Erythropoietin Protects Erythrocytes Against Oxidative Stress-Induced Eryptosis In Vitro

Excessive eryptosis has been found in maintained hemodialysis or peritoneal dialysis patients. Signaling of triggering eryptosis includes oxidative stress, increased cytosolic Ca2+-activity, and ceramide. Erythropoietin (EPO) possesses the property of an antioxidant. The aim of this study was to investigate the ability of hydrogen peroxide (H2O2) on erythrocytes in vitro, and to assess the possible effects of recombinant human erythropoietin (rhEPO) on eryptosis.

One percent erythrocyte suspension was cultured in vitro in three kinds of media: Control group (Group C), H2O2 group (Group H), and EPO group (Group E). Erythrocytes were sampled at 24 hours and 60 hours. Phosphatidylserine (PS) was estimated with annexin-V, reactive oxygen species (ROS) with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA) and cytosolic Ca2+ activity ([Ca2+]i) with Fluo3.

Eryptosis in Group C increased as the incubating time extended (2.05 ± 0.06 at 24 hours, and 10.00 ± 0.08 at 60 hours). Eryptosis increased in Group H compared with Group C (10.86 ± 0.06 at 24 hours, p < 0.01; 12.46 ± 0.14 at 60 hours, p < 0.01, respectively), while it decreased in Group E compared with Group H (8.80 ± 0.08 at 24 hours, p < 0.01; 11.29 ± 0.04 at 60 hours, p < 0.01, respectively). Meanwhile, ROS increased in Group H compared with Group C (9.37 ± 0.04 versus 5.49 ± 0.09 at 24 hours, p < 0.01;19.82 ± 0.05 versus 13.51 ± 0.10 at 60 hours, p < 0.01). [Ca2+]i increased in Group H compared with Group C (10.91 ± 0.12 versus 2.53 ± 0.06 at 24 hours, p < 0.01;14.55 ± 0.05 versus 4.63 ± 0.08 at 60 hours, p < 0.01). ROS decreased in Group E compared with Group H (6.80 ± 0.05 at 24 hours, p < 0.01; 16.82 ± 0.06 at 60 hours, p < 0.01). [Ca2+]i decreased in Group E compared with Group H (7.63 ± 0.14 at 24 hours, p < 0.01; 10.72 ± 0.07 at 60 hours, p < 0.01).

Our research showed eryptosis was triggered by H2O2 and paralleled by increased ROS and [Ca2+]i which was partially reversed by EPO. It indicated that EPO could protect erythrocytes against oxidative stress-induced eryptosis.