Role of reactive oxygen species in high concentration glucose-induced growth inhibition of human peritoneal mesothelial cells

Abstract

Background: To investigate the effects and mechanism of high concentration glucose (HG), exogenous hydrogen peroxide (H 2 O 2 ), and antioxidants on the cell growth (cell proliferation) of human peritoneal mesothelial cells (HPMCs).Methods: All tests were conducted on cultured HPMCs (HMrSV5) in vitro. Various concentrations of glucose (0.1%, 1.35%, and 3.86%), H 2 O 2 (0.5 and 0.1 mmol/L), and antioxidants (pyruvate and catalase) were used in cell culture. Moreover, in order to study the interaction between these factors, HG and H 2 O 2 , HG and antioxidants, HG, H 2 O 2 , and antioxidants, were used respectively. After 12-24 h, phase-contrast microscopy was used to examine the morphological changes of HPMCs. DNA synthesis was detected by 3 H-thymidine incorporation to measure cell proliferation, and flow cytometry was used to evaluate the proportion of cells in G 1 phase. Furthermore, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to determine the mRNA expression of p21 Waf1 and p27 Kip1 [cyclin-dependent kinase inhibitors (CKIs)], while immunocytochemistry (ICC) and Western blotting were employed to measure the protein expression of p21 Waf1 and p27 Kip1 .Results: HG or low-dose exogenous H 2 O 2 resulted in hypertrophy and senescence of HPMCs, resulting in similar morphological changes. Both HG and exogenous H 2 O 2 (0.5 mmol/L) inhibited the proliferation of HPMCs and led to G1 phase arrest of HPMCs. The proportion of cells in G 1 phase increased. Moreover, HG enhanced the toxic effects of exogenous H 2 O 2 . Both HG and exogenous H 2 O 2 increased the expression of p21 Waf1 and p27 Kip1 . The addition of an antioxidant in HG medium arrested cells in the G 1 phase and improved the inhibited cell proliferation.Conclusions: Both HG and exogenous H 2 O 2 treatments can induce growth inhibition of HPMCs by arresting cell cycle progression, which is partially due to the increased expression of p21 Waf1 and p27 Kip1 . Thus, the effects of HG might be associated with endogenous reactive oxygen species (ROS), and it might be beneficial to use antioxidants in peritoneal dialysis (PD).