低氧应激促红细胞生成素生成效应及其影响因素

低氧应激促红细胞生成素生成效应及其影响因素大连大学医学院（大连，１１６０１２）孟宪法，于洪九青海医学院（西宁，８１０００１）许存和，耿排力天津医科大学（天津，３０００７０）姜如冈生理条件下，促红细胞生成素（ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ，ＥＰＯ）是维持...

Desferoxamine preconditioning protects against cerebral ischemia in rats by inducing expressions of hypoxia inducible factor 1α and erythropoietin

Objective To investigate whether desferoxamine （DFO） preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1 α （HIF- 1α） and erythropoietin （EPO） in vivo and in vitro. Methods Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation （OGD）, and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1 and EPO. Results The protective effect induced by DFO （decreasing the infarction volume and ameliorating the neurological function） appeared at 2 d after administration ofDFO （post-DFO）, lasted until 7 d and disappeared at 14 d （P 〈 0.05）; the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD： neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO （P 〈 0.05）. Immunofluorescent staining found that HIF-1 α and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1 α and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO. Conclusion DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF- 1 α and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF- 1 α and EPO.

Protective effect of erythropoietin against 1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12 cells

Objective The neuroprotective effect of erythropoietin （EPO） against 1-methyl-4-phenylpyridinium （MPP^＋）- induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. Methods PC12 ceils impaired by MPP^＋ were used as the cell model of Parkinson＇s disease. Methyl thiazolyl tetrazolium （MTT） was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay was used to analyze the apoptosis ratio of PC 12 cells. The expression of Bcl-2 and Bax in PC 12 cells were examined by Western blot, and the reactive oxygen species （ROS）, the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. Results Treatment of PC12 cells with MPP^＋ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP＋ significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. Conclusion The inhibitive effect of EPO on the MPP^＋ -induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson＇s disease.

Plasma erythropoietin levels in anaemic and non-anaemic patients with chronic liver diseases

AIM: To investigate the serum erythropoietin (Epo) levels in patients with chronic liver diseases and to compare to subjects with iron-deficiency anaemia and healthy controls.METHODS: We examined 31 anaemic (ALC) and 22 non-anaemic (NALC) cirrhotic patients, 21 non-anaemic subjects with chronic active hepatitis (CAH), 24 patients with iron-deficiency anaemia (ID) and 15 healthy controls. Circulating Epo levels (ELISA; R&D Systems, Europe Ltd, Abingdon,UK) and haemoglobin (Hb) concentration were determined in all subjects.RESULTS: Mean±SD of Epo values was 26.9±10.8 mU/mL in ALC patients, 12.5±8.0 mU/mL in NALC subjects,11.6±6.3 mU/mL in CAH patients, 56.4±12.7 mU/mL in the cases of ID and 9.3±2.6 mU/mL in controls. No significant difference (P>0.05) was found in Epo levels between controls, CAH and NALC patients. ALC individuals had higher Epo levels (P<0.01) than these groups whereas ID subjects had even higher levels (P<0.001) than patients suffering from ALC.CONCLUSION: Increased Epo values in cirrhotics, are only detectable when haemoglobin was lesser than 12 g/dL.Nevertheless, this rise in value is lower than that observed in anaemic patients with iron-deficiency and appears blunted and inadequate in comparison to the degree of anaemia.

Anemia and inflammatory bowel diseases

Too often anemia is considered a rare or unimportant manifestation in inflammatory bowel disease (IBD). However, over the last 10 years a number of studies have been conducted and the most relevant conclusions obtained are: (1) anemia is quite common in IBD; (2) although in many cases anemia parallels the clinical activity of the disease, many patients in remission have anemia, and iron, vitamin B12 and/or folic acid deficiency; (3) anemia, and also iron def iciency without anemia, have important consequences in the clinical status and quality of life of the patient; (4) oral iron can lead to gastrointestinal intolerance and failure of treatment; (5) intravenous iron is an effective and safe way to treat iron deficiency; (6) erythropoietin is needed in a significant number of cases to achieve normal hemoglobin levels. Thus, the clinician caring for IBD patients should have a comprehensive knowledge of anemia, and apply recently published guidelines in clinical practice.

Functional significance of erythropoietin receptor on tumor cells

Erythropoietin (Epo) is the regulator of red blood cell formation. Its receptor (EpoR) is now found in many cells and tissues of the body. EpoR is also shown to occur in tumor cells and Epo enhances the proliferation of these cells through cell signaling. EpoR antagonist can reduce the growth of the tumor in vivo. In view of our current knowledge of Epo, its recombinant forms and receptor, use of Epo in cancer patients to enhance the recovery of hematocrit after chemotherapy treatment has to be carefully evaluated.

Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

Is iron overload in alcohol-related cirrhosis mediated by hepcidin?

In this case report we describe the relationship between ferritin levels and hepcidin in a patient with alcohol-related spur cell anemia who underwent liver transplantation.We demonstrate a reciprocal relation-ship between serum or urinary hepcidin and serum ferritin,which indicates that inadequate hepcidin production by the diseased liver is associated with elevated serum ferritin.The ferritin level falls with increasing hepcidin production after transplantation.Neither inflammatory indices(IL6)nor erythropoietin appear to be related to hepcidin expression in this case.We suggest that inappropriately low hepcidin production by the cirrhotic liver may contribute substantially to elevated tissue iron stores in cirrhosis and speculate that hepcidin replacement in these patients may be of therapeutic benefi t in the future.

The effect of erythropoietin to the proliferation of cancer cell lines

Objective： To investigated whether erythropoietin （EPO） would promote the proliferation of human cancer cell line MCF-7, Panc-1 and HepG2 so as to determine whether EPO would result a worse prognosis to the patients with malignant diseases. Methods： We examined the growth inhibitory or promotional effect of EPO to MCF-7, Panc-1 and HepG2 by MTT assay. Then we examined the cell cycle of those cancer cell lines which was cultured with EPO and without EPO by FCM. Results： The results showed there was no significant growth inhibitory or promotional effect of EPO to MCF-7, Panc-1 and HepG2. The cell cycle of those cancer cell lines cultured with EPO also has no significant different to that of those cancer cell lines cultured without EPO. Conclusion： It is concluded that EPO has no promotion effect to the cancer cell lines＇ proliferation, and maybe will not result a worse prognosis to the patients with malignant diseases.

Erythropoietin -induced proliferation of gastric mucosal cells

AIM： To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS： Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine （BrdU）. RESULTS： Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of antierythropoietin antibody （P〈 0.01）. CONCLUSION： These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Prognostic significance of erythropoietin and erythropoietin receptor in gastric adenocarcinoma

AIM: To investigate the expression of Erythropoietin (Epo) and its receptor (EpoR) in gastric adenocarcinoma (GAC) and the correlation with angiogenesis and clinicopathological features. METHODS: The expressions of Epo, EpoR and vascular endothelial growth factor (VEGF), as well as mi-crovessel density were evaluated in 172 GAC biopsies by immunohistochemical staining. The correlations between these parameters and patient’s clinicopathological features were analyzed statistically. RESULTS: The proportion of Epo and EpoR alterations in GAC was higher than that in adjacent normal mucosa (P = 0.035 and 0.030). Epo high-expression was associ-ated with EpoR high-expression, Lauren type, extensivelymph node metastasis and advanced stage of GAC (P = 0.018, 0.018, 0.004 and 0), while EpoR expression was linked with older age, World Health Organization type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.013, 0.008 and 0.001). VEGF high expression was significantly correlated with EpoR low-expression, Lauren type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.001, 0.001 and 0.007). The expression of Epo or EpoR was associated with microvessel density (P = 0.004 and 0.046). On multivariate analysis, only lymph node metastasis, abnormal Epo expression and tumor nodes metastases stage were independently associated with survival. In addition, a strong association with the immunohistochemical expression of EpoR and the angiogenic protein, VEGF, was noted. CONCLUSION: Increased expression of Epo and EpoR may play a signif icant role in the carcinogenesis, angiogenesis and progression of GAC. Epo may be an inde-pendent prognostic factor.

Does recombinant human erythropoietin accelerate correction of post-ulcer-bleeding anaemia?A pilot study

AIM:Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron,but patients usually face a two-month recovery period from post- haemorrhage anaemia.This prospective,randomised,open, pilot study was designed to investigate whether recombinant human erythropoietin(Epoetin)therapy accelerate haematocrit increase in the post-bleeding recovery period. METHODS:We studied hospitalised patients admitted because of acute ulcer bleeding or haemorrhagic gastritis, who had a haematocrit of 27-33% and did not receive blood transfusions.One day after the endoscopic confirmation of cessation of bleeding,they were randomised either to erythropoietin(20 000 IU Epoetin alfa subcutaneously,on days 0,4 and 6)plus iron(100 mg im,on days 1-6,(G_1)or iron only(G_2).Haematocdt was measured on days 0,6,14, 30,45,and 60,respectively. RESULTS:One patient from G_1 and two from G_2 were lost to follow-up.Therefore,14 and 13 patients from G_1 and G_2 respectively were analysed.Demographic characteristics,serum iron,ferritin,total iron binding capacity,reticulocytes,and haernatoait were not significantly different at entry to the study. Median reticulocyte counts were significantly different between groups on day six(G_1:4.0,3.0-6.4 vs G_2:3.5,2.1-4.4%, P=0.03)and median haematocrit on day fourteen [G_1:35.9, 30.7-41.0 vs G_2:32.5,29.5-37.0%(median,range),P=0.04]. CONCLUSION:Erythropoietin administration significantly accelerates correction of anemia after acute ulcer bleeding. The haematocrit gain is equivalent to one unit of transfused blood two weeks after the bleeding episode.

STAT1 is involved in signal transduction in the EPOinduced HEL cells

Erythropoietin (EPO) is the ma jor regulator of mammalian erythropoisis, which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor (EPO-R). Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells. our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase (Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells. Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the binding of EPO to HEL cells.Furthermore, the binding of both STAT1 and STAT5 proteins to specific DNA elements (SIE and PIE elements) is revealed in an EPO-dependent manner. Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature erythroid cell from the spleen of mice infected with anemia strain of Friend virus.

Erythropoietin in liver cirrhosis: Two questions without answers

TO THE EDITORIn a recent paper[1], and in a subsequent letter[2], Tacke et al. reported the investigation of plasma erythropoietin (Epo) levels in patients affected by chronic liver disease of various aetiologies. The authors also compared[2] their data to our previous work[3]. The results show a substantial agreement but also some important differences between the two works. We would like to highlight, from our point of view, this issue. Both the papers demonstrated increased Epo values in anaemic cirrhotic subjects when compared to healthy controls and non-anemic patients with liver disease.

Erythropoietin： From a Biosimilar to Innovative Products Protected by Industrial Property Assets

Aim： EPO （erythropoietin） is a hormone that stimulates the erythropoiesis and is mainly produced by the kidneys. In the early 1990s among the emerging biotech drugs, the recombinant human EPO （rhEPO） was the best-selling product worldwide, reaching nearly three billion dollars annually. The CIM （center of molecular immunology） produced and sold the rhEPO as commercial strategy to recover the investment made in its new facilities. This work summarizes the inventions that protect the innovative products developed by three Cuban institutions, starting from rhEPO, and the industrial property strategy followed by them. Methods： The information was obtained from the United States Patent, Trademark Office （USPTO） database, Patentscope, Espacenet, patent databases of Center for Pharmaceutical Research and Drug Development （CIDEM） and Cuban Industrial Property Office. Conclusions： The manufacturing process of CIM＇s EPO has its own patent family. From a manufacturing by product an innovative formulation protected by patent was obtained. There is a patent family around the nasal formulation and it continues enlarging. From a biosimilar pharmaceutical innovative products impacting on human health have been obtained.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF ERYTHROPOIETIN AND ITS RECEPTOR EXPRESSION IN POSTNATAL RAT RETINA DEVELOPMENT

Objective To investigate the distribution of erythropoietin （EPO） and erythropoietin receptor （ EPOR ） expression in the postnatal rat retina development. Methods Forty-two male Sprague-Dawley rats were divided into 7 groups according to their various postnatal days： postnatal 1 d （D1 group）, 3 d （D3 group）, I week （W1 group）, 2 weeks （W2 group）, 3 weeks （W3 group）, 4 weeks （W4 group） and8 weeks （W8 group） （ n = 6 ）. Single eye was randomly chosen from each rat for the study. The retinal sections were stained with hematoxylin and eosin （HE） and used for the retina development observation. Immunohistochemical staining was used to localize EPO and EPOR expressions in retinas.of differentstages of development, and the expression intensities were determined by an image plus 4 program~~ Results The retinal inner nuclear layer （INL） and outer nuclear layer （ONL） were mixed together and had not yet fully differentiated in D1 and D3 groups. The INL and ONL formed their own independent regions and the outer plexiform layer （OPL） appeared between two layers in W1 group. With the postnatal retinal development, the inner plexiform layer （ IPL ） , rods and cones layer （ RCL ）, and OPL were gradually widened and stabilized in W2 to W3 groups. EPO/EPOR expressions located prominently in the inner part of the postnatal rat developing retinas. The expression of EPO in GCL and INL gradually increased from DI to W4, then the expression decreased in W8. Expression of EPOR in GCL gradually increased from DI to WI , then decreased in W2 ; and it gradually increased again from W3 to W8. Expression of EPOR in INL gradually increased from D1 to W1, then decreased in W2 ; and it continued to decrease from W3 to W8. Expression of EPOR in the external segment of RCL gradually increased from D1 to W8. However, expression in the internal segment of RCL gradually decreased from D1 to W3 , then no obvious expression was seen in the internal segment of RCL in W4 and W8. Conclusion EPO/EPOR expressions locate prominently in the inner part of the postnatal rat developing retina. And EPO/EPOR expressions in the rat retinas exist the dynamic changes during the postnatal retina development period.

炎性肠病相关贫血的研究进展

贫血是炎性肠病（IBD）患者常见且严重的并发症．贫血可致该类患者生活质量的下降及住院时间和住院次数的增加。大量研究及相关结论证实：（1）贫血情况在IBD中普遍存在；（2）尽管在很多情况下贫血与IBD患者疾病活动性相关．但许多缓解期患者同样存在贫血和铁、维生素B12和（或）叶酸的缺乏：（3）贫血或单纯缺铁即可对患者病情转归和生活质量造成严重影响：（4）口服铁剂受患者低吸收率、耐受差和肠道病变部位氧化应激反应的影响而疗效不佳；（5）静脉补铁有效率高，且可明显改善患者生活质量；（6）为达到理想的血红蛋白水平。联合应用促红细胞生成素（EPO）可使血红蛋白水平快速显著提高．进一步改善病情。因此．对于IBD患者．临床医生对贫血状况应该有全面认识以进一步指导临床工作。

Comparative study on different derivatization procedures for analysis of recombinant human erythropoietin by capillary electrophoresis with laser-induced fluorescence detection

Human erythropoietin （hEPO）, an endogenous glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant human erythropoietin （rhEPO） has made it possible for its abuse in competitive sports. In this work, pre-capillary and on-capillary derivatization by 5-furoylquinoline-3-carboxaldehyde （FQ） and fluorescein isothiocyanate （FITC） for the detection of rhEPO by capillary electrophoresis with laser-induced fluorescence detection （CE-LIF） were compared. FQ pre-capillary labeling improves sensitivity but degrades the glycoforms separation due to the inhomogeneity of the reaction products from multiple labeling. Compared with FITC pre-capillary derivatization with the excess fluorescent background, the on-capillary FQ derivatization method can provide shorter analysis time, lower background, and better selectivity. It is demonstrated that, through optimizing reaction conditions of FQ on-capillary derivatization, both high sensitivity and satisfactory resolution for the analysis of the be used for the glycoforms profiling and quality control of rhEPO doping control analysis. glycoforms of rhEPO could be obtained. This method can It may be used as a candidate method for fast screening in

Recombinant human erythropoietin counteracts cisplatin-induced visceral hyperalgesia

Objective Cisplatin exerts its cytotoxic effect through distinct DNA lesions,leading to peripheral neuropathy.The risk of sensory neuropathy is a common problem during cancer treatment with cisplatin,leading to somatic hyperalgesia.Yet,data focussing on cisplatin-induced impairment of the autonomic nervous system are limited.The present study was aimed to investigate the effect of recombinant human erythropoietin（rhEPO）on cisplatin-induced visceral hyperalgesia.Methods C57BL/6 mice were treated either with cisplatin（2 mg/kg,once per week）or with cisplatin（2 mg/kg,once per week）plus rhEPO （40μg/kg,3 times per week）for 8 weeks.Controls were treated with saline.To quantify the visceromotor response（VMR）at week 9,standardized electrodes were implanted into the external oblique musculature for electromyographic recordings.After that,animals were decapitated and dorsal root ganglia（DRG）was removed for transmission electron microscopy studies.Results Cisplatin-treated mice showed a significant increase of VMR compared to the controls[（7080±969）vs（2864±279）;P0.001],while rhEPO dramatically counteracted this effect[（2962±336）vs（7080±969）;P0.001）].Transmission electron microscopy revealed cisplatin-induced structural lesions of nuclear membrane in DRG cells,which could be ameliorated by rhEPO.Conclusion Erythropoietin can significantly ameliorate the cisplatin-induced visceral hyperplasia and DRG nuclear membrane structure damage in mice,indicating a neuroprotective role of erythropoietin.