Autophagy Can Alleviate Oxidative Demage Induced By High Glucose In Lens Epithelial Cells

BackgroundDiabetic cataracts remain the main cause of visual impairment and blindness in diabetic patients.Some studies have pointed out that the increase of free radical levels is the most critical factor in the development of diabetic cataracts,and it has been newly reported that autophagy also involves the formation of diabetic cataracts,but the interrelationship between the two in the process of diabetic cataract formation is not complete.clear.OBJECTIVETo detect the expression of autophagy-related factors(LC3B,P62),antioxidant enzyme catalase CAT and superoxide dismutase SOD2(Mn SOD)in lens epithelial cells(LECs)of diabetic mice.And the mouse lens epithelial cells were cultured in vitro by autophagy enhancer and autophagy inhibitors to investigate the effect of autophagy on oxidative stress of autophagic lens epithelial cells under high glucose conditions.MethodsA total of 80 male C57BL/6 mice of the same batch of 6-8 weeks were used.50 mice of type I diabetes were induced by continuous low-dose intraperitoneal injection of streptozotocin(STZ)(50 mg/Kg/d).In the experimental group,the remaining 30 mice were intraperitoneally injected with a suitable dose of citrate buffer as a control group.After 3 months,the fasting blood glucose in the experimental group was detected.Transmission electron microscopy was used to observe the morphological changes of autophagosome in lens epithelial cells in mice of the experimental group and the control group;immunohistochemistry was used to detect LC3 B,P62 protein,and CAT and SOD2 in lens anterior capsule tissue of two groups of mice.Expression and localization;Fluorescence quantitative PCR was used to detect the m RNA expression of LC3 B,P62,CAT and SOD2 in lens anterior capsule of two groups;Western blot was used to compare autophagy and anti-oxidation related protein LC3 B in lens anterior capsule of the two groups.P62,relative expression of CAT and SOD2.Anterior lens capsules of male C57BL/6 mice were taken for primary culture in vitro for 6-8 weeks and divided into 4 groups: normal glucose group(NG,5 m M),high glucose group(HG,30 m M)and high sugar added.Enhancer group(HG+RAPA).The levels of autophagy-related proteins LC3 B and P62 and oxidative stress-related proteins CAT and SOD2 were detected by Western blot.The fluorogenic probe Mito SOX Red detects three groups of mitochondrial oxidative stress levels.RESULTSTransmission electron microscopy revealed that the volume of autophagosomes in the lens epithelial cells of the experimental group was greater than that of the control group,and that multiple mitochondria were contained in the lens epithelial cells.Immunohistochemistry showed that compared with the control mice The expression of LC3 B and P62 protein in the anterior lens capsule of the experimental group was enhanced,but the expression of SOD2 protein was decreased.The relative expression of LC3 B and P62 m RNA in the lens anterior capsule of the experimental group was high in the experimental group.In control mice(1.10±0.05 vs.2.62±0.15;1.01±0.01 vs.1.89±0.20),the relative expression of SOD2 m RNA was lower than that of control mice(1.00±0.00 vs.0.53±0.08).All were statistically significant(t=14.25,P<0.05;t=6.14,P<0.05;t=8.00,P<0.05);western blot showed that the relative expression of LC3 B and P62 protein in the experimental group was higher than that of the control group.(1.00±0.00 vs.1.24±0.09;1.00±0.00 vs.3.19±1.04),the difference was statistically significant(t=6.10,P<0.05;t=3.65,P<0.05),while the expression of SOD protein was Lower than the control group(1.00±0.00 vs.0.63±0.05),the difference was statistically significant(t=11.88,P<0.05).In lens epithelial cells of mice cultured in vitro,we found that the ratio of LC3II/LC3 I increased,the content of P62 protein increased,but the SOD2 and CAT protein levels decreased(all P<0.05)under high glucose conditions,and treated with rapamycin.After the cells,the ratio of LC3II/LC3 I further increased,the level of P62 decreased,and the levels of SOD2 and CAT proteins increased(both P<0.05).The Mito SOX Red fluorescence probe showed that the mitochondrial ROS level was higher in the high glucose group than in the normal glucose group.Compared with high glucose group,mitochondrial ROS level in rapamycin group was decreased.ConclusionAutophagy and anti-oxidation proteins are abnormal in lens epithelial cells of diabetic mice.Autophagy dysfunction and oxidative damage may all play an important role in the formation of diabetic cataract;a certain degree of autophagy upregulation may reduce high glucose induction.The oxidative stress level of the lens epithelial cells thus inhibits their damage,and the in-depth study of their interaction mechanism has important clinical and social significance for the prevention and treatment of diabetic cataracts.