氨甲酰化促红细胞生成素的制备及鉴定

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（CEPO）。方法：用氰酸钾使促红细胞生成素（EPO）氨甲酰化，对CEPO的蛋白酶（endoproteinase Lys-C）酶切产物进行电泳鉴定。分别给予成年KM小鼠腹腔注射EPO、CEPO和生理盐水，每周3次，于3、7和14d测定小鼠红细胞（RBC）、网织红细胞（Ret）数及Ret百分数，观察其动态变化。另外，给予不同种属的成年C57／B6小鼠腹腔注射增加1倍剂量的EPO、CEPO和生理盐水，隔日1次，30d后测定各组小鼠上述的血细胞指标，以证明CEPO不具有促红细胞生成作用。结果：经SDS-PAGE电泳鉴定，EPO已成功地氨甲酰化为CEPO，并且有较高的纯度。KM小鼠和C57／B6小鼠分别接受EPO注射后，RBC数、Ret数、Ret百分数均随给药次数的增多而显著增加，而接受CEPO和生理盐水组均未见有明显的改变。结论：经氨甲酰化反应后，EPO成功地转化为CEPO，并消除了促RBC生成的作用。     

促红细胞生成素对脑缺血再灌注大鼠海马CA1区Bcl-xl表达和认知功能的影响

目的 探讨促红细胞生成素(EPO)对脑缺血再灌注(IR)大鼠海马CA1区Bcl-xl表达和认知功能的影响.方法 将雄性SD大鼠随机分为假手术组、IR组、EPO组,并制作IR模型;制模前3 h,EPO组及IR组大鼠脑室立体定向分别注射重组人EPO(rHu-EPO)及生理盐水.制模24 h后,应用免疫组化法检测各组大鼠海马CA1区Bcl-xl蛋白表达;4周时电迷宫测验各组大鼠学会逃避及记忆再现所需的电击次数.结果 与假手术组比较,制模24 h时,EPO组和IR组海马CA1区Bcl-xl蛋白表达明显减少,但EPO组明显多于IR组 (均P<0.01);制模4周后,EPO组及IR组大鼠学会逃避所需的电击次数明显增多,但EPO组明显少于IR组 (均P<0.01);EPO组及IR组大鼠记忆再现次数明显减少,但EPO组多于IR组(均P<0.01).结论 EPO预处理可以促进IR大鼠海马CA1区Bcl-xl蛋白表达,改善IR大鼠的认知功能损害.     

氨甲酰化促红细胞生成素对毛果芸香碱癫(癎)模型小鼠脑保护作用的研究

目的:研究氨甲酰化促红细胞生成素(CEPO)对毛果芸香碱诱导小鼠癫(癎)模型海马神经元的保护作用.方法:C57/B6小鼠随机分为正常未干预组、对照组和CEPO干预组.正常未干预组小鼠不作任何处理;对照组和CEPO干预组小鼠用毛果芸香碱350 mg·kg-1腹腔注射诱导癫(癎)发作,并于癫(癎)发作30 min后用地西泮...     

促红细胞生成素对血管性痴呆大鼠行为学及脑组织形态学的影响

目的永久性结扎双侧颈总动脉（2-VO）建立血管性痴呆（VaD）动物模型,观察EPO治疗对VaD大鼠行为学及脑组织形态学的影响。方法选用14～15月龄Wistar大鼠,应用水迷宫进行空间定向学习训练,将达到学会标准者随机分为3组：假手术（Sham）组、VaD组、VaD＋EPO腹部皮下注射治疗（EPO）组。2-VO制作VaD模型,应用水迷宫检测空间定向学习能力;行HE染色,透射电镜观察海马组织病理学变化。结果与Sham组比较,VaD组、EPO组2-VO后8周学习记忆能力均明显下降;VaD组2-VO术后8周海马CA1区锥体神经元细胞稀疏、肿胀,与VaD组相比,EPO组脑组织损伤程度明显减轻,学习记忆能力增强。结论2-VO可引起大鼠学习记忆能力下降,EPO治疗的VaD大鼠学习记忆能力明显增强;海马CA1区神经元的缺血损伤减轻可能是EPO改善VaD大鼠认知功能障碍的组织病理学基础。     

EPO对Alzheimer样大鼠记忆能力及海马synapsin1蛋白表达的影响

目的探讨促红细胞生成素(EPO)对Alzheimer样大鼠记忆能力和海马synapsin 1蛋白表达的影响及作用机制。方法 48只雄性Wistar大鼠随机分为正常对照组、生理盐水(NS)组、模型组、EPO治疗组,每组12只。其中NS组双侧海马注射NS各5μl;模型组和EPO治疗组双侧海马注射凝聚态Aβ各5μl。EPO治疗组从造模当天按5000 IU/kg腹腔注射EPO,隔天一次。术后第10天Y迷宫法检测各组大鼠空间定向学习和记忆能力,透射电镜观察海马线粒体和突触结构的改变,使用Western blot技术检测各组大鼠synapsin 1蛋白水平。结果 (1)与NS组比较,模型组学习记忆能力显著下降(P<0.05);与模型组比较,EPO治疗组Y迷宫作业尝试次数减少,错误反应次数减少,全天总反应时间缩短,差异有统计学意义(P<0.05)。(2)透射电镜结果显示,正常对照组和NS组神经细胞结构完整,线粒体膜、脊完整,神经轴突和神经突触结构完整;EPO治疗组大鼠海马线粒体和神经突触基本正常;模型组大鼠海马线粒体结构破坏,线粒体肿胀,脊断裂,神经突触密度降低,突触膜增厚,突触间隙不清,突触小泡数量减少。(3)与模型组相比,EPO治疗组大鼠海马synapsin 1蛋白表达增强,差异均有统计学意义(P<0.05)。结论 EPO可以显著改善AD样大鼠的空间记忆能力,减轻Aβ对大鼠海马超微结构的破坏,其机制可能与提高synapsin1蛋白表达有关。     

促红细胞生成素对β淀粉样蛋白诱导大鼠海马CA_1区锥体细胞线粒体细胞色素C释放的影响

目的探讨促红细胞生成素(EPO)对β淀粉样蛋白诱导大鼠海马CA1区神经元凋亡的影响及可能机制。方法采用大鼠海马内注射β淀粉样蛋白(Aβ)制作阿尔茨海默病(AD)模型。将SD大鼠随机分为假手术组、生理盐水组及EPO处理组。Aβ大鼠海马内注射造模,然后在生理盐水组大鼠行脑室立体定向注射生理盐水,EPO处理组则行脑室立体定向注射重组人促红细胞生成素(rHu-EPO)。观察术后24小时海马CA1区神经元细胞色素C(Cyt C)的变化,以及术后7天海马CA1区细胞凋亡变化。结果 EPO处理组大鼠海马CA1区神经元Cyt C的表达明显高于生理盐水组(P<0.01),EPO处理组大鼠海马CA1区凋亡细胞较生理盐水组减少(P<0.01)。结论 EPO可以通过抑制Aβ1-40诱导海马CA1区锥体细胞线粒体Cyt C释放来抑制神经元凋亡。     

促红细胞生成素对β淀粉样蛋白诱导大鼠海马CA1区细胞凋亡的保护作用及机制

目的 探讨促红细胞生成素(Erythropoietin,EPO)对阿尔茨海默病(Alzheimer disease,AD)大鼠脑损伤的保护机制.方法 采用大鼠海马内注射B淀粉样蛋白制作AD模型.将SD大鼠随机分为假手术对照组、生理盐水对照组及EPO处理组.大鼠海马内注射Aβ1-40加造模,然后在生理盐水对照组大鼠行脑室立体定向注射生理盐水,EPO处理组则行脑室立体定向注射重组人促红细胞生成素(rHu-EPO).观察手术后24h大鼠海马CA1区抗凋亡蛋白Bcl-xl表达变化,以及术后7d海马CA1区细胞凋亡变化.结果 EPO处理组和生理盐水组海马CA1区大鼠Bcl-xl蛋白表达较假手术对照组减少,但是EPO处理组Bcl-xl蛋白表达高于生理盐水(P<0.05).生理盐水组海马CA1区凋亡细胞明显多于EPO组(P<0.05).结论 EPO可以抑制β淀粉样蛋白诱导海马CA1区细胞凋亡,与其抑制Bcl-xl蛋白表达下降有关.     

促红细胞生成素对大鼠脑缺血海马CA1区神经元凋亡和细胞色素C释放的影响

目的　探讨促红细胞生成素 (Erythropoietin ,EPO)的神经保护机制。方法　采用 4 VO法制作大鼠全脑缺血模型。将SD大鼠随机分为假手术组、生理盐水组、EPO组。全脑缺血前 3h ,EPO组大鼠脑室立体定向注射重组人促红细胞生成素 (recombinantHumanErythropoietin ,rHuEPO) ,生理盐水组则给予生理盐水 ,假手术组只进行假手术处理。观察缺血后 2 4h海马CA1区细胞色素C(CytochromeC ,CytC)的变化 ,及缺血后 72h海马CA1区细胞凋亡情况。结果　EPO组海马CA1区呈现点状分布的CytC表达较生理盐水组增强 (P <0 .0 1) ,并且较生理盐水组呈现较少的凋亡细胞 (P <0 .0 1)。结论　EPO预处理可以抑制海马CA1区CytC从线粒体向胞浆释放及减少神经元凋亡。     

局灶性脑缺血大鼠海马CA3区EPO和STAT-5的表达

目的观察局灶性脑缺血后大鼠海马CA3区促红细胞生成素（EPO）和信号传导转录活化因子5（STAT-5）的表达规律，探讨EPO和STAT-5在脑缺血损伤过程中的作用。方法采用线栓法制备大鼠大脑中动脉局灶性脑缺血模型，SD大鼠随机分为假手术组和脑缺血组，后者又分为2、12、24h三个亚组，免疫组化法检测各组大鼠海马CA3区EPO和STAT-5的表达水平。结果（1）脑缺血组各时间点大鼠海马CA3区EPO和STAT-5的表达水平均较假手术组明显增高（P％0．01）；（2）脑缺血组大鼠海马CA3区EPO和STAT-5阳性细胞数在2h明显增加；12h达到高峰，24h下降；（3）脑缺血组各时间点EPO和STAT-5表达水平同步增减。结论EPO有可能是通过EPOR-JAK2-STAT-5信号转导途径对缺血性脑损伤发挥保护性作用。     

康复训练联合促红细胞生成素对脑出血大鼠神经 修复及海马胶质原纤维酸性蛋白表达的影响

目的 康复训练联合促红细胞生成素（EPO）对脑出血大鼠神经修复及海马组织胶质原纤 维酸性蛋白（GFAP）表达的影响。方法 健康雄性SD 大鼠30 只随机分成3 组：模型组、EPO 组和训练组， 每组各10 只大鼠。3 组均建立脑出血模型，EPO 组与训练组在造模成功24 h 后均腹腔注射EPO，训练组 大鼠给予康复训练，模型组注射与EPO 组及训练组所注射EPO等量的生理盐水。观察与检测大鼠神经 修复及海马GFAP表达情况。结果 所有大鼠均造模成功，EPO 组与训练组造模后7 d、14 d 和28 d 的 Tarlov 评分高于模型组（P＜ 0.05），训练组也显著高于EPO 组（P ＜ 0.05）。造模后28 d，EPO 组与训练组 的脑含水量、血清IL-6 和TNF-α 值、海马组织GFAP蛋白相对表达水平显著低于模型组（P＜ 0.05），训练 组也低于EPO 组（P ＜ 0.05）。结论 康复训练联合EPO在脑出血大鼠中的应用能抑制海马GFAP表达 与血清炎性因子的释放，减轻脑水肿，从而促进神经功能恢复正常。     

Carbamylated erythropoietin ameliorates hypoxia‐induced cognitive and behavioral defects with the generation of choline acetyltransferase‐positive neurons

Carbamylated erythropoietin (CEPO) is attracting widespread interest because of its neuroprotective effects without influencing erythropoiesis. Here we show that CEPO, unlike EPO, does not stimulate erythropoiesis. Both CEPO and EPO inhibit the death/apoptosis of neurons in the hypoxic model of primary neurons and induce neuron proliferation and differentiation in hypoxic mice. Hypoxic mice show apparent memory deficits at 3 and 30 days after hypoxia. The administration of CEPO/EPO significantly improves cognitive and behavioral defects after hypoxic insults. Further investigation shows that CEPO/EPO induces neuron proliferation and differentiation and promotes the generation of choline acetyltransferase (ChAT)⁺ neurons in hypoxic mice. Phosphorylated AKT was colabeled with ChAT⁺ neurons and coexpressed in bromodeoxyuridine‐positive cells, suggesting that the PI3K/AKT pathway may play a pivotal role in CEPO/EPO‐cholinergic neuron generation. These results reveal that CEPO/EPO ameliorates hypoxia‐induced cognitive and behavioral defects possibly through the generation of ChAT‐positive neurons. © 2012 Wiley Periodicals, Inc.     

促红细胞生成素对血管性痴呆大鼠模型海马神经细胞凋亡的影响

目的:探讨促红细胞生成素(EPO)对血管性痴呆(VaD)模型大鼠海马神经细胞凋亡的影响。方法:将经行为学筛选合格的Wistar大鼠分为假手术组、VaD组、EPO组,采用永久性结扎双侧颈总动脉的方法建立VaD模型。应用TUNEL染色检测大鼠海马的凋亡细胞数;通过免疫组化学和RT-PCR方法,检测大鼠海马Bcl-2和Bax蛋白以及其mRNA表达水平的变化。结果:EPO能明显抑制海马神经细胞凋亡,上调Bcl-2表达,抑制Bax表达。结论:EPO可能通过调控VaD大鼠海马Bcl-2和Bax表达,从而抑制神经细胞凋亡。     

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 microg/kg) or CEPO (40 microg/kg). Results indicated that 3 days post-injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase-mediated dUTP nick-end labelling (TUNEL)-labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker beta-amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the beta common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.     

Neuroprotective potential of erythropoietin and its derivative carbamylated erythropoietin in periventricular leukomalacia

Periventricular leukomalacia (PVL) is the predominant pathology in premature infants, characterized by prominent cerebral white matter injury, and commonly caused by hypoxia–ischemia and inflammation. Activated microglia trigger white matter damage and play a major role in the development of PVL. Erythropoietin (EPO) and its derivative carbamylated erythropoietin (CEPO) have been shown to be neuroprotective in several brain disease models. Here we investigated whether EPO and CEPO could provide protection in mouse models of PVL induced by hypoxia–ischemia or hypoxia–ischemia-inflammation. We administered EPO or CEPO to mice with PVL, and found that both EPO and CEPO treatments decreased microglia activation, oligodendrocyte damage and myelin depletion. We also noted improved performance in neurological function assays. Inhibited disease progression in PVL mice by EPO or CEPO treatment was associated with decreased poly-(ADP-ribose) polymerase-1 (PARP-1) activity. PARP-1 activity was increased dramatically in activated microglia in untreated mice with PVL. Furthermore, we demonstrated that the neuroprotective properties of EPO and CEPO were diminished after PARP-1 gene depletion. The therapeutic doses of EPO and CEPO used in this study did not interfere with normal oligodendrocyte maturation and myelination. Together, our data demonstrate that EPO and CEPO are neuroprotective in cerebral white matter injury via a novel microglial PARP-1 dependent mechanism, and hold promise as a future treatment for PVL and other hypoxic–ischemic/inflammatory white matter diseases.     

血管性痴呆小鼠海马胆碱乙酰转移酶mRNA变化特征及喜得镇的影响

目的:观测喜得镇对血管性痴呆小鼠海马神经元胆碱乙酰转移酶mRNA变化的影响,以探讨胆碱乙酰转移酶mRNA变化在血管性痴呆发病中的作用及喜得镇对此变化的机制。方法:双侧颈总动脉线结.反复缺血-再灌注法制备模型,药物组用喜得镇溶液灌胃,利用跳台试验和水迷宫试验观测其行为学改变,采用原位杂交技术观测小鼠海马神经元胆碱乙酰转移酶mRNA的表达变化。结果:喜得镇组小鼠学习、记忆成绩优于模型组(P<0.01),其海马胆碱乙酰转移酶mRNA表达也明显增高(P<0.01)。结论:喜得镇改善血管性痴呆小鼠学习、记忆成绩与其恢复海马低水平的胆碱乙酰转移酶mRNA有关。     

促红细胞生成素及其衍生物在局灶性脑缺血中的神经保护作用

目的 探讨氨甲酰促红细胞生成素(CEPO)对缺血性脑损伤的保护作用及机制,并与促红细胞生成素(EPO)进行比较. 方法 用腔内线栓法制造小鼠脑缺血90min再灌注模型,根据颈动脉注射药物的不同分为对照组(注射生理盐水)、EPO 5 μg/kg组、EPO 50 μg/kg组、CEPO 50μg/kg组,每组6只,用脑血流激光多普勒监测脑缺血过程中脑血流的变化;用甲酚紫染色法显示脑梗死区并用imageJ软件计算梗死体积和脑水肿体积;用TUNEL法观察凋亡细胞;用免疫组织化学方法 观察脑缺血再灌注后诱导型一氧化氮合酶(iNOS)的改变. 结果与对照组比较,EPO 50μg/kg组和CEPO 50μg/kg组的脑梗死体积、脑水肿体积、神经功能缺损评分明显减少,差异均有统计学意义(P＜0.05).EPO 50μg/kg组、CEPO 50 μg/kg组缺血皮层iNOS阳性细胞数与对照组比较,差异均有统计学意义(P＜0.05).EPO 50μg/kg组的凋亡细胞[(43.6±10.1)个]、CEPO 50 μg/kg组的凋亡细胞[(40.5±9.8)个]与对照组[(94.2±15.2)个]比较,差异均有统计学意义(P＜0.05). 结论 低剂量的EPO(5μg/kg)无脑保护作用,CEPO 50 μg/kg与较高剂量EPO(50μg/kg)具有相似的增加脑缺血再灌注后的脑血流、减少神经功能缺损评分、减少梗死体积、缩小脑水肿体积和抗细胞凋亡作用,它们通过减少iNOS的表达而发挥神经保护作用.     

血管性痴呆小鼠海马NMDA受体mRNA表达特征及喜得镇的影响

目的观察喜得镇对血管性痴呆(vascular dementia,VD)小鼠海马神经元N-甲基D-天门冬氨酸(N-methyl-D-aspartate,NMDA)受体原位杂交变化的影响,并探讨NMDA受体原位杂交变化特征及喜得镇对其变化的影响.方法双侧颈总动脉结扎,反复缺血-再灌注法制备模型,药物组用喜得镇溶液灌胃,跳台试验和水迷宫试验观察其行为学改变,采用原位杂交技术观察小鼠海马神经元NMDA受体的表达变化.结果药物组小鼠学习、记忆成绩优于模型组(P＜0.01),其海马N-甲基-D-天门冬氨酸受体表达也明显增高(P＜0.01).结论喜得镇能改善血管性痴呆小鼠模型的学习、记忆功能,这可能与N-甲基D-天门冬氨酸受体mRNA水平正常化有关.     

Hippocampal neuron loss is correlated with cognitive deficits in SAMP8 mice

The objective of this study is to examine whether neuron loss occurs in SAMP8 and whether neuron loss is correlated with cognitive deficits of these mice. Neuronal loss is considered as one of the most important pathological hallmarks of Alzheimer disease (AD). In addition to the early-onset, irreversible, severe deficits of learning and memory, SAMP8 mice show spontaneous age-related neurodegenerative changes and other characteristics seen in AD patients, such as amyloid plaques and neurofibrillary tangles. However, it is still unknown whether neuron loss occurs in SAMP8 and whether neuron loss is correlated with cognitive deficits of these mice. We employed 8-month-old SAMP8 and SAMR1 mice to investigate the cognitive function and neuron numbers. The behaviors were examined by the grading score of senescence and Morris water maze (MWM) test, the neuron number in hippocampus was estimated by the optical fractionator technique. The grading score of senescence and MWM test demonstrated that SAMP8 exhibited notable age-related changes in appearance and cognitive function. Moreover, severe hippocampal neuron loss was found in SAMP8 as determined by the optical fractionator stereological method. Compared to SAMR1, the neuron number of CA1, CA3 and DG in SAMP8 was reduced by 15.6, 19.8 and 20.2 %, respectively, and the neuron loss in hippocampus was associated with cognitive deficits. Collectively, these results suggest that hippocampal neuronal loss is well correlated with learning and memory deficits in SAMP8 and SAMP8 represents an important mouse model for AD.     

氨甲酰化促红细胞生成素经PI3-K/Akt信号通路抗脑缺血损伤

目的观察脑缺血后氨甲酰化促红细胞生成素(CEPO)的神经保护作用并探讨其可能机制。方法健康雄性SD大鼠随机分为6组(n=10):(1)假手术组;(2)缺血组;(3)EPO组;(4)CEPO组;(5)LY(LY294002)组;(6)CEPO+LY组。应用大脑中动脉线栓法(MCAO)制作大鼠局灶性脑缺血模型,评定大鼠神经功能并计算脑梗死体积,Western blot方法检测PI3-K/Akt活性变化。结果 EPO组与CEPO组脑梗死体积均明显缩小,神经功能显著改善,磷酸化Akt(p Akt)水平明显增高,且两组之间无明显差异,但CEPO的神经保护作用及对Akt磷酸化的诱导效应均可被PI3-K抑制剂LY294002部分抵消。结论 CEPO具有与EPO相当的缺血后脑保护作用,其机制可能与PI3-K/Akt信号通路激活有关。