Inhibitory effect on cerebral inflammatory agents that accompany traumatic brain injury in a rat model: a potential neuroprotective mechanism of recombinant human erythropoietin (rhEPO)

Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.     

重组人促红细胞生成素对人脐静脉内皮细胞CyclinD1表达的影响

目的探讨重组人促红细胞生成素(rHuEPO)对人脐静脉内皮细胞(HUVEC)细胞周期蛋白CyclinD1的影响。方法采用细胞培养及免疫组织化学方法检测不同剂量(10、20、40U/mL)rHuEPO作用下HUVEC中CyclinD1表达的变化。结果对组照及10、20、40U/mlrHuEPO实验组内皮细胞CyclinD1表达阳性百分率(%)分别为26±3、41±5、69±3、65±4,与对照组相比,rHuEPO作用后HUVEC中CyclinD1表达明显增强(P﹤0.05),且随rHuEPO剂量增加作用增强,呈剂量效应关系。结论rHuEPO能明显促进体外培养内皮细胞CyclinD1表达,提示rHuEPO具促内皮细胞增殖作用。     

促红细胞生成素促进内皮祖细胞增殖的体外研究及机制探讨

目的：研究人重组促红细胞生成素（rhEPO）对人内皮祖细胞（EPCs）数量和功能的影响并探讨其作用机制。方法: 以不同浓度rhEPO分别作用于15例处于肾衰竭期的患者以及15例健康志愿者EPCs，MTT法检测其增殖，Annexin-V法检测其凋亡，Western blotting法检测Akt蛋白激酶。结果: 100 U/L、600 U/L及1 200 U/L rhEPO均能提高实验组和对照组EPCs的数量及增殖能力，呈剂量依赖性，实验组EPCs数量及功能均显著低于对照组。1 200 U/L rhEPO能显著降低EPCs的凋亡率，提高 Akt蛋白激酶的表达。加入Wortmannin后能阻断这一效应。结论: rhEPO可以显著提高健康人群和肾衰竭患者EPCs的数量与功能，且这一效应与PI3K/Akt信号通路有关。     

Effects of prolonged low doses of recombinant human erythropoietin during submaximal and maximal exercise

The aim of this study was to characterise the effect of prolonged low doses of recombinant erythropoietin (r-HuEPO) on the responses to submaximal and maximal exercise. Volunteer recreational athletes (n=21) were divided into three groups: r-HuEPO+intravenous iron (EPO+IV, n=7), r-HuEPO+oral iron (EPO+OR, n=9) and placebo (n=5). During the 12 week study, r-HuEPO or saline injections were given three times a week for the first 8 weeks and for the final 4 weeks the subjects were monitored but no injections were administered. The r-HuEPO doses were 50 IU·kg^–1 body mass for 3 weeks and 20 IU·kg^–1 body mass for the next 5 weeks. An exercise test comprising three submaximal intensities and then increments to elicit maximal aerobic power ( ) was conducted during weeks 0, 4, 8 and 12. During week 0, the mean intensity of the submaximal stages was 60%, 72% and 81% . Blood taken at rest was analysed twice a week for haematocrit (Hct). The relative increases in at weeks 4, 8 and 12 were 7.7%, 9.7% and 4.5%, respectively, for the EPO+IV group; 6.0%, 4.7% and 3.1% for the EPO+OR group; and –0.5%, –0.1% and –1.0% for the placebo group, where the improvements at week 12 for the EPO+IV and EPO+OR groups remained significantly above week 0 values. The Hct was significantly elevated by 0.06 and 0.07 units at week 3 in the EPO+IV and EPO+OR groups, respectively, and was stable during the 5 weeks of low-dose r-HuEPO. After 8 weeks of r-HuEPO use, plasma lactate concentration tended to be lower at exercise intensities ranging from 60% to 100% . This study confirmed the ability of low doses of r-HuEPO to maintain Hct and at elevated levels. Electronic Publication     

Production and characterization of anti-recombinant human erythropoietin (rhEPO) monoclonal antibody

Anti-rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti-rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme-linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS-PAGE, and Western-blotting. Experimental results showed that the subclass and the light chain of anti-rhEPO McAb was IgG1 and kappa light chain. The molecular weight of anti-rhEPO McAb was 166,000 Daltons. The affinity constant (K(aff)) of anti-rhEPO McAb with coated antigen was 5.0 x 10(5)L/mol.     

促红细胞生成素对大鼠脊髓损伤后caspase-3表达及神经细胞凋亡的影响

目的通过观察重组人红细胞生成素（recombinant human erythropoietin，rHu—EPO）对大鼠急性脊髓损伤后Caspase-3表达及凋亡的影响，探讨其脊髓保护的作用机制。方法60只SD大鼠随机分成3组：假手术组，对照组和治疗组，每组20只，采用Allen’s打击法制成急性脊髓损伤模型。观察药物治疗前后大鼠的神经功能行为，用免疫组化染色检测caspase-3表达，原位脱氧糖核苷酸末端转移酶介导的缺口末端标记法（Tunel法）标记凋亡细胞；比较各组间差别。结果HE，免疫组化染色示EPO治疗组脊髓损伤程度小，神经元细胞破坏少；caspase-3在各时相点的表达明显降低，8h和7dTunel标记凋亡阳性细胞显著减少；大鼠神经功能恢复显著优于对照组（P〈0．01）。结论EPO能下调Caspase-3表达，抑制脊髓神经细胞凋亡，对继发性脊髓损伤有保护作用。     

重组人促红细胞生成素对大鼠皮肤深Ⅱ°烫伤创面愈合的影响

目的研究重组人促红细胞生成素(rHuEPO)对大鼠皮肤深Ⅱ°烫伤愈合的影响。方法健康SD大鼠随机分成低剂量组(50U/mL rHuEPO)、中剂量组(100U/mL rHuEPO)、高剂量组(200U/mL rHuEPO)和生理盐水(NS)对照组,建立深Ⅱ°烫伤动物模型,并于伤后3、5、7、14、21d取创面皮肤标本,HE染色和免疫组织化学方法染色分别检测创面愈合情况及微血管密度。结果与对照组相比,rHuEPO治疗组创面愈合时间缩短,同一时相内大鼠烫伤愈合率高于对照组,微血管密度明显增加。结论rHuEPO能促进深Ⅱ°烫伤创面微血管形成,加速创面愈合。     

Binding of human recombinant interleukin 2 to murine erythrocytes is erythropoietin receptor mediated

Murine red blood cells (RBC) incubated with recombinant human interleukin 2 (rIL2) bound 10%–20% of the added cytokine with 41% ± 5% of the population positive for bound cytokine as determined by fluorescence activated cell scanning (FACS) analysis. Since a high degree of homology exists between the erythropoietin receptor (EPOR) and the IL2R chain (based both on amino acid sequence and hydrophobicity alignment), we hypothesised that the rIL2 was binding to residual EPOR on murine RBC. Upon bioassay, it was found that reticulocytes (RET), which express a higher percentage of EPOR than normal RBC, bound 400% more rIL2 as compared to normal RBC. FACS analysis using fluorescently labelled rIL2 revealed a log higher fluorescence intensity on RET compared to RBC. Therefore, the population of immature or young RBC in circulation which are expressing residual EPOR are binding rIL2 due to cross-reactivity between the EPOR and IL2R.Mention of a tradename, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

促红细胞生成素预处理对大鼠急性缺血再灌注肾的保护作用

目的：探讨促红细胞生成素（rHuEPO）预处理对急性缺血再灌注肾（IR）的保护作用。方法：SD大鼠分为3组:假手术组、IR组、rHuEPO+IR组。除假手术组外，其余两组用动脉夹夹闭双侧肾动脉45 min后恢复灌注。24 h前静脉注射rHuEPO。术后24 h处死各组大鼠。检测血肌酐（SCr）、血尿素氮（BUN）、血红蛋白（Hb）、血球容积比（Hct）。肾组织以过碘酸雪夫（PAS）染色、细胞凋亡以TUNEL assay测定。正常大鼠在给予rHuEPO后，在不同时点处死，利用免疫印迹和免疫组化检测热休克蛋白-70的表达。结果：IR组表现为肾功能下降、肾组织坏死性改变、TUNEL阳性细胞数增多。rHuEPO预处理使上述各项均逆转，各组间的Hb和Hct无显著差异。正常大鼠给予rHuEPO明显增加热休克蛋白-70的表达，呈时间依赖性，24 h达高峰。结论：rHuEPO预处理提高了大鼠对急性肾IR的耐受性，有显著的肾保护作用而不依赖于其抗贫血功能。     

红细胞生成素3'-增强子片段对内皮细胞缺氧时环加氧酶-2基因表达的影响

目的和方法：体外合成红细胞生成素（Ｅｐｏ）３′－增强子野生型及突变型片段，借助脂质体，转入内皮细胞，用半定量ＲＴ－ＰＣＲ方法测定分离细胞在常氧或缺氧条件下培养４ｈ的环加氧酶２（ＣＯＸ－２）ｍＲＮＡ。结果：（１）大鼠主动脉内皮细胞、肺微血管内皮细胞在常氧下培养，有ＣＯＸ－２基因表达；（２）缺氧能诱导这两种细胞ＣＯＸ－２基因表达增加；（３）野生型Ｅｐｏ３′－增强子片段能阻断缺氧对内皮细胞ＣＯＸ－２基因表达的诱导作用，而突变片段则无此作用。结论：在ＣＯＸ－２基因序列中，可能存在Ｅｐｏ３′－端增强子片段，它参与了内皮细胞的缺氧反应     

Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia

The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (>1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC(50)) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities.     

Increased platelet volume — Sign of impaired thrombopoiesis in diabetes mellitus

Summary Increased functional properties of diabetic platelets might be already conditionated during thrombopoiesis in the stem cell system. This hypothesis was studied by recording the distribution characteristics of the peripheral platelet pool in 218 diabetic patients versus 51 controls. Furthermore, platelet membrane coating with the stem cell marker glycoprotein IB was analyzed in 41 diabetic subjects and compared to 23 healthy volunteers. A consistant, significant shift of the volume distribution to larger platelets was found in diabetics: Mean platelet volume (MPV) — 7.9±0.9 versus 7.2±0.8 [fl]; Megathrombocyte index (MTI) — 20.4±2.8 versus 18.1±2.5 [fl]. These deviations were present in all patient subsets, however did not correlate to parameters of glucose metabolism. Whole blood platelet count was increased in the patient group: 195.0±59.5 versus 184.0±37.5×10³ plts/ul. Coating with glycoprotein IB receptors correlated significantly to platelet size in platelets of both controls and diabetics (r _normal=0.52±0.07;r _diabetic=0.46±0.1). The quantitativ expression of glycoprotein IB was significantly enhanced in the diabetic group: 54500×1.28^±1 versus 39100×1.3^±1 molecules per platelet. In conclusion, these findings strongly support the assumption of diabetic stem cell dysfunction of the megakaryocytic series and progenitor cells resulting in platelets with primarily increased potency to adhere and aggregate in diabetes mellitus.Abbreviations F(ab)₂ fragment (ab)₂ - g gravitation force - fl femtolitre - log logarithmus to 10 - nm nanometer - PBS phosphate buffered saline - µl microliter - µM micromolar - MPV mean platelet volume (geometric mean) - MTI megathrombocyte index (=95% percentile) - GPIB glycoprotein IB - plts platelets     

Cerebrospinal fluid erythropoietin (EPO) in amyotrophic lateral sclerosis

A candidate neuroprotective agent for neurodegenerative disorders is erythropoietin (EPO). We measured EPO in cerebrospinal fluid (CSF) and serum of patients with amyotrophic lateral sclerosis (ALS). Patients with ALS (N=60), Alzheimer's disease (AD, N=20) and age-matched controls (N=33) were included. Patients with ALS included 30 patients who showed a rapid progression of disease, and 30 patients that showed a slower progression. EPO was measured using ELISA technique. We found CSF EPO levels to be lower in ALS as compared to AD and controls (p<0.05), while no differences were found with regard to serum levels. Patients with ALS who showed a rapid disease progression had lower CSF EPO levels compared to those who progressed more slowly (p=0.03). Low CSF EPO in ALS may imply that the EPO-associated capacity to protect neurons from degeneration is impaired in ALS. Low concentrations of CSF EPO seem to point towards a rapid progression of disease that may be associated with a poorer prognosis.     

Plasma bioactive erythropoietin in mice with dietary Iron deficiency anaemia: Enhanced reticulocytosis and recombinant erythropoietin administration

Plasma erythropoietin (EPO) activity was determined by an erythroid colony-forming microassay during the development of and recovery from iron deficiency anaemia (IDA) in mice. Growing mice were made iron deficient by feeding a low-iron diet. During the development of IDA, the plasma EPO level increased as the haematocrit (Hct) value decreased in the time-dependent fashion. Likewise, reticulocytosis was enhanced depending on the severity of anaemia or the plasma EPO level. After 9 weeks of the feeding, the development of IDA was confirmed by blood analyses. During the recovery from IDA after switching to an iron-rich diet and/or parenteral iron administration, the plasma EPO levels rapidly declined to normal level, while the Hct values increased and completely returned to normal on day 6. Administration of recombinant human EPO (rHuEPO) in addition to the iron-rich diet and iron brought about the supranormal levels of Hct after day 6. Thus, rHuEPO had no therapeutic role in IDA. The plasma EPO titres were inversely correlated with the Hct values during the progression and reversal of IDA. This result supports a generally accepted concept that the EPO production in anaemia is primarily regulated by Hct feedback control mechanism.     

人促红细胞生成素对肾缺血再灌注损伤的保护作用

目的 探讨促红细胞生成素(erythropoietin, Epo)对失血性休克大鼠肾损伤的保护作用.方法 建立失血性休克大鼠肾损伤模型,分为对照组、休克组及Epo治疗组3组,进行组织学观察,并检测血丙二醛(MDA)、肌酐(Cr)、素氮(BUN)和肾组织匀浆超氧化物歧化酶(SOD)、白介素-6(IL-6)水平.结果 Epo治疗组血浆MDA、Cr、BUN水平较失血性休克组组显著下降(P＜0.05);肾组织匀浆SOD显著升高、IL-6显著降低(P＜0.05).结论 Epo可提高SOD,降低IL-6,对肾缺血再灌注损伤具有保护作用.     

Effects of recombinant erythropoietin (r-HuEPO) on plasma glucose concentration in endurance-trained rats

This study examines the effect of training intensity on the activity of enzymes in m. vastus lateralis. Elite junior cross-country skiers of both sexes trained 12–15 h weeks^–1 for 5 months at either moderate (60–70% of VO _2max, MIG) or high training intensity (80–90% of the VO _2max, close to the lactate threshold; HIG). Muscle biopsies for enzyme analyses and fibre typing were taken before and after the training period. Histochemical analyses on single fibres were done for three enzymes (succinate dehydrogenase [SDH], hydroxybutyrate dehydrogenase [HBDH], glycerol-3-phosphate dehydrogenase [GPDH]), while the activity of citrate synthase [CS] and phosphofructokinase [PFK] was measured on whole biopsies. The activity of GPDH was low in ST fibres and high in FT fibres. The activity of SDH and HBDH was high in both ST and FTa fibres but low in the FTb fibres. The HIG increased their performance more than the MIG did during the training period as judged from scores on a 20-min run test. The SDH activity rose by 6% for the HIG (P < 0.02). No effects of training were found in the activities of CS, HBDH or GPDH, neither in the two training groups nor for the two genders (P ≥ 0.16). The PFK activity fell by 10% for the HIG (P=0.02), while no change was found for the MIG. For GPDH, CS and SDH the women’s activity was ≈20% less than the value for the men (P < 0.03). For PFK and HBDH there was no sex difference (P ≥ 0.27). There were positive correlations between the activity of three of the enzymes (CS, SDH and GPDH) and the performance parameters (VO _2max, cross-country skiing and running performance; r ≥ 0.6, P < 0.01). No correlations were found between the PFK or HBDH activities and the performance parameters (r ≤ 0.16, P > 0.05). This study suggests that intensities near the lactate threshold affect biochemical and physiological parameters examined in this study as well as the performance of elite skiers, and that the rate-limiting enzymes may be more sensitive to training than non-rate-limiting enzymes.     

重组人促红细胞生成素与骨髓间充质干细胞的迁移及黏附

背景：促红细胞生成素可促进内皮祖细胞的增殖分化并增强其黏附性。 目的：观察重组人促红细胞生成素干预对人骨髓间充质干细胞迁移和黏附能力及相关细胞信号传导通路的影响。 方法：体外培养人骨髓间充质干细胞,使用重组人促红细胞生成素干预第6代人骨髓间充质干细胞。分别用PI3K/Akt通路特异抑制剂Ly294002或p38MAPK通路特异激动剂anisomycin或ERK1/2通路特异抑制剂U0126预处理。 结果与结论：重组人促红细胞生成素可使人骨髓间充质干细胞的PI3K/Akt通路磷酸化水平升高,抑制p38MAPK通路磷酸化水平,对人骨髓间充质干细胞的ERK通路和总Akt、总p38MAPK水平无明显影响。重组人促红细胞生成素作用组中迁移细胞数目显著高于对照组(P < 0.01),黏附细胞数亦明显增加(P < 0.01),PI3K/AKT通路特异抑制剂Ly294002预处理后重组人促红细胞生成素对迁移能力的作用消失,p38MAPK通路特异激动剂anisomycin预处理对两种作用影响均不明显。提示重组人促红细胞生成素具有增强人骨髓间充质干细胞迁移和黏附能力的作用,其增强迁移能力的作用与激活人骨髓间充质干细胞的PI3K/Akt通路有关,但是它对黏附能力的作用与PI3K/Akt和p38MAPK通路均无关。     

氨基胍对内毒素诱发的兔急性肺损伤的影响

目的:观察氨基胍(AG)对内毒素(ET)诱发的兔急性肺损伤(ALI)血流动力学及肺血管壁通透性的影响。方法:24只健康成年兔被均分为4组,生理盐水组、ET组、AG组和ET+AG组。ET+AG组先静脉注射ET,复制ALI模型,再以25mg/kg的剂量静脉滴注AG,共维持3h,观察不同时点平均动脉压(MAP)、平均肺动脉压(MPAP)和动脉血液气体参数的变化。实验结束后行支气管肺泡灌洗,测肺湿重/干重比率并常规留取肺标本。结果:ET组MAP下降,MPAP明显升高,动脉血氧分压下降;持续静滴AG后MAP无明显变化,但MPAP明显降低,血红蛋白氧合状况明显改善;AG可减少支气管肺泡灌洗液(BALF)中细胞数量;虽然BALF中蛋白含量无显著变化,但BALF电泳分析表明AG可明显减少小分子量蛋白的渗出;ET+AG组肺湿重/干重比率较ET组明显小;病理组织化学观察表明ET+AG组兔肺内炎症细胞渗出较少,肺水肿较ET组减轻。结论:AG可改善兔ALI血流动力学,减轻肺损伤。