Table 1.
List of primers for qPCR analysis.
Fig 1.
Rat LIPC sera increased the cells’ viability of H2O2-injured HUVECs.
HUVECs were pretreated with 5% different sera for 12 h and followed by 2 h incubation with 1 mMH2O2. Cells’ viability was measured by MTT assay and expressed as the ratio of optical density (OD) with OD value of the control as 100%. The data (mean ± SEM) were obtained from at least three independent experiments, **P < 0.01 vs model. Control: cultured with normal medium without any intervention throughout the experiment. Model: cultured with normal medium for 12 h and then incubated with 1mM H2O2 for 2 h. NPS, EPS and DPS: cultured with normal medium containing either 5% NPS (serum derived from rats after sham LIPC), 5% EPS (serum derived from rats 20 min after LIPC) or 5% DPS (serum derived from rats 24 h after LIPC) respectively for 12 h and followed by 2 h incubation with 1mM H2O2. The figure legends are same in following figures unless illustrated elsewhere.
Fig 2.
Rat LIPC sera decreased ROS in H2O2-injured HUVECs.
ROS level was determined by measuring the intensity of dihydroethidium (DHE) fluorescence. The relative fluorescence intensities of DHE were analyzed by Image-Pro Plus software taking the fluorescence intensity of the control as 100%. A: original images of the cells preloaded with DHE. B: pooled data are expressed as mean ± SEM, n = 5, **P < 0.01 vs model.
Fig 3.
Rat LIPC sera reduced MDA and increased the activities of antioxidases in H2O2-injured HUVECs.
Medium MDA concentration was measured spectrophotometrically at 532 nm. The activities of CAT, GSH-Px and total SOD were analyzed spectrophotometrically at 240 nm, 340 nm and 550 nm respectively. All data are expressed as mean ± SEM, n = 6, *P < 0.05, **P < 0.01 vs model.
Fig 4.
Rat LIPC sera upregulated the mRNA expression of antioxidases in HUVECs.
CAT, SOD-1, SOD-2 and GSH-Px-1 mRNA levels in HUVECs were detected by real-time PCR. The mRAN expression of antioxidases was presented by normalizing the antioxidases expression with GAPDH and taking control as 100%. All data are expressed as mean ± SEM, n = 6, *P < 0.05, **P < 0.01 vs model.
Fig 5.
Rat LIPC sera enhanced Nrf2 localization into nucleus inH2O2-injured HUVECs.
Localization of Nrf2 was performed by immunofluorescence and confocal microscopy. Nrf2 was stained with an anti-Nrf2 antibody and visualized with a secondary antibody conjugated with FITC (green). The nuclei were counterstained with DAPI (4’,-diamidino-2-phenylindole) staining indicating the location of the nucleus (blue). The merged image showed the nuclear location of Nrf2 protein.
Fig 6.
Effects of rat LIPC sera on Nrf2 translocation and cellular viability were affected by neither PI3K/Akt inhibitor nor MEK/ERK inhibitor in H2O2-injured HUVECs.
The cells were cultured in medium for 24 h, after which the inhibitors U0126 (U0, 26 μM) and LY294002 (LY, 25 μM) were added 1 h before the especial serum and H2O2 treatments. A: nucleus localization of Nrf2 determined by immunofluorescence and confocal microscopy. B: cells’ viability detected using MTT method. The data (mean ± SEM) were obtained from at least three independent experiments, **P < 0.01 vs model.