AGE-BSA activated NADPH oxidase in HUCLs.

<p>HUCLs were treated with AGE-BSA (200 µg/ml) for 1, 3 and 6 h. (A) NADPH oxidase activity was assessed. (B) Western blot analysis was used to detect p-p47phox expression. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group.</p

JNK and p38 MAPK mediated AGE-BSA-induced apoptosis in HUCLs.

<p>HUCLs were pretreated with JNK inhibitor (SP600125, 20 µM) or p38 MAPK inhibitor (SB203580, 20 µM) for 1 h respectively. Subsequently they were treated with AGE-BSA (200 µg/ml) for 24 h. (A) Bax protein expression was analyzed by Western blot. (B) Apoptosis was analyzed by flow cytometer. HUCLs were pretreated with ROS scavengers NAC (20 µM) for 1 h. Then the cells were stimulated with AGE-BSA (200 µg/ml) for 24 h, followed by Western blot analysis for total and phosphorylated JNK (C) and p38 MAPK (D). Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group; #, <i>P</i><0.05 vs. AGE-BSA group.</p

AGE-BSA induced JNK and p38 MAPK phosphorylation in HUCLs.

<p>HUCLs were incubated with AGE-BSA (200 µg/ml) for 6, 12 and 24 h. The total and phosphorylation of JNK (A) and p38 MAPK (B) were analyzed by Western blot. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group.</p

AGE-BSA induced RAGE expression in HUCLs.

<p>Expression of RAGE protein was determined by Western blot analysis. (A) HUCLs were incubated with AGE-BSA (200 µg/ml) for 6, 12, 24 and 48 h. (B) HUCLs were treated with 50, 100 and 200 µg/ml of AGE-BSA for 24 h. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group.</p

AGE-BSA induced Bax protein expression and inhibited Bcl-2 protein expression in HUCLs.

<p>HUCLs were incubated with AGE-BSA (200 µg/ml) for 6, 12 and 24 h, Western blot analysis of Bax and Bcl-2 levels in the whole cell extracts. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group.</p

AGE-BSA induced apoptosis of cultured HUCLs.

<p>Apoptosis was determined by flow cytometer. (A) HUCLs were incubated with AGE-BSA (200 µg/ml) for 6, 12, 24 and 48 h. (B) HUCLs were treated with 50, 100 and 200 µg/ml of AGE-BSA for 24 h. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group.</p

AGE-BSA increased NADPH-dependent intracellular ROS production in HUCLs.

<p>Intracellular ROS production was assessed using a DCFH-DA fluorescence. (A) HUCLs were treated with AGE-BSA (200 µg/ml) for 3, 6, 12 and 24 h, Hydrogen peroxide (1 mM) was added to cells as a positive control. (B) Intracellular ROS generation was visualized under a laser scanning confocal microscope. Scale bar  = 50 µm. (C) HUCLs were pretreated with DPI (NADPH oxidase inhibitor, 10 µM), apocynin (NADPH oxidase inhibitor, 300 µM), allopurinol (xanthine oxidase inhibitor, 10 µM), rotenone (inhibitor of mitochondrial electron transport complex I, 5 µM) or neutralizing anti-RAGE antibodies (20 µg/ml) for 1 h. and then incubated with AGE-BSA (200 µg/ml) for 12 h. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group; #, <i>P</i><0.05 vs. AGE-BSA group.</p

Intracellular ROS generation was required for AGE-BSA-induced apoptosis in HUCLs.

<p>(A) HUCLs were pretreated with DPI (10 µM) or apocynin (300 µM) for 1 h, and Bax protein expression was analyzed 24 h after AGE-BSA (200 µg/ml) treatment by Western blot. (B) HUCLs were pretreated with DPI (10 µM), apocynin (300 µM), NAC (20 µM) or neutralizing anti-RAGE antibodies (20 µg/ml) for 1 h, and apoptosis was analyzed 24 h after AGE-BSA (200 µg/ml) treatment by flow cytometer. Data are representative of three independent experiments. *, <i>P</i><0.05 vs. medium alone group; #, <i>P</i><0.05 vs. AGE-BSA group.</p

Flow diagram of the participants in the plasma study.

Flow diagram of the participants in the plasma study.</p

Clinical characteristic of patients and control group.

Clinical characteristic of patients and control group.</p