Effect of C-peptide on advanced glycation end products-induced oxidative stress in rat mesangial cells

Abstract

Objective To investigate the effect of C-peptide on advanced glycation end products (AGE)-induced oxidative stress in rat mesangial cells and its mechanism. Methods Rat mesangial cells were cultured in the normal medium (Control group), or medium with 200 mg/L AGEs (AGEs group), or medium with 200 mg/L AGEs and 5 μmol/L C-peptide (AGEs+C-peptide group) or medium with 10 μmol/L H89 (added in advance, and incubated for 30 min) and 200 mg/L AGEs and 5 μmol/L C-peptide (AGEs+C-peptide + H89 group). The intracellular reactive oxygen species (ROS) was detected with fluorescence method, and the supernatant nitric oxide (NO) level was detected by Griess reaction. Real-time PCR and Western blotting were used to detect the expression of the receptor for advanced glycation endproducts (RAGE), protein kinase A (PKA), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and inducible nitric oxide synthase (iNOS). Non-parametric Kruskal-Wallis H test and Mann-Whitney U test were used to compare data between groups, and two groups, respectively. Results Compared with control group, the level of ROS and NO increased in AGEs group (193.7±6.4 vs 136.1±4.9; and 27.2±4.7 vs 15.5±0.7, respectively, all U=0, P<0.05). C-peptide could suppress the production of ROS and NO. In AGEs+C-peptide group, ROS and NO reduced than AGEs group (136.9±14.3 vs 193.7±6.4 and 16.0±2.1 vs 27.2±4.7 respectively, all U=0, P<0.05). Compared with control group, AGEs could up-regulate the expression of RAGE (0.565±0.027 vs 0.148±0.006,0=0,P<0.05) but down-regulate PKA (0.085±0.035 vs 0.518±0.019, U=0, P<0.05). And AGEs increased the expression of NOX4 and iNOS (0.912±0.055 vs 0.105±0.012, and 0.279±0.003 vs 0.126±0.004 respectively, all U=0, P<0.05). Compared with AGEs group, C-peptide down-regulated RAGE (0.159±0.003 vs 0.565±0.027,0=0,P<0.05), up-regulated PKA (0.594±0.079 vs 0.085±0.035, U=0, P<0.05), and down-regulated NOX4 and iNOS (0.085±0.005 vs 0.912±0.055, and 0.071±0.016 vs 0.279±0.003 respectively, all U=0, P<0.05) Compared with C-peptide group, H89 groupinhibited the activation of PKA, increased the production of ROS and NO (195.7±9.3 vs 149.7±11.7 and 22.2±1.1 vs 16.4±2.1, all U=0, P<0.05). Moreover, compared with C-peptide group, the expression of NOX4 and iNOS in H89 group was increased (0.455±0.018 vs 0.085±0.005 and 0.296±0.013 vs 0.071±0.016 respectively, all U=0, P<0.05). Conclusion C-peptide inhibited oxidative stress in mesangial cells through PKA signaling pathway. Key words: C-peptide; Mesangial cells; Oxidative stress; Protein kinase A; Advanced glycation end products