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

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).