Objective: To investigate the roles of DNA double stains damage repairing mechanisms in high glucose-induced cellular senescence.
Methods: Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of glucose (5.5 mmol/L, 11 mmol/L, 22 mmol/L and 33 mmol/L) for 72 hrs before the assay of senescence-associated beta-galactosidase staining. The superoxides were detected by flow cytometry. The levels of NO were detected by enzyme assay. Gamma-H2AX and phosphorylated P53 protein were measured by Western blot. Changes after co-incubation with KU55993 (an inhibitor of ATM) were examined with methods mentioned above.
Results: Compared with control group, percentage of positive cells of senescence-associated beta-galactosidase staining increased significantly in high glucose groups. The corresponding levels of reactive oxygen increased and NO decreased in a concentration-dependent manner. Intra-cellular levels of gamma-H2AX and phosphorylated P53 protein were significantly increased in high glucose groups. Statistical significances were revealed between high-glucose group and control group, as well as among different high-glucose groups, but no significant difference was observed between mannitol and control group. KU55993, an inhibitor of ATM, significantly reduced the levels of gamma-H2AX, phosphorylated P53 protein, and positive rate of senescence-associated beta-galactosidase staining.
Conclusion: High glucose may promote DNA double strains damage by enhancing oxidative stress and decreasing NO, and thus accelerate cellular senescence. ATM-P53 pathway, the key proteins related to DNA double strain damage repairing mechanisms, may play an important role in high glucose induced cellular senescence and atherosclerosis.