Downregulated Recycling Process but Not De Novo Synthesis of Glutathione Limits Antioxidant Capacity of Erythrocytes in Hypoxia

Abstract

Red blood cells (RBCs) are susceptible to sustained free radical damage during circulation, while the changes of antioxidant capacity and regulatory mechanism of RBCs under different oxygen gradients remain unclear. Here, we investigated the changes of oxidative damage and antioxidant capacity of RBCs in different oxygen gradients and identified the underlying mechanisms using an in vitro model of the hypoxanthine/xanthine oxidase (HX/XO) system. In the present study, we reported that the hypoxic RBCs showed much higher oxidative stress injury and lower antioxidant capacity compared with normoxic RBCs. In addition, we found that the disturbance of the recycling process, but not de novo synthesis of glutathione (GSH), accounted for the significantly decreased antioxidant capacity of hypoxic RBCs compared to normoxic RBCs. We further elucidated the underlying molecular mechanism by which oxidative phosphorylation of Band 3 blocked the hexose monophosphate pathway (HMP) and decreased NADPH production aggravating the dysfunction of GSH synthesis in hypoxic RBCs under oxidative conditions.

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“…Previous studies have confirmed that Band 3 has a vital role in maintaining the structural stability and mechanical properties of RBCs by linking phospholipid bilayer and spectral-based skeletal networks ( 23 , 33 ). Band 3 cluster caused by OS is usually accompanied by a decrease in the deformability of RBCs ( 19 , 34 ).…”

“…ab150077; Abcam). After being washed three times in PBS, fluorescence images were captured using an Olympus IX71 fluorescence microscope (Olympus Corporation) ( 23 ).…”

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“…P0006; Beyotime Institute of Biotechnology). Western blot analyses were performed as previously described ( 20 ). Briefly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by heating the samples for 8 min at 100˚C and loading 10 µg membrane proteins on to a 5-15% linear acrylamide gradient gel (10 µg protein/lane).…”

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