Abstract:

Objective   To prepare carbamylated erythropoietin(CEPO)， and to investigate protective effects and possible mechanisms of CEPO and EPO on SH-SY5Y cells injured by hydrogen peroxide(H2O2).  Methods   After EPO was carbamylated by reaction with potassium cyanate, CEPO was prepared and identified by using sodium dodecly sulfate polyacrylamide gel electrophoresis(SDS-PAGE), which could not be digested by endoproteinase Lys-C. In order to observe their protective effects against oxygen-derived free radical damage, different concentrations of CEPO and EPO were added to the SH-SY5Y cell culture after exposure to 300 and 400μmol/L of H2O2 for 12 hours. Then living cellswere measured by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide(MTT) and lactate dehydrogenase, apoptosis was measured by Annexin V/PI immunofluorescent staining and laser scanning confocal microscopy, and Bcl-2 and Caspase-8 were tested by RT-PCR. Results   The electrophoretic pattern showed that EPO was successfully prepared and had a high purity． Both EPO and CEPO supplementation groups displayed a significant increase in MTT and a decrease in LDH.  Also, there were striking decreases in apoptosis and expression of Caspase-8 and an increase in expression of Bcl-2. There was no difference between the effects of EPO and CEPO. When concentrations of EPO and CEPO reached 100IU/mL， the dose-dependent protection of EPO was maximized． Conclusion   Both CEPO and EPO can evidently protect SH-SY5Y cells against oxygen-derived free radical damage， and there is no difference between them. The dose-dependent protective effects of CEPO and EPO are increased with the concentration．

Key words:  Carbamylated erythropoietin; Erythropoietin; Hydrogen peroxide; SH-SY5Y cells; Apoptosis