A Study On The Roles Of Carbamylated Erythropoietin In Hypoxia Injury

Partâ… Preparation and Identification of Carbamylated ErythropoietinObjective:To prepare carbamylated erythropoietin(CEPO),which has no hematopoietic bioactivity in vivo.Methods:The EPO was carbamylated to CEPO by the reaction of EPO and potassium cyanate.The product of CEPO digested with endoproteinase Lys-C was identified by using sodium dodecly sulfate polyacrylamide gel electrophoresis(SDS-PAGE).For evaluation of the CEPO hematopoiesis,the KM male mice were examined by intraperitoneal injection of EPO,CEPO and normal saline 3 times a week,respectively.The numbers of red blood cells(RBC) and reticulocyte(RET)numbers as well as per cent of RET were accounted at day 3,day7 and day 14.In addition,the C57/B6 mice also were tested by intraperitoneal injection of EPO,CEPO and normal saline every other day with double doses,respectively. The hematopoietic indexes were measured at day 30.Results:The electrophoretic pattern indicated that the carbamylated erythropoietin was prepared successfully and had a high yield(74%) Our results demonstrate that EPO dose dependently promoted the generation of RBC,RET,RET%,but CEPO did not in KM mice and C57/B6 mice.Conclusion:The EPO was successfully transferred to CEPO by the reaction of carbamylation,which loses the ability of hematopoiesis.Partâ…¡The role of carbamylated erythropoietin in mouse hypoxia model1.The establishment of the model of cerebral hypoxia in miceObjective:To elucidate the effect of hypoxia in learning and memory as well as the loss of hippocampus neurons.Methods:Mice were exposed to hypoxia(8%O₂) in a special box for 0.5h,1.5h,3h,and 6h,respectively.Mice under normoxic condition were used for controls.We utilized Y maze to test the role of hypoxia in learning and memory.In addition,NeuN stain was used to detect the loss of hippocampus neurons after hypoxia.Results:Y maze test demonstrated a time dependent learning ability deficit after hypoxia,especially in hypoxia 3h and 6h group.When tested 30 days after reoxygen,Y maze test showed that the mice in hypoxia 6h group had a significant higher error number in 10 tests.However,the number of NeuN positive neurons in CA1 was significantly decreased after hypoxia 6h/reoxygenation 24h,as compared to the normoxia group.At 72h after reoxygen,the neuron loss was more severe in all regions of the hippocampus,especially in hypoxia 6h group.Conclusion: Under 6h hypoxia(8%O₂),we observe the deficit of learning and memory ability and loss of hippocampus in mice.2.The protective role of carbamylated erythropoietin in mouse hypoxia modelObjective:To study whether the carbamylated erythropoietin still remains the capability of neuroprotective on hypoxia-induced cerebral injury in mice.Methods: In this experiment,we choose these mice that can pass the Y maze test.At 6h after hypoxia,EPO,CEPO and normal saline were i.p.injected,respectively.Mice under normoxic condition were used for controls.By using Y maze test,the error number, total react time and active avoidance response were recorded at 10d and 30d after reoxygen.NeuN stain was used to detect the loss of hippocampus neurons.The number of proliferative cells labeled with BrdU was counted.Simultaneously,BrdU was double stained with DCX,NeuN,GFAP and F4/80 to identify the type of proliferated cells.Results:Y maze test showed that both CEPO and EPO can improve the ability of learning and memory compared to saline group which was declined significantly at 10d and 30d after hypoxia.The number of NeuN positive neurons decreased significantly in CA1 in normal saline group than in EPO and CEPO group.Both EPO and CEPO induced BrdU positive cells in SVZ and DG area under both normoxia and hypoxia at 3d after hypoxia.Double staining of BrdU and DCX showed that proliferated cells were neural progenitor cells.At 14d after hypoxia, BrdU positive cells migrated into dentate gyrus,which was double stained with NeuN, indicating the generation of mature neurons.The number of BrdU positive cells stained with F4/80 in corpus callosum was also higher in EPO and CEPO group than in normal saline group at 7d after hypoxia.Conclusion:CEPO,Like EPO,protects the hypoxia-induced cerebral injury,enhances the neurogenesis and the differentiation and may influence microglia proliferation in mouse brain following hypoxia. Partâ…¢The protective effects of carbamylated erythropoietin on the cells undergoing oxygen deprivation1.The protective effects of carbamylated erythropoietin on the neurons undergoing oxygen and glucose deprivationObjective:To confirm the effect of carbamylated erythropoietin in anti-apoptosis and neurogenesis via the neuron OGD model.Methods:LDH assay was adopted to detect cytotoxicity at 1.5 and 3h after OGD.Apoptosis and death cells were analysis by TUNNEL staining and flow cytometry.Cell viability was measured after OGD by MTT.Results:Cytotoxicity was increased at 1.5 or 3h after OGD,and was lower in CEPO group after 3h of OGD followed by 4h of reoxygenation compared to controls. The percent of cell apoptosis and death was significant lower in both CEPO and EPO group by using TUNNEL staining and flow cytometry after 3h of OGD followed by 4h of reoxygenation.Cell viability significantly decreased after 3h of OGD,but both CEPO and EPO increased cell viability at 24h after reoxygen.Conclusion:CEPO remains the ability of anti-apoptosis and cell proliferation.2.The effect of carbamylated erythropoietin on the mircoglia undergoing oxygen deprivationObjective:To determine the effect of CEPO on inflammatory cytokines released from microglia undergoing oxygen deprivation.Methods:Microglia were applied onto oxygen deprivation 3h,and inflammatory cytokines IL-6 and IL-1βwere checked in culture supernatants by ELISA kit.Results:1L-6 but not IL-βhad a time dependent increase after oxygen deprivation in cultured microglia.CEPO can significantly inhibit the release of IL-βand IL-6 at 24h after oxygen deprivation.Conclusion: CEPO significantly reduces the release of inflammatory cytokines(IL-6 and IL-1β) from cultured microglia undergoing oxygen deprivation.Partâ…£The protective mechanism of carbamylated erythropoietinObjective:To elucidate the underlying mechanism of carbamylated erythropoietin in anti-apoptosis and neurogenesis.Methods:Expression of JAK-2 was identified by immunocytochemistry staining,while expression of MAPK phosphorylation was determined by Western-blot.We also defined the role of PI3K/Akt on cell proliferation by LY294002,an inhibitor of PI3K/Akt activity.Results:The immunocytochemistry staining showed that CEPO did not stimulate JAK-2. Compared to the normoxia group,the expression of ERK,JNK and P38 phosphorylation was higher in OGD group.EPO can significantly reduce phosphorylated JNK and P38,whereas CEPO had no effect on MAPK pathway. CEPO also promoted cell proliferation significantly by BrdU staining,the pretreatment with LY294002,an inhibitor of PI3K/Akt,obviously abolished the proliferative ability of CEPO.Conclusion:The neuroprotective ability of CEPO probablely though PI3K/Akt,but not JAK-2,MAPK pathway.Conclusion1.In contrast to EPO,carbamylated erythropoietin has no hematopoietic bioactivity.2.Hypoxia(8%O₂) 6h results in deficit of learning and memory and loss of hippocampus neurons in mice.CEPO,Like EPO,protects from hypoxia-induced cerebral injury(learning and memory as well as neuron loss in hippocampus), enhances neurogenesis and cell differentiation,and may influence microglia functions in vivo hypoxia brain.3.In vitro neuron OGD model,CEPO exhibits the ability of anti-apoptosis and cells proliferation.CEPO significantly reduces the release of inflammatory cytokines (IL-6 and IL-1β) from cultured microglia undergoing oxygen deprivation.4.The neuroprotective ability of CEPO probablely though PI3K/Akt but not JAK-2 or MAPK pathway.