Injection Of Recombinant Human Erythropoietin Research Articles

Correction of hyperphosphatemia in a patient with end-stage chronic kidney disease and a complex case of anemia. A clinical case

Iron supplements and/or blood transfusions in combination with recombinant human erythropoietin injections are routinely used to correct renal anemia, which is a common complication of end-stage renal disease. However, massive blood transfusions and inadequate intravenous iron supplementation can lead to iron overload syndrome, and anemia is not always nephrogenic. A rare clinical case of a patient with end-stage chronic kidney disease with complex genesis of anemia, who is on hemodialysis, is presented. The case is interesting because the prescription of first-line drugs was life-threatening in nature, and sevelamer was the drug chosen for the correction of mineral-bone disorders.

A novel alternative for pyrogen detection based on a transgenic cell line

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted ‘reduction, replacement and refinement’ principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

Efficacy observation of erythropoietin on sepsis complicated with acute respiratory distress syndrome

This study is to evaluate the efficacy of erythropoietin in treating sepsis-associated acute respiratory distress syndrome (ARDS). One hundred patients with sepsis-related ARDS were randomized into the placebo group and Erythropoietin (EPO) group. Patients in the placebo group received saline as placebo on the standard therapy, while the EPO group received recombinant human erythropoietin injections on the standard therapy. It was found that the heart rate and mean arterial pressure did not differ significantly between days 7 and 14 after treatment initiation (p > 0.05). The partial pressure of oxygen (PaO2) and oxygenation index levels measured on days 7 and 14 were significantly higher than the placebo group and partial pressure of carbon dioxide (PaCO2) was significantly lower than the placebo group (p < 0.05). Lung capacity and functional residual capacity (and FRC) increased significantly in tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and C-reactive protein (CRP) concentrations (p < 0.05). In the EPO group, the duration of mechanical ventilation was significantly shorter and the mortality rate was significantly reduced (Log-Rank test, χ2 = 4.651, p = 0.031). The results confirm that EPO significantly improves lung function and blood gas parameters, reduces serum levels of inflammatory markers, and reduces the risk of death in sepsis-induced ARDS patients, highlighting the potential therapeutic role of EPO in the management of this disease.

Efficacy and Mechanism of Roxadustat plus Oral Iron in the Treatment of Elderly Chronic Kidney Disease with Anemia.

Objective For investigating the efficacy and mechanism of Roxadustat + oral iron in the treatment of elderly chronic kidney disease (CKD) complicated with anemia. Methods A total of 100 elderly patients with CKD and anemia admitted to our hospital between April 2020 and December 2021 were enrolled as research subjects, and the patients were assigned to control group (Con group, n = 50) or experimental group (Exp group, n = 50). The patients in the Con group were given oral iron, and those in the Exp group were given Roxadustat capsule based on the Con group. Both groups were given subcutaneous injection of recombinant human erythropoietin. The clinical efficacy, anemia indexes, iron metabolism indexes, inflammatory indexes, and adverse reactions were compared between the two groups. Results The Exp group showed a notably higher treatment effective rate than the Con group (P < 0.05). After treatment, the anemia indexes, iron metabolism indexes, and inflammatory indexes in the Exp group were notably better than those in the Con group (P < 0.05). The Exp group showed a notably lower incidence of adverse reactions during treatment than the Con group (P < 0.05). Conclusion Roxadustat plus oral iron yields a pronounced clinical efficacy in the therapy of elderly patients with CKD and anemia.

Efficacy and safety of darbepoetin alfa injection replacing epoetin alfa injection for the treatment of renal anemia in Chinese hemodialysis patients: A randomized, open-label, parallel-group, noninferiority phase III trial.

BackgroundThis study was to explore the clinical efficacy and safety of darbepoetin alfa injection replacing epoetin alfa injection (recombinant human erythropoietin injection, rHuEPO) for the treatment of anemia associated with chronic kidney failure in Chinese patients undergoing hemodialysis.MethodThis study was a multicenter, randomized, open‐label, intergroup parallel control phase III noninferiority trial from April 19, 2013 to September 9, 2014 at 25 sites. In this study, the members of the darbepoetin alfa group underwent intravenous administration once per week or once every two weeks. The members of the control drug epoetin alfa group underwent intravenous administration two or three times per week. All subjects underwent epoetin alfa administration during the 8‐week baseline period. After that, subjects were randomly assigned to the darbepoetin alfa group or epoetin alfa group. The noninferiority in the changes of the average Hb concentrations from the baseline to the end of the evaluation period (noninferiority threshold: −1.0 g/dl) was tested between the two treatments. The time‐dependent hemoglobin (Hb) concentration and the maintenance rate of the target Hb concentration (the proportion of subjects with Hb concentrations between 10.0 and 12.0 g/dl) were also evaluated. Iron metabolism, including changes in the serum iron, total iron‐binding capacity, ferritin, transferrin saturation, and comparisons of the dose adjustments between the two groups during the treatment period were analyzed further. Adverse events (AEs) were also observed and compared, and the safety was analyzed between the two treatment groups. The conversion rate switching from epoetin alfa to darbepoetin alfa was also discussed. SAS® software version 9.2 was used to perform all statistical analyses. Descriptive statistics were used for all efficacy, safety, and demographic variable analyses, including for the primary efficacy indicators.ResultsFour hundred and sixty‐six patients were enrolled in this study, and ultimately 384 cases were analyzed for safety, including 267 cases in the darbepoetin alfa group and 117 cases in the epoetin alfa group. There were 211 cases in the per‐protocol set, including 152 cases in the darbepoetin alfa group and 59 cases in the epoetin alfa group. The changes in the average Hb concentrations from the baseline to the end of the evaluation period were −0.07 and −0.15 g/dl in the darbepoetin alfa group and epoetin alfa group respectively. The difference between the two groups was 0.08 g/dl (95% confidence interval [CI]: −0.22 to 0.39), and the lower limit of the 95% CI was −0.22 > −1.0 g/dl. The average Hb concentrations of the two groups were 10.88–11.43 g/dl (darbepoetin alfa) and 10.91–11.38 g/dl (epoetin alfa) during the study period of Weeks 0–28, with the maintenance rates of the target Hb concentration ranging within 71%–87% and 78%–95% in the darbepoetin alfa group and epoetin alfa group respectively. During the period of comparison between the two groups, the incidence of AEs in the darbepoetin alfa group was 61.42%, while in the epoetin alfa group it was 56.41%. All of the adverse events and reactions in the study were those commonly associated with hemodialysis.ConclusionThe overall efficacy and safety of darbepoetin alfa for the treatment of Chinese renal anemia patients undergoing hemodialysis are consistent with those of epoetin alfa.

Darbepoetin alfa injection versus epoetin alfa injection for treating anemia of Chinese hemodialysis patients with chronic kidney failure: A randomized, open-label, parallel-group, non-inferiority Phase III trail.

BackgroundErythropoietin is a glycoprotein that mainly regulates erythropoiesis. In patients with chronic renal failure with anemia, darbepoetin alfa can stimulate erythropoiesis, correct anemia, and maintain hemoglobin levels. This study was designed to demonstrate the efficacy and safety of darbepoetin alfa injections as being not inferior to epoetin alfa injections (Recombinant Human Erythropoietin injection, rHuEPO) when maintaining hemoglobin (Hb) levels within the target range (10.0–12.0 g/dL) for the treatment of renal anemia.MethodsNinety‐five patients were enrolled in this study from April 15, 2013 to April 10, 2014 at 25 sites. In this study, patients (n = 95) aged 18–70 years were randomized into a once per week intravenous darbepoetin alfa group (n = 56) and a twice or three times per week intravenous epoetin alfa group (n = 39) for 28 weeks, who had anemia with hemoglobin levels between 6 g/dL and 10 g/dL due to chronic kidney disease (CKD) and were undergoing hemodialysis or hemofiltration with ESA‐naive (erythropoiesis stimulating agent‐naive). The primary efficacy profile was the mean Hb level (the non‐inferiority margin was −1.0 g/dL, week 21–28); the secondary efficacy profiles were the Hb increase rate (week 0–4), the target Hb achievement cumulative rate and time, the change trends of the Hb levels, and the target Hb maintenance ratio. Adverse events (AEs) were observed and compared, and the efficacy and safety were analyzed between the two treatment groups. Additionally, the frequencies of dose adjustments between the darbepoetin alfa and epoetin alfa groups were compared during the treatment period. SAS® software version 9.2 was used to perform all statistical analyses. Descriptive statistics were used for all efficacy, safety, and demographic variable analyses, including for the primary efficacy indicators.ResultsThe mean Hb level was 11.3 g/dL in the darbepoetin alfa group and 10.7 g/dL in the epoetin alfa group, respectively; the difference of the lower limits of the 95% confidence intervals (CI) between the two groups was 0.1 g/dL (>−1.0 g/dL), and non‐inferiority was proven; the Hb levels started to increase in the first four weeks at a similar increase rate; no obvious differences were observed between the groups in the target Hb achievement cumulative rates, and the Hb levels as well as the target Hb level maintenance rate changed over time. The incidence of AEs was 62.5% in the darbepoetin alfa group and 76.9% in the epoetin alfa group. All the adverse events observed in the study were those commonly associated with hemodialysis.ConclusionDarbepoetin alfa intravenously once per week can effectively increase Hb levels and maintain the target Hb levels well, which makes it not inferior to epoetin alfa intravenously twice or three times per week. Darbepoetin alfa shows an efficacy and safety comparable to epoetin alfa for the treatment of renal anemia.

Comparison of the effects of systemic and subcutaneous injection of recombinant human erythropoietin on the tissue-healing process in BALB/c Mice

Introduction: Erythropoietin (EPO) is a glycoprotein hormone that is secreted for biological responses and is regulated by the interaction between EPO and specific cytokine receptors. The EPO is a mediator of angiogenesis and erythropoiesis and can increase several growth factors. The current research aimed to compare the effects of systemic and subcutaneous injection of recombinant human EPO on the tissue-healing process. Methods: For the purposes of the study, 30 male BALB/c mice were selected and divided into three groups. The first one received EPO treatment with systemic injection (n=10) while the second one received EPO treatment with subcutaneous injection (n=10). The third group was the control or placebo group without any treatment (n=10). The biopsies were studied on days 4, 7, 10, and 14 through staining by hematoxylin-eosin and Mallory's trichrome. In each group, 3 mice were sacrificed due to exposure to high doses of anesthetic on days 4 and 7. Moreover, 2 other mice were sacrificed after 10 and 14 days. The collected data and images were analyzed in SPSS (version 18.0) and ImageJ software, respectively. Furthermore, they were analyzed using Wilcoxon signed-rank and analysis of variance tests in. During all the analytical processes, a p-value of &lt;0.05 was considered statistically significant Results: According to the results, the density of inflammatory cells significantly decreased in EPO-treated groups, compared to the control group. Moreover, the EPO-treated groups underwent an increase regarding the number of fibroblast cells, new capillary formation, and epithelial thickness in the margins of the wound, unlike the control group. Conclusions: Findings of the present study indicated that the systemic injection of EPO resulted in a better physiological response regarding the acceleration of tissue-healing in comparison with the subcutaneous injection. Furthermore, the systemic injection of EPO improved the parameters related to skin wound repair.

Detection of Stimulated Erythropoiesis by the RNA-Based 5'-Aminolevulinate Synthase 2 Biomarker in Dried Blood Spot Samples

Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.

Tubular immaturity causes erythropoietin-deficiency anemia of prematurity in preterm neonates

Kidneys are physiologically hypoxic due to huge oxygen consumption for tubular reabsorption. The physiological hypoxia makes the kidney an appropriate organ for sensitively detecting oxygen levels and producing erythropoietin (EPO). In preterm neonates, immature kidneys cannot produce sufficient EPO, which results in anemia of prematurity (AOP). The cause of EPO insufficiency in AOP has been unclear, therefore current therapeutic options are transfusion and injection of recombinant human EPO. This report shows that the cause of insufficient EPO production in AOP is elevated renal oxygen levels due to poor oxygen consumption by immature tubules. Neonatal mice with AOP showed low tubular transporter expression and elevated renal oxygen levels compared with those without AOP. Enhancing transporter expression in AOP mice induced renal hypoxia and EPO production. In preterm neonates, red blood cell counts, hemoglobin levels, and hematocrit levels correlated with tubular function, but not with serum creatinine, gestational age, or birth weight. Furthermore, pharmacological upregulation of hypoxia signaling ameliorated AOP in mice. These data suggest that tubular maturation with increased oxygen consumption is required for renal EPO production.

FGF23 expression in rodents is directly induced via erythropoietin after inhibition of hypoxia inducible factor proline hydroxylase.

Plasma levels of FGF23 are increased in patients with chronic kidney disease. Beside its role in phosphate homeostasis, iron deficiency and anemia are associated with increased FGF23 plasma levels. Recently, FGF23 plasma levels were shown to be increased in mice after treatment with hypoxia inducible factor-proline hydroxylase (HIF-PH) inhibitors which are strong inducers of erythropoietin and erythropoiesis and are known to modulate iron uptake and availability. Therefore we investigated a potential context between expression of FGF23 and stimulation of erythropoiesis using a HIF-PH inhibitor and erythropoietin in rats. FGF23 plasma levels are induced at peak levels 2 h after intravenous injection of recombinant human Erythropoietin (rhEPO). Likewise induction of endogenous EPO using a HIF-PH inhibitor (BAY 85–3934) is followed by an increase of FGF23 plasma levels. In contrast to rhEPO the HIF-PH inhibitor induces lower peak levels of FGF23 applying equivalent hematopoietic doses. Bone and bone marrow were identified as sources of EPO-induced FGF23. Immediate induction of FGF23 mRNA was also detected in EPO receptor positive murine hematopoietic BAF3 cells after treatment with rhEPO but not after treatment with the HIF-PH inhibitor. Pretreatment of mice with a neutralizing anti-EPO antibody abrogated FGF23 induction by the HIF-PH inhibitor. Thus, direct impact on FGF23 expression by HIF-PH inhibition in vivo via hypoxia mimicking and modulation of iron metabolism appears unlikely. Collectively, the findings point to an EPO dependent regulation pathway of FGF23 gene expression which might be important in the context of erythropoiesis stimulating therapies in patients with renal anemia.

Abstract 4068: HH-002, a derivative of Homoharringtonine, show promsing in diverse cancer models in preclinical characterization

Abstract Homoharringtonine (HHT) is an anti-cancer drug used to treat imatinib resistant CML, and a reversible inhibitor of translation acting upon the early steps in polypeptide chain elongation. BS-HH-002.SA is a derivative of HHT. BS-HH-002.SA was shown to decrease the phosphorylation of Janus protein tyrosine kinase 2 (JAK2) as well as downstream targets, such as signal transducers and activators of transcription 3 and 5 (STAT3 and STAT 5), it also reduced Myeloid cell leukemia-1 (Mcl-1) protein level significantly and resulted in apoptosis. Evaluation of BS-HH-002.SA efficacy. was test in HEL, an erthroleukemia cell line carrying a JAK2V617F mutation which is seen in 90% of patients with polycythemia vera (PCV) and half of those with essential thrombocythemia or primary myelofibrosis. HH-002.SA showed was highly potent (at low nM concentration) to HEL. Efficacy of BS-HH-002 was tested in a recombinant human erythropoietin injection (rhEpo)-induced murine model of polycythemia Vera, Treatment with BS-HH-002.SA BID for 4 days showed a significant inhibitory effect on rhEpo induced increase in reticulocytes, hematocrit and splenomegaly, as well as in red blood cell, white blood cell and platelet counts. Furthermore, histology evaluations revealed that BS-HH-002.SA exerted an inhibitory effect on rhEpo induced extramedullary hematopoiesis in spleen, and decreased cellularity in bone marrow with more predominant effect on erythroid cells than on myeloid cells. Also, BS-HH-002.SA also show potent in vitro and in vivo efficacy in pancreatic cancer cell line KP-4 and non-small cell lung cancer (NSCLC) NCI-H1975, BS-HH-002.S inhibited STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression Mcl-1 protein levels in both two cancer cell lines. Note: This abstract was not presented at the meeting. Citation Format: Hank Rui. HH-002, a derivative of Homoharringtonine, show promsing in diverse cancer models in preclinical characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4068. doi:10.1158/1538-7445.AM2017-4068

Erythropoietin (EPO) ameliorates obesity and glucose homeostasis by promoting thermogenesis and endocrine function of classical brown adipose tissue (BAT) in diet-induced obese mice

Erythropoietin (EPO), clinically used as a hematopoietic drug, has received much attention due to its nonhematopoietic effects. EPO reportedly has beneficial effects on obesity and diabetes mellitus. We investigated whether interscapular brown adipose tissue (iBAT: main part of classical BAT) could play a role in EPO’s anti-obesity and anti-diabetic effects in diet-induced obese mice. Four-week-old male C57BL/6J mice were fed a high-fat diet (HFD-Con), and half were additionally given an intraperitoneal injection of recombinant human EPO (200 IU/kg) (HFD-EPO) thrice a week for four weeks. At 8 weeks, EPO-injected mice showed significantly reduced body weight with reduced epididymal and subcutaneous white fat mass and unchanged caloric intake and locomotor activity. HOMA-IR (insulin resistance index) and glucose levels during intraperitoneal glucose tolerance test (IPGTT) were significantly lower in HFD-EPO mice than in HFD-Con mice. EPO-injected mice also showed increased oxygen consumption, indicative of metabolic rate, and skin temperature around iBAT tissue masses. EPO significantly upregulated the PRD1-BF1-RIZ1 homologous domain containing 16 (PRDM16), a transcriptional factor with a crucial role in brown adipocyte differentiation. EPO significantly increased phosphorylated signal transducer and activator of transcription 3 (STAT3), which is downstream of erythropoietin receptor (EpoR) and known to stabilize PRDM16. EPO’s suppression of myocyte enhancer factor 2c (Mef2c) and microRNA-133a (miR-133a) via β3-adrenergic receptor caused PRDM16 upregulation. EPO-mediated enhancement of EpoR/STAT3 and β-adrenergic receptor/Mef2c/miR-133 pathways dramatically increases total uncoupling protein 1 (UCP1), an essential enzyme for BAT thermogenesis. Furthermore, EPO activated BAT’s endocrine functions. EPO facilitated fibroblast growth factor 21 (FGF21) production and excretion in iBAT, associated with reduction of liver gluconeogenesis-related genes. Thus, EPO’s improvement of obesity and glucose homeostasis can be attributed to increased iBAT thermogenic capacity and activation of BAT’s endocrine functions.

DNA methylation is necessary for erythropoietin to improve spatial learning and memory in SAMP8 mice

ObjectiveTo reveal the role of Dnmts in the improvement of spatial learning and memory induced by erythropoietin (EPO) in SAMP8 mice. MethodsThe Morris water maze (MWM) was used to assess spatial learning and memory. Mice were administered by intraperitoneal (i.p.) injection of recombinant human EPO and hippocamppi infusion (IH) of 5-aza-2′-deoxycytidine (5-AZA). The expression of genes Dnmt1, Dnmt3a and Dnmt3b in the hippocampus was detected by real-time qPCR. The level of proteins DNMT1, DNMT3A and DNMT3B was measured by Western blotting. ResultsSpatial learning and memory in SAMP8 were promoted after i.p. injection of EPO (5000IU/kg/day) and expression of Dnmt3b mRNA and DNMT3B proteins in the hippocampus increased. The improved memory by EPO was blocked after IH 5-AZA. ConclusionDNA methylation is necessary for EPO to enhance spatial learning and memory in SAMP8 mice.

Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor.

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.

Protective effects of recombinant human erythropoietin on oligodendrocyte after cerebral infarction

To study biological effect of recombinant human erythropoietin (RhEPO) on the expression of oligodendrocyte in the neuron glia antigen 2(NG2), Nogo receptor-interacting protein 1(LINGO-1), myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to explore the protective mechanism of RhEPO for oligodendrocyte after cerebral infarction. Experimental rats were randomly divided into the treatment group (RhEPO at a dose of 3 000 U/kg) or saline control group. Both groups received intraperitoneal injection of RhEPO after cerebral ischemia in 30 min, 3 h, 6 h, 12 h and 24 h, which was administered daily for 7 days. The modified neurological severity score (mNSS) and histology were analyzed, and immunohistochemistry was used to detect the protein expression of NG2, MAG, MBP and LINGO-1. The overall mNSS of RhEPO treatment group significantly decreased compared with the saline control group on the seventh day after cerebral infarction (P<0.05). Such treatment effect was more obvious in the treatment group at 30 min and 3 h (P<0.01). Compared with the saline control group, the numbers of NG2 positive cells increased in RhEPO treatment group. In contrast, the expression of LINGO-1 protein significantly decreased (P<0.05), with a dramatic decrease observed at 30 min and 3 h (P<0.01). However, the expression of MBP protein decreased more significantly in saline control group, while the level of the MAG protein expression increased. The differences were statistically significant (P<0.05), especially at 30 min (P<0.01). After cerebral ischemia, RhEPO promotes the proliferation of NG2 positive cells, and inhibits the expression of LINGO-1 and MAG proteins. RhEPO improves the proliferation and differentiation of oligodendrocyte precursor cells, which in turn protects neuronal function, particularly at the early phase of ischemia.

Erythropoietin enhances whole body lipid oxidation during prolonged exercise in humans.

Animal studies have suggested that erythropoietin, besides its well-known hematopoietic effects, can modulate metabolism and prevent fat accumulation. We investigated the effects of repeated injections of recombinant human erythropoietin (EPO) on the balance of substrate oxidation during aerobic exercise in humans. Twelve healthy aerobically trained males received subcutaneously either moderate dose of EPO (50 U/kg, EPO) or saline injections (NaCl 0.9 %, control) three times a week for 4 weeks. Body weight, % fat, maximal aerobic capacity, and substrate utilization during exercise were assessed before and after treatment, while hemoglobin and hematocrit were monitored regularly during the treatment. Carbohydrate and fat oxidation were evaluated via indirect calorimetry, during a submaximal exercise performed at 75 % of the participants' maximal aerobic capacity (V̇(O2max)) for 60 min. Results showed that 4 weeks of EPO treatment significantly enhanced fat oxidation (+56 % in EPO versus -9 % in control) during exercise, independent of its effects on hematological parameters or V̇(O2max). This study shows that EPO can modulate substrate utilization during exercise, leading to enhanced fat utilization and lower use of carbohydrates. This opens new research directions exploring whether systemic EPO levels, in physiological conditions, participate to the modulation of fat oxidation.

Wound Healing - A Proteomic Analysis of the Effect of Erythropoietin on Granulation Tissue Isolated from ePTFE Implants

Background: Erythropoietin is a multifunctional cytokine, with recent studies indicating a positive effect on wound healing in models of incisional and burn wounds. Objective: The objective of this study was to investigate the changes in protein expression after daily injections of recombinant human erythropoietin in granulation tissue isolated from subcutaneous implants of expanded polytetrafluoroethylene (ePTFE tube). Methods: ePTFE tubes were implanted subcutaneously in twelve C57bl6 mice. Six of the mice were treated with daily subcutaneous injection of recombinant human erythropoietin of 1000 IU/kg. The remaining six mice, the control group, were treated with injections of 0.9% saline. The implants were removed on day 9 after implantation. The granulation tissue was isolated and analyzed for protein expression using a proteomic approach including two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Some of the results were confirmed with Western Blotting. Results: We identified 33 differentially expressed proteins in the rhEPO-treated group. Three proteins involved in the glycolytic pathway — Glyceraldehyde-3-phosphate dehydrogenase, Alpha-enolase, and Triosephosphate isomerase — were altered in the granulation tissue from the rhEPO-treated group. Conclusion: Daily injection of recombinant human erythropoietin of 1000 IU/kg alters the protein expression of GAPDH, ENOA and TPIS in granulation tissue from wounds on postoperative day 9. The successful combination of proteomic analysis of wound tissue and the ePTFE wound model could advance our knowledge of the complex healing process.

Study to establish the role of JAK2 and SMAD1/5/8 pathways in the inhibition of hepcidin by polysaccharides from Angelica sinensis

Ethnopharmacological relevanceAngelica sinensis polysaccharide (ASP) is one of the major active ingredients in Angelica sinensis (Oliv.) Diels. This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases. Aim of the studyPrevious studies have suggested that ASP from the roots of Angelica sinensis (Oliv.) Diels suppresses hepcidin expression, but the underlying molecular mechanisms are not known. The present study was designed to establish the role of the janus-kinases 2 (JAK2) and son of mothers against decapentaplegic 1/5/8 (SMAD1/5/8) pathways in the inhibition of hepcidin by polysaccharides from Angelica sinensis in normal rats. Materials and methodsASP was administered orally (0.3, 0.6 and 1.2g/kg body weight) to male Sprague–Dawley rats every day for 20 days. Intraperitoneal injections of recombinant human erythropoietin (rhEPO; 800 and 2000U/kg body weight) were given to the positive control group every day for 3 days. After administration, hepcidin levels, blood parameters, serum iron status and non-heme iron concentrations in the liver were examined. Western blot analyses were used to investigate the expression of five relevant signaling proteins in the liver. ResultsRhEPO injection significantly stimulated erythropoiesis and expression of the serum transferrin receptor (sTfR), and decreased serum iron status and non-heme iron concentrations in the liver. However, blood parameters barely changed in the ASP groups. sTfR, serum iron, and liver iron levels altered only in the ASP high-dose group (1.2g/kg body weight). rhEPO and ASP significantly reduced hepcidin expression by inhibiting the expression of phospho-SMAD1/5/8 and JAK2 in the liver, but not through transmembrane protease serine 6 (TMPRSS6) and extracellular signal-regulated kinase 1/2 (ERK1/2). ConclusionsThese data suggested that ASP can interrupt the JAK2 and SMAD1/5/8 pathways, which eventually results in lower expression of hepcidin.

Acute, NO‐mediated, effects of erythropoietin are not associated with its cardioprotective properties

Our serendipitous observation of acute reduction of blood pressure associated with a systemic injection of recombinant human erythropoietin (rhEPO) in the rat prompted a systematic evaluation of the acute effects of rhEPO on cardiovascular function and cardioprotection. Four groups of rats (Wistar, male, 3‐mo old) received bolus i.v. injection of one of the following: physiological saline, rhEPO (3000 U/kg), saline+L‐NAME (15 mg/kg), or rhEPO+L‐NAME. Two hours after injections rats were anesthetized with Isoflurane and subjected to detailed analyses of cardiovascular performance via open chest pressure‐volume loops analyses. In the second experiment four groups of rats were subjected to a permanent ligation of descending coronary artery immediately followed by the same i.v. injections as in the first experiment. Twenty four hours following coronary ligation hearts were harvested and myocardial infarct size (MI) was measured histologically. A significant reduction of arterial blood pressure two hours after a rhEPO injection was accompanied with a reduction of left ventricular systolic function (Table). L‐NAME completely suppressed this acute cardiovascular response to rhEPO. In contrast, the 50% reduction of MI size observed 24 hrs after coronary ligation in rhEPO‐treated vs untreated rats was not affected by L‐NAME treatment. Thus, these novel NO‐mediated acute hemodynamic effects of rhEPO are not associated with EPO‐associated cardioprotection. Parameters Saline rhEPO ESP 85±5 66±5* dP/dt+ 7136±530 5420±382* PRSW 51.8±3.9 34.6±2.5* BPs 90±6 70±3* BPd 55±5 38±3 BPm 66±6 49±3* ‐p&lt;0.05

Effects of erythropoietin on angiogenesis after myocardial infarction in porcine

Erythropoietin (EPO) has recently been shown to confer cardioprotective effects via angiogenesis and antiapoptosis. The administration of EPO after myocardial infarction (MI) reduces infarct size and improves cardiac function in small animals. The purpose of this study is to investigate the protective effects of EPO in porcine MI. Each animal in the EPO group received four injections of recombinant human EPO (rhEPO; 6000 U per injection) at 2-day intervals, starting after coronary occlusion. Animals in the control group received saline. Left ventriculography was performed just after coronary occlusion and at 28 days. Time-course changes in serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF) were measured. The number of vessels was calculated, and the mRNA expressions of VEGF and insulin-like growth factor (IGF) were examined. Left ventricular function was similar between the groups. The numbers of cells positive for anti-α-smooth muscle actin, von Willebrand factor, and c-kit were significantly higher in the EPO group than in the controls (P < 0.05). The EPO group exhibited significantly higher HGF and FGF concentrations (P < 0.05) and higher expression of VEGF and IGF mRNA (P < 0.05) compared with the controls. In conclusion, EPO accelerates angiogenesis via the upregulation of systemic levels such as HGF and FGF, and the local expression of VEGF and IGF, in porcine MI.