Neuroprotective action of erythropoietin against cell death induced by hypoxia on SHSY5Y cells

WENKER S; CHAMORRO ME; VOTA D; VITTORI D; NESSE A

Buzios, Brasil

Congreso; Congress Ibro/larc of Neurosciences for Latin America, Caribbean and Iberian Peninsula,; 2008

OBJECTIVES: On exposure to hypoxia, brain tissue becomes sensitive to cell injuryand death, which could cause neurodegenerative outcomes. Erythropoietin (Epo) hasemerged as a multifunctional factor that could play a significant role in tissues outsidethe hematopoietic system. We decided to investigate whether Epo might be able toprevent deleterious effects of hypoxia. METHODS: Human neuroblastoma SH-SY5Ycells exposed to hypoxia were used. Treatments with Epo were made before and afterhypoxia injure. Morphological alterations were observed by scanning electronmicroscopy (SEM). Cell viability was determined by MTT assay. Apoptosis wasdetected by DNA laddering and by nuclear fluorescence microscopy (Hoechst).Expression of Bcl-2 members was analyzed by RT-PCR. RESULTS: Hypoxia exposurefor 16 or 24 h caused morphological changes like lost of neuritogenesis and celldetachment. Under this condition, a significantly reduced cell viability dependent on theexposure-length (H16 71±9%, H24 28±15%; P<0.05 with respect to controls; n=4) anda significant increase in cell apoptosis were detected (H16 35±2% vs. controls P<0.05;n=5). These results are in accordance with cell morphology alterations and membraneblebbing observed by SEM. The effects triggered by hypoxia in this undifferentiatedneuroblastoma cell line were totally prevented by a previous treatment with Epo (25U/ml, 9 h) as shown by data of cell viability: H16 71±7%, E9/H16 100±4%; P<0.05;n=5, and percentage of apoptotic cells: H16 28±1%, E9/H16 7±2%; P<0.001; n=5. Apost treatment with Epo up to 48 h after 16 h of hypoxia did not overcome the injures.Cell exposure to hypoxia showed downregulation of RNAm levels of the antiapoptoticmember Bcl-xL (RT-PCR) to near 45±12% of the control levels (P<0.05; n=3), while theprevious treatment with Epo has the opposite effect (Bcl-xL RNAm: 147±12%; P<0.05).CONCLUSIONS: these results demonstrate that short treatments with Epo could notreverse the damages caused by SH-SY5Y cell cultures under low oxygen condition.However, Epo prevented the apoptosis induced by hypoxia, suggesting aneuroprotective action partially dependent on the modulation of Bcl-xL.