An iron-based beverage, HydroFerrate fluid (MRN-100), alleviates oxidative stress in murine lymphocytes in vitro

Abstract

Background: Several studies have examined the correlation between iron oxidation and H(2)O(2) degradation. The present study was carried out to examine the protective effects of MRN-100 against stress-induced apoptosis in murine splenic cells in vitro. MRN-100, or HydroFerrate fluid, is an iron-based beverage composed of bivalent and trivalent ferrates.

Methods: Splenic lymphocytes from mice were cultured in the presence or absence of MRN-100 for 2 hrs and were subsequently exposed to hydrogen peroxide (H(2)O(2) ) at a concentration of 25 microM for 14 hrs. Percent cell death was examined by flow cytometry and trypan blue exclusion. The effect of MRN-100 on Bcl-2 and Bax protein levels was determined by Western blot.

Results: Results show, as expected, that culture of splenic cells with H(2)O(2) alone results in a significant increase in cell death (apoptosis) as compared to control (CM) cells. In contrast, pre-treatment of cells with MRN-100 followed by H(2)O(2) treatment results in significantly reduced levels of apoptosis.In addition, MRN-100 partially prevents H(2)O(2) -induced down-regulation of the anti-apoptotic molecule Bcl-2 and upregulation of the pro-apoptotic molecule Bax.

Conclusion: Our findings suggest that MRN-100 may offer a protective effect against oxidative stress-induced apoptosis in lymphocytes.

Figure 1

Flow cytometery analysis of MRN-100-treated splenic lymphocyte apoptosis. Splenic lymphocytes were cultured in the presence of H₂O₂, MRN-100, MRN-100 + H₂O₂, or control (CM). The percentage of dead cells was examined by the propidium iodide (PI) technique using flow cytometry. Shown are representative plots from three independent experiments.

Figure 2

Cell death analysis of splenic lymphocytes by trypan blue assay. Splenic lymphocytes were cultured in the presence of H₂O₂, MRN-100, MRN-100 + H₂O₂, or control (CM). At 16 hrs, cells from the different groups were treated with trypan blue stain for 5 minutes and counted. The percent of dead cells was calculated out of a total of 300 cells. * indicates p < 0.02 as compared to cells treated with H₂O₂. Data shown is representative of three independent experiments.

Figure 3

Effect of MRN-100 on Bcl-2 and Bax protein levels. Splenic cells were cultured in the presence of H₂O₂, MRN-100, MRN-100 + H₂O₂, or control (CM). Cell lysates were subjected to Western blot using anti-Bcl-2 or anti-Bax antibodies. Fig. 3A and 3B are representative blots showing Bcl-2 and Bax protein levels, respectively. Lane 1: Control (CM); Lane 2: Splenic cells exposed to H₂O₂; Lane 3: Splenic cells treated with MRN-100; Lane 4: Splenic cells pre-treated with MRN-100 and exposed to H₂O₂. Depicted below the blots is the ratio of Bcl-2/β-Actin (Fig. 3A) or Bax/β-Actin (Fig. 3B), respectively, as calculated using densitometry. Fig. 3C depicts the percent Bcl-2 decrease (calculated using densitometry) as compared to the respective control: H₂O₂ was compared to control (CM), and MRN-100 + H₂O₂ was compared to MRN-100 control. The value for H₂O₂ treatment was calculated by dividing the value of Lane 2 over the Lane 1 from Fig. 3A. The value for MRN-100 + H₂O₂ treatment was calculated by dividing the value of Lane 4 over Lane 3 from Fig. 3A. *highly significant (p < 0.001). Fig. 3D shows the Bcl-2/Bax ratio for each treatment. The Bcl-2 bands were measured using densitometry and were compared to Bax bands (data not shown). The ratio of Bcl-2/Bax is taken from a representative experiment.