diabetes and the potential mechanism. Methods MES-13 cells were divided into low glucose control group (LG), hyper osmotic pressure control group (HOP), high glucose group (HG), 3-methyladenine+high glucose group (HG+3-MA)and chloroquine+high glucose group (HG+CQ).The groups were respectively incubated with low glucose DMEM, 30 mmol/L mannitol hypertonic control medium, 30 mmol/L high glucose medium, 5 mmol/L 3-MA+30 mmol/L high glucose medium and 10 mmol/L CQ+30 mmol/L high glucose medium for 24 hours. CCK-8 assay and Western blot were performed. In vivo experiment: Male C57BL/6J mice were induced diabetes for model development by intra-peritoneal injection of STZ 60 mg/kg for five consecutive days. After two weeks of injection, the blood glucose was measured. Animals with blood glucose level higher than 16.7 mmol/L (250 mg/dL) were randomly divided into diabetes control group (DM), 3-MA intervention group (DM+3-MA) and CQ intervention group (DM+CQ), then were fed under the same conditions as normal control group (NC) mice. The DM+3-MA group was given 10 mg/kg of 3-MA aqueous solution by gavage every day, the DM+CQ group was given 50 mg/kg of CQ by intraperitoneal injection every three days, the NC group and DM group were given the same amount of normal saline and killed after 6 weeks. The kidneys were stripped for kidney/body weight ratio determination, periodic acid-schiff staining (PAS), MASSON staining microscopy and Western blot. Results In vitro experiment: Compared to the LG group, the cell viability, PCNA expression, ratio of phosphorylated Akt to total Akt (p-Akt/Akt) and ratio of phosphorylated rpS6 to total rpS6 (p-rpS6/rpS6) were significantly increased in the HG group (P＜0.05). Compared with HG group, the cell viability, PCNA and p-Akt/Akt ratio of HG+3-MA group and HG+CQ group were significantly decreased and p-rpS6/rpS6 ratio of HG+3-MA group was significantly decreased(P＜0.01). In vivo experiment: Compared to NC group, the kidney/body weight ratio, glomerular volume, renal tubular injury index, PCNA, fibronectin, COL1A1, p-Akt/Akt, p-rpS6/rpS6 in DM group were all significantly up-regulated(P＜0.05). Compared with DM group, the kidney/body weight ratio, glomerular volume, renal tubular injury index, PCNA, fibronectin, COL1A1, p-Akt/Akt, p-rpS6/rpS6 of DM+3-MA group mice were all significantly decreased(P＜0.05). Conclusions 3-MA can improve glomerular mesangial cell proliferation and renal ECM deposition in early diabetes nephropathy (DN). The improvement of 3-MA in early DN may be related to the inhibition of Akt/rpS6 signaling pathway.

Key words: 3-methyladenine, diabetes nephropathy, mesangial cell, extra cellular matrix