The effects of IV iron preparations on the phosphorylation of p38 MAPK p38 MAPK

HUVEC were cultured in complete endothelial growth medium containing 50 µg/ml of IV iron sucrose or IV FCM for 15 min, 30 min, 60 min, 2, 6 and 24hr.

Antibodies against phosphorylated p38 MAPK were used to detect p38 MAPK phosphorylation in the cell lysates. Results from repeated protein assays showed the protein concentration in the cell lysates from confluent 6 well plates as shown below 0.9 mg/ml (Figure 4.6).

Figure 4.7: Graph of Protein assay standard curve shows the absorbencies and concentrations of the standards. A serial of dilution of BSA in PBS was prepared and absorbance was read at 750 nm. The absorbance of this sample was 0.13 and when it is plotted on the graph, protein concentration showed to be 0.9 mg/ml.

154 concentration of 50 µg/ml for increasing t times (15 min, 30 min, 60 min, 2, 6 and 24hr). All figures are representative of 3 different experiments from three different donors. The band densities were quantified by the ratios of phosphorylated p38 to total p38 MAP kinase expression in the cells using Image J and the results are shown as the mean ratio from 3 different experiments (mean +/- SD) (Figures 4.8B and 4.9B)

HUVEC grown in complete endothelial growth media, were considered as a negative control and showed a less intense band compared with IV iron sucrose or IV FCM-treated HUVEC, These results showed low phosphorylation of p38 MAP kinase in non-treated cells (Figures 4.8A and 4.9A). However, cells grown in endothelial growth media containing 50 µg/ml IV iron sucrose or IV FCM at 15 min-6hr showed obvious increased phosphorylation of p38 MAP kinase compared with non-treated cells (Figures 4.8A and 4.9A). HUVEC grown in media containing 50 µg/ml IV iron sucrose or IV FCM for 15 and 30 min showed a slight increase in phosphorylation of p38 MAP kinase compared with non-treated cells (Figures 4.8A and 4.9A).

However, this increase was not statistically significant (Figures 4.7B and 4.9B), while IV iron sucrose at 60 min and 2hr showed a statistically significant increase in p38 MAPK activation versus non-treated cells (p<0.01) (Figure 4.8B). In addition, p38 MAPK was significantly activated in cells

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treated with IV FCM for 60 min and 2hr compared with non-treated cells (p<0.05) (Figure 4.8B). The p38 MAPK showed to be activated over 6hr, but was not significant compared with non-treated cells and slowly declined (Figures 4.10B and 4.11B). Chapter 3 clearly showed that, IV iron sucrose or IV FCM induced ROS generation in HUVEC. Thus, in order, to investigate whether the phosphorylation of p38 MAPK by IV iron sucrose or IV FCM was ROS dependent. HUVEC were pre-treated with ROS scavenger, NAC (2 mM) and then treated with IV iron sucrose or IV FCM for different time course as described in section (4.2.2.1.6.7). The results showed that in HUVEC treated with NAC and IV iron sucrose or IV FCM at 60 min and 2hr, p38 MAPK phosphorylation was significantly reduced compared with HUVEC treated with sucrose or IV FCM alone at 60 min and 2hr (p<0.05) (Figure 4.10B and 4.11B). P-p38 and total p38 MAPK exposure time under UV using Chemidoc machine was kept constant between experiments.

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Figure 4.8: Shows the effect of IV iron sucrose on activation of p38 MAPK in HUVECsas determined by p38 phosphorylation. HUVECs were cultured in 6 well plates until confluence, then cells were cultured in the absence or presence of 50 µg/ml IV iron sucrose for 15, 30, 60 min, 2hr, 6hr and 24hr and lysed. 30-40 µg protein from each sample was loaded on an SDS-PAGE gel and used in the Western blot. (A): representative blots; the phospho-p38 and total p38 MAPK proteins were detected using specific antibodies and were identified by Western blot as a band of approximately 43 kDa. (B): the phospho-p38/total p38 ratio were quantified by densitometry and expressed as the ratio, compared to the control. All values are expressed as mean (+/- SD) from three different experiments (n=3). **p<0.01 compared with non-treated cells.

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Figure 4.9: Shows the effect of IV FCM on activation of p38 MAPK in HUVECs. HUVECs were cultured in 6 well plates until confluence, then cells were treated in the absence or presence of 50 µg/ml IV FCM for 15, 30, 60 min, 2hr, 6hr and 24hr and lysed. 30-40 µg protein from each sample was loaded on an SDS-PAGE gel and used in the Western blot. (A):

representative blots; the phospho-p38 and total p38 MAPK proteins were detected using specific antibodies and were identified by Western blot as a band of approximately 43 kDa. (B): the phospho-p38/total p38 ratio were quantified by densitometry and expressed as the ratio, compared to the control. All values are expressed as mean (+/- SD) from three different experiments (n=3).*p<0.05 compared with non-treated cells.

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Figure 4.10: Shows the effects of NAC and IV iron sucrose on p38 MAPK phosphorylation in HUVECs. HUVECs were cultured in 6 well plates until confluence, then cells were incubated in the absence or presence of 2 mM NAC and 50 µg/ml IV iron sucrose for 60 min and 2hr, and lysed. 30-40 µg protein from each sample was loaded on an SDS-PAGE gel and used in the Western blot. (A): representative blots; the phospho-p38 and total p38 MAPK proteins were detected using specific antibodies and were identified by Western blot as a band of approximately 43 kDa. (B): the phospho-p38/total p38 ratio were quantified by densitometry and expressed as the ratio compared to the control. All values are expressed as mean (+/- SD) from three different experiments (n=3). **p<0.01 versus non-treated cells and

#p<0.05 for IV iron sucrose and NAC at 60 min and 2hr compared with IV iron sucrose alone at 60 min and 2hr.

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Figure 4.11: Shows the effects of NAC and IV FCM on p38 MAPK phosphorylation in HUVECs. HUVECs were cultured in 6 well plates until confluence, and then cells were incubated in the absence or presence of 2 mM NAC and 50 µg/ml of IV FCM for 60 min and 2hr, and lysed. 30-40 µg protein from each sample was loaded on an SDS-PAGE gel and used in the Western blot. (A): representative blots; the phospho-p38 and total p38 MAPK proteins were detected using specific antibodies and were identified by Western blot as a band of approximately 43 kDa. (B): the phospho-p38/total p38 ratio were quantified by densitometry and expressed as the ratio compared to the control. All values are expressed as mean (+/-SD) from three different experiments (n=3). *p<0.05 versus non-treated cells and

#p<0.05 for IV FCM and NAC at 60 min and 2hr compared with IV FCM alone at 60 min and 2hr .

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4.3.3.1.2. The effect of IV iron preparations on expression of Bcl-2 and