Mitochondrial Mechanisms Of The Inhibitory Effect Of EPO On Hydrogen Peroxide-induced Human Retinal Pigment Epithelium Cell Apoptosis

[Objective] To explore the effect of cytochrome C and apoptosis-inducing factor in the signal Pathway of apoptosis on oxidative injury of human retinal pigment epithelial cell induced by hydrogen peroxide and to explore the possible protection mechanism of EPO . [Methods] The human retinal pigment epithelium cultured with different level of Hydrogen peroxide for 24hs to establish oxidative injury model. The protective effect of EPO on apoptosis of cultured hRPE cells was assessed by Giemsa staining. Immunocytochemical staining and Western blot were used to determine the expression of AIF and CytC. [Results ] (1) The result of Giemsa stain showed that apoptosis bodies were found in the oxidative injury groups, and the increasing of the apoptotic cells showed dose-dependent of Hydrogen peroxide. While the situation is much better in the EPO treatment groups compared with that in the oxidative injury groups. (2) CytC was expressed in mitochondrion of hRPE cells which showed light brown pigmenting in cytoplasm. Immunohistochemistry staining showed that there was no significant difference between the 200μmol/L injury groups and the control groups(p>0.05) . With the level of Hydrogen peroxide increased, the expression of CytC protein raised. Among it the expression of CytC protein reached the peak at H₂O₂ 800μmol/L injury groups. Significant difference (P<0.01) was showed between the H₂O₂ 800μmol/L group and the control group ,The expression of CytC in therapeutic groups was lower than that of injury ones. No significant difference was got between 200μmol/L subgroups (p>0.05). There was significant difference between others subgroups with the same H₂O₂ level (P<0.01) . AIF was detected in the cytoplasm of normal hRPE cells which showed light brown yellow pigmented. The difference between 200μmol/L injury groups and the normal ones is of no significance (P>0.05). With the level of Hydrogen peroxide increased, the expression of positive cells rised, and showed deep brown yellow pigmented. In 800μmol/L injury groups, the expression of AIF reached the peak, which significantly increased as compared with that in control group (P < 0. 001). Compared with that of injury group, the expression of AIF protein in therapeutic one was significantly lower (P<0.01) .except the difference between 200μmol/L subgroup(P>0.05). (3) Western blot analysis showed that CytC was expressed at a low level in the normal hRPE cells' cytoplasm. The expression of CytC protein of injury group was higher compared with that in the control group (P<0.05). While the expression of CytC in EPO therapeutic group was decreased obviously than that of injury groups (P<0.05) . We could not detect AIF protein in the nucleus of normal hRPE, With the level of Hydrogen peroxide increased, the expression of AIF protein in the nucleus significantly increased. Compared with the injury group, the expression of AIF protein in the nucleus of EPO therapeutic group was significantly lower (P<0.01) . [ Conclusions ] Hydrogen peroxide induced RPE cell apoptosis by mitochondrial pathway. We inferred that EPO protected hRPE from apoptosis by inhibiting the release of CytC , AIF from mitochondrion.