Abstract
BackgroundRecombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved.MethodsLoss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting.ResultsIn contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA.ConclusionTogether, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.
Highlights
Recombinant human erythropoietin alpha has been reported to protect the liver of rats and mice from ischemia-reperfusion injury
Cultured rat hepatocytes were pre-incubated in the presence or absence of Recombinant human erythropoietin alpha (rHu-erythropoietin alpha (EPO)) (100 U/ml) in L-15 medium (37°C) for 20 hours at normoxia (74% N2/21% O2/ 5% CO2)
Effect of rHu-EPO on cold-induced apoptosis of cultured rat hepatocytes In former studies we have demonstrated that the incubation of cultured rat hepatocytes at 4°C leads to an increased cellular "chelatable iron pool" which later mediates the opening of the mitochondrial permeability transition pore eventually triggering apoptotic cell death [25,28,39]
Summary
Recombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. Treatment of rats and mice with recombinant human erythropoietin alpha (rHu-EPO) significantly reduced ischemia-reperfusion injury as indicated by decreases in histopathological scores, release of liver enzymes, markers of apoptosis, injurious intracellular signalling, and reactive oxygen species formation [4,5,6,7,8,9]. A variety of additional signal transduction pathways have been proposed In this context, signalling via signal transducer and activator of transcription 3 (STAT-3) is considered to be involved in transducing EPO-dependent cell protection in non-erythroid tissues [16,17,18]. B-cell lymphoma 2 (Bcl-2) is among the target genes that confer the anti-apoptotic action of EPO in hematopoietic progenitor cells [19]
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