Effect of bone marrow-derived mesenchymal stem cells on endotoxin-induced oxidation of plasma cysteine and glutathione in mice

Abstract

Bone marrow-derived mesenchymal stem cells (BMDMSC) are emerging as a therapeutic modality in various inflammatory disease states, including acute lung injury (ALI). A hallmark of inflammation, and a consistent observation in patients with ALI, is a perturbation in the systemic redox environment. However, little is known about the effects of BMDMSC on the systemic redox status. The objective of the present study was to determine whether exogenously infused BMDMSC protect against endotoxin-induced oxidation of plasma cysteine (Cys) and glutathione (GSH) redox states. To determine the effect on the redox state if BMDMSC, mice received endotoxin intraperitoneally (1 mg/kg), followed by intravenous infusion of either 5 × 10(5) BMDMSC or an equal volume of saline solution. Control mice received intraperitoneal endotoxin followed by 5 × 10(5) lung fibroblasts given intravenously. Cys, cystine (CySS), GSH, and glutathione disulfide (GSSG) concentrations were determined by HPLC. Results showed sequential preservation of plasma Cys and GSH levels in response to BMDMSC infusion. The data show that BMDMSC infusion leads to a more reducing Cys and GSH redox state. The findings are the first to demonstrate that BMDMSC have antioxidant effects in vivo, and add to our understanding of the systemic effects of BMDMSC in lung injury.

Figure 1

Characterization of BMDMSC by flow cytometry and differentiation capacity. Flow cytometry of BMDMSC demonstrated that cells did not express the hematopoietic markers, CD11b and CD45 ((a)-(b); blue; BMDMSC; grey; isotype control, red; alveolar macrophages). BMDMSC stained positive for Sca-1 ((c); blue; BMDMSC; grey; isotype control). In (d)–(f), cells were examined for differentiation capacity. Fat droplets in cell preparations incubated in adipogenic medium for 14 days were stained with Oil Red O (d). Cells incubated with osteogenic medium for 21 days were stained Alizarin red to detect Calcium (e). Cartilage pellets were stained with Alcian blue to detect mucopolysachharides, after 14 days in chondrogenic medium (f).

Figure 2

Effect of BMDMSC infusion on endotoxin-induced inflammatory response. C57BL/6J mice were treated with 1 mg/kg i.p LPS followed by i.v infusion of either 500,000 BMDMSC (LPS/BMDMSC), or 0.1 mL Saline (LPS/Saline) alone. At 2 hours, 6 hours, and 24 hours mice were sacrificed and plasma was collected for analysis of TNF-α (a), IFN-γ (b), and IL-1β (c). Data are expressed as means + SEM. ^aValues in LPS/BMDMSC group significantly different from time-matched LPS/Saline group.

Figure 3

Effect of BMDMSC infusion on endotoxin-induced weight loss. C57BL/6J mice were treated with 1 mg/kg i.p LPS or with saline. LPS-treated animals were either given intravenous Saline (LPS/Saline) or BMDMSC (LPS/BMDMSC). Saline treated mice were given intravenous BMDMSC. Body weight was measured at 0 hours and 24 hours post treatment.

Figure 4

Effect of BMDMSC infusion on endotoxin-induced depletion of plasma GSH and oxidation of GSH/GSSG redox state. C57BL/6J mice were treated with 1mg/kg i.p LPS followed by i.v infusion of either 500,000 BMDMSC (LPS/BMDMSC), Lung fibroblasts (LPS/Lung fibroblast), or 0.1 mL Saline (LPS/Saline) alone. At 2 hours, 6 hours, and 24 hours mice were sacrificed and plasma was collected for HPLC analysis of GSH (a) and GSSG (b).   E _h GSH/GSSG (c) was calculated from the GSH and GSSG concentrations using the Nernst equation. Data are expressed as means + SEM. ^aValues in LPS/BMDMSC group significantly different from time-matched LPS/Saline group. ^bValues in LPS/BMDMSC group significantly different from time-matched LPS/Lung fibroblast group.

Figure 5

Effect of BMDMSC infusion on endotoxin-induced depletion of plasma Cys and oxidation of Cys/CySS redox state. C57BL/6J mice were treated with 1 mg/kg i.p LPS followed by i.v infusion of either 500,000 BMDMSC (LPS/BMDMSC), Lung fibroblasts (LPS/Lung fibroblast), or 0.1 mL Saline (LPS/Saline) alone. At 2 hours, 6 hours, and 24 hours mice were sacrificed and plasma was collected for HPLC analysis of Cys (a) and CySS (b). In (c), E _h Cys/CySS was calculated from the Cys and CySS concentrations using the Nernst equation. Data are expressed as means + SEM. ^aValues in LPS/BMDMSC group significantly different from time-matched LPS/Saline group. ^bValues in LPS/BMDMSC group significantly different from time-matched LPS/Lung fibroblast group.