Figure 8. Oxidative stress is involved in the AGEs-RAGE axis-induced inhibition of HSP60 expression in β-cells.

RINm5f cells were treated with AGE-BSA (0.5-50 μg/ml) and non-glycated BSA (50 μg/ml) for 24 hours. A. The levels of cellular H₂O₂ were detected by ELISA. Data are presented as means ± SEM (n ≥ 5). *P < 0.05, versus BSA. B. ROS production was also determined by flow cytometric assay. NAC, N-acetyl-L-cysteine. C. RINm5f cells were treated with AGE-BSA and non-glycated BSA (10 μg/ml) for 24 hours in the presence or absence of RAGE neutralizing antibody. The levels of cellular H₂O₂ were detected by ELISA. D. Effect of antioxidant N-acetyl-L-cysteine (NAC) on HSP60 protein expression in AGE-BSA-treated RINm5f cells. After pretreatment with NAC (2 mM) for 1 hour, the cells were treated with AGE-BSA or non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expression of HSP60 was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Data are presented as means ± SEM (n = 4). *P < 0.05, versus BSA, ^#P < 0.05, versus AGE-BSA. In some experiments, the H₂O₂ productions in islets of db/db and db/m+ mice were measured E.. Data are presented as means ± SEM (n ≥ 10). **P < 0.01, versus db/m+ mice.