促红细胞生成素氨甲酰化衍生物在缺氧性脑损伤中的作用研究
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摘要
第一部分促红细胞生成素氨甲酰化衍生物的制备及鉴定
目的:制备不具有促红细胞生成作用的氨甲酰化促红细胞生成素(erythropoietin,EPO)衍生物(carbamylated erythropoietin,CEPO)。方法:用氰酸钾使EPO氨甲酰化,对氨甲酰化EPO经赖氨酸C端蛋白酶(endoproteinase Lys-C)酶切产物进行电泳鉴定。分别给予成年KM小鼠EPO、CEPO和生理盐水每周三次腹腔注射,于3d、7d和14d动态测定动物血红细胞(RBC)、网织细胞(RET)以及网织细胞百分数(RET%)。另外,利用成年C57/B6小鼠,EPO、CEPO和生理盐水剂量增加一倍时间延长至30d后测定各组动物上述的血细胞指标。结果:经酶解电泳鉴定,氨甲酰化为CEPO不能被赖氨酸C端蛋白酶水解,并且有74%的得率。KM小鼠接受EPO注射后,RBC数随给药次数的增加而显著增加,而接受CEPO和NS组均未见有意义的改变。当给药剂量和时间增加一倍后,EPO显著增加了C57/B6小鼠RBC的数目,CEPO组的红细胞数仍未增加。结论:EPO经氨甲酰化反应后,成功地转化为CEPO,并丧失了促红细胞生成的能力。
第二部分促红细胞生成素氨甲酰化衍生物抗低氧神经损伤的在体观察
一.低氧小鼠模型的建立
目的:研究低氧处理对小鼠学习和记忆功能的影响,及对小鼠海马区神经元的损伤与程度。方法:使用小鼠专用缺氧箱,分别给予小鼠8%氧气0.5h、1.5h、3h和6h。同时,设置常氧组小鼠作为对照。各组小鼠于复氧后1d起同时进行连续6d的Y迷宫训练,记录每天20次训练中的错误反应次数。以上各组小鼠在复氧30d再次进行Y迷宫检测,记录10次测试中的错误反应次数和总反应时间。另外,采用NeuN染色检测低氧处理复氧24h和72h小鼠海马神经元脱失情况。结果:Y迷宫结果显示:小鼠吸入8%氧气后学习能力明显下降,以3h和6h组为明显。低氧6h组在复氧后30d测试中错误反应次数最高。复氧24h后CA1区NeuN阳性神经元即有明显脱失,以低氧6h组最明显。复氧72h后,低氧处理各组海马各亚区神经元均有明显脱失,以低氧6h组最为明显。结论:吸入8%氧气6h将严重损伤小鼠海马神经元,降低小鼠学习和记忆能力。
二.促红细胞生成素氨甲酰化衍生物对低氧脑损伤保护作用的研究
目的:研究CEPO对低氧所致小鼠海马损伤的保护作用。方法:给予已学会Y迷宫的小鼠8%氧气6h后即刻给予EPO、CEPO和生理盐水,隔日一次腹腔注射,设置常氧组小鼠作为对照。于复氧10d、30d重复进行小鼠Y迷宫测试,记录小鼠在10次测试中的错误反应次数、总反应时间和主动回避率。分别以BrdU、DCX、NeuN、GFAP、F4/80免疫组化染色,观察EPO组、CEPO组及生理盐水组复氧72h后脑海马神经元脱失、增殖,星型胶质细胞和小胶质细胞的情况。结果:复氧第10d、30d的Y迷宫测试显示,CEPO组和EPO组不仅错误反应次数均明显低于NS组,还具有较高的主动凹避率。NeuN染色提示低氧后给予CEPO、EPO组的海马神经元脱失均少于NS组。常氧及低氧处理后小鼠给予CEPO或EPO均可明显地提高海马DG区以及SVZ区BrdU阳性细胞数。应用免疫组化多重染色显示:随时间推移,CEPO治疗组的DG区新生神经元分化为成熟神经元,同时也促胼胝体区小胶质细胞增生的作用,也具有激活星型胶质细胞的作用。结论:CEPO可保护低氧所致学习和记忆的损伤;减少海马神经元脱失;促DG区神经前体细胞增殖,并分化成为成熟神经元。同时也有促进小胶质细胞增生分化的作用。
第三部分促红细胞生成素氨甲酰化衍生物对缺氧损伤保护作用的离体研究
一.促红细胞生成素氨甲酰化衍生物对缺糖、缺氧神经元损伤的保护作用
目的:在体外培养的神经元OGD模型上观察CEPO的抗凋亡和促细胞增殖的作用,并初步探讨其机理。方法:测定体外培养的神经元OGD 1.5h或3h复氧后不同时间点细胞LDH释放率,比较OGD 3h后给予EPO、CEPO或未干预组的细胞损伤情况。采用TUNEL、流式细胞仪Annexin V/PI染色进一步验证OGD 3h复氧4h时各组细胞凋亡和死亡情况。以MTT法测定OGD对细胞生存率的影响,并比较OGD 3h后各干预组细胞生存率。以BrdU标记增殖细胞,检测OGD 1.5h复氧48h后各组细胞增殖情况。以免疫组化法检测并比较各组细胞JAK-2表达;以免疫印迹法检测OGD后各组MAPK通路磷酸化蛋白的表达情况;以及采用PI3K/Akt途径抑制剂LY-294002预处理后,计数并比较OGD1.5h复氧48h后各组的BrdU阳性细胞数。结果:OGD1.5h或3h后细胞LDH释放率明显增高,OGD 3h复氧4h后CEPO和EPO组细胞死亡率较对照组明显下降。TUNEL法和Annexin V-FITC/PI流式细胞仪计数,均验证OGD3h复氧4h后CEPO干预组细胞凋亡和死亡率较对照组明显下降。MTT法的结果显示CEPO组可显著提高复氧24h后的细胞生存率。OGD1.5h复氧48h后,CEPO及EPO组BrdU阳性细胞显著高于对照组及常氧组。JAK-2免疫组化染色显示CEPO的神经保护没有激活JAK-2通路。免疫印迹法检测显示OGD后MAPK途径的磷酸化ERK、JNK、P38表达量均有明显增高;EPO可以显著降低JNK和P38的磷酸化,CEPO对上述磷酸化蛋白的表达无明显影响。BrdU计数也显示,CEPO促进OGD后的细胞增殖,在给予PI3K/Akt抑制剂LY-294002后完全被阻断。结论:体外神经元OGD模型证实,CEPO可减轻OGD所致细胞损伤,促神经元增殖,其作用不通过JAK-2和MAPK途径,而可能通过PI3K/Akt途径。
二.促红细胞生成素氨甲酰化衍生物对无氧小胶质细胞的作用
目的:研究CEPO对小胶质细胞无氧损伤后炎性因子分泌的影响。方法:培养原代小胶质细胞并鉴定。给予小胶质细胞无氧处理3h,检测复氧后各时间点及加入CEPO、EPO后各组炎性因子IL-6和IL-1β的分泌情况。结果:无氧处理后小胶质细胞IL-6分泌明显增高,IL-1β增高不明显。给予CEPO后复氧24h各组IL-6和IL-1β均较无氧组明显下降,而EPO组无明显下降。结论:CEPO可以明显减少缺氧后小胶质细胞炎性因子IL-6和IL-1β的分泌。
第四部分促红细胞生成素氨甲酰化衍生物神经保护作用的可能机制探讨
目的:在体外培养的神经元OGD模型上初步探讨CEPO作用机理。方法:以免疫组化法检测并比较各组细胞JAK-2表达;以免疫印迹法检测OGD后各组MAPK通路磷酸化蛋白的表达情况;以及采用PI3K/Akt途径抑制剂LY-294002预处理后,计数并比较OGD1.5h复氧48h后各组的BrdU阳性细胞数。结果:JAK-2免疫组化染色显示CEPO的神经保护没有激活JAK-2通路。免疫印迹法检测显示OGD后MAPK途径的磷酸化ERK、JNK、P38表达量均有明显增高;EPO可以显著降低JNK和P38的磷酸化,CEPO对上述磷酸化蛋白的表达无明显影响。BrdU计数也显示,CEPO促进OGD后的细胞增殖,在给予PI3K/Akt抑制剂LY-294002后完全被阻断。结论:体外神经元OGD模型证实,CEPO作用不通过JAK-2和MAPK途径,而可能通过PI3K/Akt途径。
结论
1.EPO的氨甲酰化衍生物——CEPO,完全不具有促红细胞生成的作用。
2.给予8%氧气6h将严重损伤小鼠海马神经元,CEPO可保护低氧所致学习和记忆的损伤,减少海马神经元脱失,促进DG区神经前体细胞增殖分化成为成熟神经元,还具有促小胶质细胞分化的作用。
3.CEPO具有减轻OGD所致神经细胞损伤,促神经元增殖的作用,可以明显减少无氧后小胶质细胞炎性因子IL-6和IL-1β的分泌。
4.CEPO作用机制可能通过PI3K/Akt途径,而可能非JAK-2、MAPK途径。
PartⅠPreparation and Identification of Carbamylated Erythropoietin
Objective: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.
PartⅡThe role of carbamylated erythropoietin in mouse hypoxia model
1.The establishment of the model of cerebral hypoxia in mice
Objective: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%O_2) 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%O_2),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 model
Objective: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.
PartⅢThe protective effects of carbamylated erythropoietin on the cells undergoing oxygen deprivation
1.The protective effects of carbamylated erythropoietin on the neurons undergoing oxygen and glucose deprivation
Objective: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 deprivation
Objective: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.
PartⅣThe protective mechanism of carbamylated erythropoietin
Objective: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.
Conclusion
1.In contrast to EPO,carbamylated erythropoietin has no hematopoietic bioactivity.
2.Hypoxia(8%O_2) 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.
目的:制备不具有促红细胞生成作用的氨甲酰化促红细胞生成素(erythropoietin,EPO)衍生物(carbamylated erythropoietin,CEPO)。方法:用氰酸钾使EPO氨甲酰化,对氨甲酰化EPO经赖氨酸C端蛋白酶(endoproteinase Lys-C)酶切产物进行电泳鉴定。分别给予成年KM小鼠EPO、CEPO和生理盐水每周三次腹腔注射,于3d、7d和14d动态测定动物血红细胞(RBC)、网织细胞(RET)以及网织细胞百分数(RET%)。另外,利用成年C57/B6小鼠,EPO、CEPO和生理盐水剂量增加一倍时间延长至30d后测定各组动物上述的血细胞指标。结果:经酶解电泳鉴定,氨甲酰化为CEPO不能被赖氨酸C端蛋白酶水解,并且有74%的得率。KM小鼠接受EPO注射后,RBC数随给药次数的增加而显著增加,而接受CEPO和NS组均未见有意义的改变。当给药剂量和时间增加一倍后,EPO显著增加了C57/B6小鼠RBC的数目,CEPO组的红细胞数仍未增加。结论:EPO经氨甲酰化反应后,成功地转化为CEPO,并丧失了促红细胞生成的能力。
第二部分促红细胞生成素氨甲酰化衍生物抗低氧神经损伤的在体观察
一.低氧小鼠模型的建立
目的:研究低氧处理对小鼠学习和记忆功能的影响,及对小鼠海马区神经元的损伤与程度。方法:使用小鼠专用缺氧箱,分别给予小鼠8%氧气0.5h、1.5h、3h和6h。同时,设置常氧组小鼠作为对照。各组小鼠于复氧后1d起同时进行连续6d的Y迷宫训练,记录每天20次训练中的错误反应次数。以上各组小鼠在复氧30d再次进行Y迷宫检测,记录10次测试中的错误反应次数和总反应时间。另外,采用NeuN染色检测低氧处理复氧24h和72h小鼠海马神经元脱失情况。结果:Y迷宫结果显示:小鼠吸入8%氧气后学习能力明显下降,以3h和6h组为明显。低氧6h组在复氧后30d测试中错误反应次数最高。复氧24h后CA1区NeuN阳性神经元即有明显脱失,以低氧6h组最明显。复氧72h后,低氧处理各组海马各亚区神经元均有明显脱失,以低氧6h组最为明显。结论:吸入8%氧气6h将严重损伤小鼠海马神经元,降低小鼠学习和记忆能力。
二.促红细胞生成素氨甲酰化衍生物对低氧脑损伤保护作用的研究
目的:研究CEPO对低氧所致小鼠海马损伤的保护作用。方法:给予已学会Y迷宫的小鼠8%氧气6h后即刻给予EPO、CEPO和生理盐水,隔日一次腹腔注射,设置常氧组小鼠作为对照。于复氧10d、30d重复进行小鼠Y迷宫测试,记录小鼠在10次测试中的错误反应次数、总反应时间和主动回避率。分别以BrdU、DCX、NeuN、GFAP、F4/80免疫组化染色,观察EPO组、CEPO组及生理盐水组复氧72h后脑海马神经元脱失、增殖,星型胶质细胞和小胶质细胞的情况。结果:复氧第10d、30d的Y迷宫测试显示,CEPO组和EPO组不仅错误反应次数均明显低于NS组,还具有较高的主动凹避率。NeuN染色提示低氧后给予CEPO、EPO组的海马神经元脱失均少于NS组。常氧及低氧处理后小鼠给予CEPO或EPO均可明显地提高海马DG区以及SVZ区BrdU阳性细胞数。应用免疫组化多重染色显示:随时间推移,CEPO治疗组的DG区新生神经元分化为成熟神经元,同时也促胼胝体区小胶质细胞增生的作用,也具有激活星型胶质细胞的作用。结论:CEPO可保护低氧所致学习和记忆的损伤;减少海马神经元脱失;促DG区神经前体细胞增殖,并分化成为成熟神经元。同时也有促进小胶质细胞增生分化的作用。
第三部分促红细胞生成素氨甲酰化衍生物对缺氧损伤保护作用的离体研究
一.促红细胞生成素氨甲酰化衍生物对缺糖、缺氧神经元损伤的保护作用
目的:在体外培养的神经元OGD模型上观察CEPO的抗凋亡和促细胞增殖的作用,并初步探讨其机理。方法:测定体外培养的神经元OGD 1.5h或3h复氧后不同时间点细胞LDH释放率,比较OGD 3h后给予EPO、CEPO或未干预组的细胞损伤情况。采用TUNEL、流式细胞仪Annexin V/PI染色进一步验证OGD 3h复氧4h时各组细胞凋亡和死亡情况。以MTT法测定OGD对细胞生存率的影响,并比较OGD 3h后各干预组细胞生存率。以BrdU标记增殖细胞,检测OGD 1.5h复氧48h后各组细胞增殖情况。以免疫组化法检测并比较各组细胞JAK-2表达;以免疫印迹法检测OGD后各组MAPK通路磷酸化蛋白的表达情况;以及采用PI3K/Akt途径抑制剂LY-294002预处理后,计数并比较OGD1.5h复氧48h后各组的BrdU阳性细胞数。结果:OGD1.5h或3h后细胞LDH释放率明显增高,OGD 3h复氧4h后CEPO和EPO组细胞死亡率较对照组明显下降。TUNEL法和Annexin V-FITC/PI流式细胞仪计数,均验证OGD3h复氧4h后CEPO干预组细胞凋亡和死亡率较对照组明显下降。MTT法的结果显示CEPO组可显著提高复氧24h后的细胞生存率。OGD1.5h复氧48h后,CEPO及EPO组BrdU阳性细胞显著高于对照组及常氧组。JAK-2免疫组化染色显示CEPO的神经保护没有激活JAK-2通路。免疫印迹法检测显示OGD后MAPK途径的磷酸化ERK、JNK、P38表达量均有明显增高;EPO可以显著降低JNK和P38的磷酸化,CEPO对上述磷酸化蛋白的表达无明显影响。BrdU计数也显示,CEPO促进OGD后的细胞增殖,在给予PI3K/Akt抑制剂LY-294002后完全被阻断。结论:体外神经元OGD模型证实,CEPO可减轻OGD所致细胞损伤,促神经元增殖,其作用不通过JAK-2和MAPK途径,而可能通过PI3K/Akt途径。
二.促红细胞生成素氨甲酰化衍生物对无氧小胶质细胞的作用
目的:研究CEPO对小胶质细胞无氧损伤后炎性因子分泌的影响。方法:培养原代小胶质细胞并鉴定。给予小胶质细胞无氧处理3h,检测复氧后各时间点及加入CEPO、EPO后各组炎性因子IL-6和IL-1β的分泌情况。结果:无氧处理后小胶质细胞IL-6分泌明显增高,IL-1β增高不明显。给予CEPO后复氧24h各组IL-6和IL-1β均较无氧组明显下降,而EPO组无明显下降。结论:CEPO可以明显减少缺氧后小胶质细胞炎性因子IL-6和IL-1β的分泌。
第四部分促红细胞生成素氨甲酰化衍生物神经保护作用的可能机制探讨
目的:在体外培养的神经元OGD模型上初步探讨CEPO作用机理。方法:以免疫组化法检测并比较各组细胞JAK-2表达;以免疫印迹法检测OGD后各组MAPK通路磷酸化蛋白的表达情况;以及采用PI3K/Akt途径抑制剂LY-294002预处理后,计数并比较OGD1.5h复氧48h后各组的BrdU阳性细胞数。结果:JAK-2免疫组化染色显示CEPO的神经保护没有激活JAK-2通路。免疫印迹法检测显示OGD后MAPK途径的磷酸化ERK、JNK、P38表达量均有明显增高;EPO可以显著降低JNK和P38的磷酸化,CEPO对上述磷酸化蛋白的表达无明显影响。BrdU计数也显示,CEPO促进OGD后的细胞增殖,在给予PI3K/Akt抑制剂LY-294002后完全被阻断。结论:体外神经元OGD模型证实,CEPO作用不通过JAK-2和MAPK途径,而可能通过PI3K/Akt途径。
结论
1.EPO的氨甲酰化衍生物——CEPO,完全不具有促红细胞生成的作用。
2.给予8%氧气6h将严重损伤小鼠海马神经元,CEPO可保护低氧所致学习和记忆的损伤,减少海马神经元脱失,促进DG区神经前体细胞增殖分化成为成熟神经元,还具有促小胶质细胞分化的作用。
3.CEPO具有减轻OGD所致神经细胞损伤,促神经元增殖的作用,可以明显减少无氧后小胶质细胞炎性因子IL-6和IL-1β的分泌。
4.CEPO作用机制可能通过PI3K/Akt途径,而可能非JAK-2、MAPK途径。
PartⅠPreparation and Identification of Carbamylated Erythropoietin
Objective: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.
PartⅡThe role of carbamylated erythropoietin in mouse hypoxia model
1.The establishment of the model of cerebral hypoxia in mice
Objective: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%O_2) 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%O_2),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 model
Objective: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.
PartⅢThe protective effects of carbamylated erythropoietin on the cells undergoing oxygen deprivation
1.The protective effects of carbamylated erythropoietin on the neurons undergoing oxygen and glucose deprivation
Objective: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 deprivation
Objective: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.
PartⅣThe protective mechanism of carbamylated erythropoietin
Objective: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.
Conclusion
1.In contrast to EPO,carbamylated erythropoietin has no hematopoietic bioactivity.
2.Hypoxia(8%O_2) 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.
引文
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