Pretreatment with pPolyHb attenuates H₂O₂-induced endothelial cell injury through inhibition of JNK/p38 MAPK pathway by upregulation of heme oxygenase-1

Figures & data

Figure 1. The protective effect of pPolyHb on H₂O₂-treated HUVEC cells. (A) HUVEC cells were treated with the indicated concentrations of H₂O₂ for 2, 4, or 8 h. Cell viability was assessed by the MTT assay. The results are expressed as the mean ± SEM, n = 6, ^#P < 0.05 compared to the control group, ^##P < 0.01 compared to the control group, ^**P < 0.01 compared to the group treated with 200 μM H₂O₂, ^$$P < 0.01 compared to the group treated with 400 μM H₂O₂.(B) HUVEC cells were pretreated in serum-free medium in the presence or absence of pPolyHb (5, 25, 125 μM) for 10 h, followed by exposure to H₂O₂ (400 μM) for an additional 4 h, and then cell viability was assessed by the MTT assay. The results are expressed as the mean ± SEM, n = 6, ^##P < 0.01 compared to the control group, ^**P < 0.01 compared to the group treated with H₂O₂ alone. (C) HUVEC cells were pretreated with the indicated pPolyHb concentrations for 10 h followed by H₂O₂ exposure for an additional 4 h, and cell morphology was observed. Representative images were taken from three independent experiments (magnification, × 20).

Figure 3 Effects of pPolyHb, SnPP, SB203580 and SP600125 on LDH leakage and cell viability in H₂O₂-treated HUVECs. HUVECs were pretreated with the indicated concentrations of pPolyHb for 10 h, or with SB203580 or SP600125 for 2 h, followed by exposure to H₂O₂ (400 μM) for 4 h. For the SnPP group, SnPP (20 μM) was co-incubated with HUVEC cells for 2 h in advance of pPolyHb pretreatment, followed by exposure to H₂O₂ (400 μM) for 4 h. (A) Effects on LDH leakage. (B) Effects on cell viability. ^##P < 0.01 compared to the control, ^**P < 0.01 compared to the group treated with H₂O₂ alone, *P < 0.05 compared to the group treated with H₂O₂ alone.

Figure 4. pPolyHb induces HO-1 expression in a concentration- and time-dependent manner. (A) Cells were treated with 125 μM of pPolyHb for the indicated time (0, 2, 4, 6, 8, 10 h) and HO-1 expression was analyzed by Western blot. (B) Cells were treated for 10 h with the indicated concentrations (0, 125, 50, 25, 5 μM) of pPolyHb or pretreated with SnPP (20 μM) for 2 h, followed by treatment ith 125 μM pPolyHb, and then HO-1 expression was analyzed by Western blot. β-actin was used for normalization. ^**P < 0.01 compared to the control.

Figure 5. Effects of pPolyHb on phosphorylation of JNK and p38 mitogen-activated protein kinase (MAPK) in H₂O₂-treated HUVECs. Cells were cultured in 6-well plates until confluent, and the medium was replaced with serum-free medium and cultured in the presence or absence of pPolyHb (5, 25, and 125 μM) for 10 h. Cells were then treated with 400 μM H₂O₂ for 4 h, followed by lysis and Western blot analysis. Densitometry scanning analysis of p-JNK and p-p38 MAPK phosphorylation was performed. GAPDH was used for normalization. Western blot images are representative of three independent experiments. Data are means ± standard errors (n = 3). ^##P < 0.01 compared to the control, ^**P < 0.01 compared to the group treated with H₂O₂ alone.

Figure 6. SnPP attenuates the inhibitory effect of pPolyHb against H₂O₂-induced phosphorylation of JNK and p38 MAPK in HUVECs. Cells were treated with pPolyHb (125 μM), with or without pretreatment with SnPP (20 μM), followed by H₂O₂ (400 μM) incubation for 4 h. Western blot analysis was performed to determine the phosphorylation and protein expression of JNK and p38 MAPK. Normalization was performed with the anti-GAPDH antibody. Western blot images are representative of three independent experiments. ^##P < 0.01 compared to control, ^**P < 0.01 compared to the group treated with H₂O₂ alone, ^$$P < 0.01 compared to the pPolyHb pretreatment group.

Figure 7. Effects of pretreatment with pPolyHb on intracellular generation of reactive oxygen species (ROS). After 10 h of pretreatment with or without pPolyHb (5, 25, and 125 μM) or pretreatment with SnPP (10 μM) and pPolyHb (125 μM), HUVECs were exposed to H₂O₂ (400 μM) for 4 h. (A) Confocal microscopy images of cells fluorescently stained with DCFH-DA (magnification, × 40, scale bar = 50 μm). The microscopy images are representative of three independent experiments. (B) ROS generation was assayed using DCFH-DA fluorescence measured with a fluorometer (excitation = 485 nm, emission = 535 nm). Data are means ± standard errors (n = 3). ^##P < 0.01 compared to the control; ^**P < 0.01 compared to the group treated with H₂O₂ alone.