Attenuation of Inflammation and Apoptosis by Pre- and Posttreatment of Darbepoetin-α in Acute Liver Failure of Mice
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.061056
ISSN1525-2191
AutoresKhoi M. Le, Katja Klemm, Kerstin Abshagen, Christian Eipel, Michael D. Menger, Brigitte Vollmar,
Tópico(s)Iron Metabolism and Disorders
ResumoIn many liver disorders inflammation and apoptosis are important pathogenic components, finally leading to acute liver failure. Erythropoietin and its analogues are known to affect the interaction between apoptosis and inflammation in brain, kidney, and myocardium. The present study aimed to determine whether these pleiotropic actions also exert hepatoprotection in a model of acute liver injury. C57BL/6J mice were challenged with d-galactosamine (Gal) and Escherichia coli lipopolysaccharide (LPS) and studied 6 hours thereafter. Animals were either pretreated (24 hours before Gal-LPS exposure) or posttreated (30 minutes after Gal-LPS exposure) with darbepoetin-α (DPO, 10 μg/kg i.v.). Control mice received physiological saline. Administration of Gal-LPS caused systemic cytokine release and provoked marked hepatic damage, characterized by leukocyte recruitment and microvascular perfusion failure, caspase-3 activation, and hepatocellular apoptosis as well as enzyme release and necrotic cell death. DPO-pretreated and -posttreated mice showed diminished systemic cytokine concentrations, intrahepatic leukocyte accumulation, and hepatic perfusion failure. Hepatocellular apoptosis was significantly reduced by 50 to 75% after DPO pretreatment as well as posttreatment. In addition, treatment with DPO also significantly abrogated necrotic cell death and liver enzyme release. In conclusion, these observations may stimulate the evaluation of DPO as hepatoprotective therapy in patients with acute liver injury. In many liver disorders inflammation and apoptosis are important pathogenic components, finally leading to acute liver failure. Erythropoietin and its analogues are known to affect the interaction between apoptosis and inflammation in brain, kidney, and myocardium. The present study aimed to determine whether these pleiotropic actions also exert hepatoprotection in a model of acute liver injury. C57BL/6J mice were challenged with d-galactosamine (Gal) and Escherichia coli lipopolysaccharide (LPS) and studied 6 hours thereafter. Animals were either pretreated (24 hours before Gal-LPS exposure) or posttreated (30 minutes after Gal-LPS exposure) with darbepoetin-α (DPO, 10 μg/kg i.v.). Control mice received physiological saline. Administration of Gal-LPS caused systemic cytokine release and provoked marked hepatic damage, characterized by leukocyte recruitment and microvascular perfusion failure, caspase-3 activation, and hepatocellular apoptosis as well as enzyme release and necrotic cell death. DPO-pretreated and -posttreated mice showed diminished systemic cytokine concentrations, intrahepatic leukocyte accumulation, and hepatic perfusion failure. Hepatocellular apoptosis was significantly reduced by 50 to 75% after DPO pretreatment as well as posttreatment. In addition, treatment with DPO also significantly abrogated necrotic cell death and liver enzyme release. In conclusion, these observations may stimulate the evaluation of DPO as hepatoprotective therapy in patients with acute liver injury. Acute liver failure (ALF) is a gastrohepatointestinal emergency that continues to be a huge therapeutic challenge.1Shenoy S Liver transplantation in acute liver failure.Indian J Gastroenterol. 2006; 25: S13-S18Google Scholar This illness can rapidly progress to coma and death from cerebral edema and multiorgan dysfunction.2Ostapowicz G Fontana RJ Schiodt FV Larson A Davern TJ Han SH McCashland TM Shakil AO Hay JE Hynan L Crippin JS Blei AT Samuel G Reisch J Lee WM U.S. Acute Liver Failure Study Group Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States.Ann Intern Med. 2002; 137: 947-954Crossref PubMed Scopus (1683) Google Scholar Prognosis of ALF depends not only on primary hepatic injury but also on complications that occur during the course of disease. This also includes major liver surgery and liver transplantation, which are prone to complications by endotoxemia and sepsis.3Selvaggi G Weppler D Nishida S Moon J Levi D Kato T Tzakis AG Ten-year experience in porto-caval hemitransposition for liver transplantation in the presence of portal vein thrombosis.Am J Transplant. 2007; 7: 454-460Crossref PubMed Scopus (75) Google Scholar, 4Silva MA Tekin K Aytekin F Bramhall SR Buckels JA Mirza DF Surgery for hilar cholangiocarcinoma: a 10 year experience of a tertiary referral centre in the UK.Eur J Surg Oncol. 2005; 31: 533-539Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Infection receives particular attention because it has been established that patients with ALF are abnormally susceptible to infectious pathogens as a result of multiple immunological defects.5Rolando N Wade J Davalos M Wendon J Philpott-Howard J Williams R The systemic inflammatory response syndrome in acute liver failure.Hepatology. 2000; 32: 734-739Crossref PubMed Scopus (589) Google Scholar In line with this, established bacterial infections are reported to occur in ∼80% of cases.6O'Grady JG Acute liver failure.Postgrad Med J. 2005; 81: 148-154Crossref PubMed Scopus (151) Google Scholar Within the extremely complex cascade of pathophysiological events, representing the systemic inflammatory response, great attention has recently been brought to the contribution of apoptosis in organ dysfunction and failure.7Patel GP Gurka DP Balk RA New treatment strategies for severe sepsis and septic shock.Curr Opin Crit Care. 2003; 9: 390-396Crossref PubMed Scopus (37) Google Scholar, 8Marshall JC Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome.Crit Care Med. 2001; 29: S99-S106Crossref PubMed Scopus (351) Google Scholar Hepatocellular apoptosis represents not only a crucial step in ALF but functions also as a signal for leukocyte migration and attack on parenchymal cells,9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar, 10Jaeschke H Fisher MA Lawson JA Simmons CA Farhood A Jones DA Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model.J Immunol. 1998; 160: 3480-3486PubMed Google Scholar thereby establishing a vicious circle with aggravation of leukocytic inflammation and final cell death. It has been shown that preventing apoptosis in hepatocytes by caspase inhibition suppresses leukocyte transmigration and leukocyte-dependent liver cell necrosis.10Jaeschke H Fisher MA Lawson JA Simmons CA Farhood A Jones DA Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model.J Immunol. 1998; 160: 3480-3486PubMed Google Scholar Although controversies still exist regarding which mode of cell death, apoptosis or necrosis, predominates in various forms of acute liver injury, it becomes evident that treatment strategies should target downstream consequences of inflammatory activation.11Riedemann NC Gou RF Ward PA Novel strategies for the treatment of sepsis.Nat Med. 2003; 9: 517-524Crossref PubMed Scopus (695) Google Scholar Thereby, pleiotropic agents, exerting multiple functions, may be of particular interest because of their broad potential to interfere with relevant pathways of disease.Erythropoietin (EPO), initially known as renal glycoprotein hormone and which promotes the survival, proliferation, and differentiation of erythrocytic progenitors in hematopoietic tissues, has meanwhile been recognized as an anti-apoptotic and tissue-protective pleiotropic cytokine.12Ghezzi P Brines M Erythropoietin as an antiapoptotic, tissue-protective cytokine.Cell Death Differ. 2004; 11: S37-S44Crossref PubMed Scopus (280) Google Scholar, 13Jelkmann W Wagner K Beneficial and ominous aspects of the pleiotropic action of erythropoietin.Ann Hematol. 2004; 83: 673-686Crossref PubMed Scopus (127) Google Scholar, 14Jelkmann W Molecular biology of erythropoietin.Intern Med. 2004; 43: 649-659Crossref PubMed Scopus (284) Google Scholar Recent studies have identified multiple paracrine/autocrine functions of EPO and its long-acting analogue darbepoetin-α (DPO) that coordinate local responses to injury by attenuating both apoptotic and inflammatory causes of cell death in brain, kidney, and myocardium.15Calvillo L Latini R Kajstura J Leri A Anversa P Ghezzi P Salio M Cerami A Brines M Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling.Proc Natl Acad Sci USA. 2003; 100: 4802-4806Crossref PubMed Scopus (543) Google Scholar, 16Belayev L Khoutorova L Zhao W Vigdorchik A Belayev A Busto R Magal E Ginsberg MD Neuroprotective effect of darbepoetin alfa, a novel recombinant erythropoietic protein, in focal cerebral ischemia in rats.Stroke. 2005; 36: 1071-1076Crossref PubMed Scopus (159) Google Scholar, 17Johnson DW Pat B Vesey DA Guan Z Endre Z Gobe GC Delayed administration of darbepoetin or erythropoietin protects against ischemic acute renal injury and failure.Kidney Int. 2006; 69: 1806-1813Crossref PubMed Scopus (164) Google Scholar Encouraged by these reports, we determined whether DPO is protective in ALF by pre- and posttreating mice exposed to d-galactosamine (Gal) and Escherichia coli lipopolysaccharide (LPS).Materials and MethodsAnimal Model and Experimental GroupsMale C57BL/6J mice (Charles River Laboratories, Sulzfeld, Germany) were used at 8 to 10 weeks of age with a body weight of ∼20 g. Animals were kept on water and standard laboratory chow ad libitum. All animals received humane care according to the German legislation on protection of animals and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1985).For induction of acute liver injury, animals were injected with Gal (720 mg/kg body weight i.p.; Sigma-Aldrich, Taufkirchen, Germany) and LPS (10 μg/kg body weight i.p., serotype 0128:B12; Sigma-Aldrich). Concentrations of Gal and LPS were used in accordance with previously published work.18Klintman D Li X Thorlacius H Important role of P-selectin for leukocyte recruitment, hepatocellular injury, and apoptosis in endotoxemic mice.Clin Diagn Lab Immunol. 2004; 11: 56-62PubMed Google Scholar, 19Leist M Gantner F Bohlinger I Tiegs G Germann PG Wendel A Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models.Am J Pathol. 1995; 146: 1220-1234PubMed Google Scholar, 20Morikawa A Sugiyama T Kato Y Koide N Jiang GZ Takahashi K Tamada Y Yokochi T Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.Infect Immun. 1996; 64: 734-738Crossref PubMed Google Scholar Via retro-orbital vein puncture, animals were treated with DPO (10 μg/kg body weight, Aranesp; Amgen Europe, Breda, The Netherlands) at either 24 hours before induction of acute liver injury (n = 7, Gal-LPS/DPOpre) or 30 minutes after induction of liver injury (n = 7, Gal-LPS/DPOpost). Physiological saline-treated animals with liver injury served as Gal-LPS controls (n = 7, Gal-LPS). Sham-treated animals, without induction of acute liver injury and receiving only isotonic saline, served as sham controls (n = 7, control). Six hours after Gal-LPS exposure, in vivo analysis of the hepatic microcirculation as well as blood and tissue sampling were performed in the four groups mentioned above.To achieve specific information on the relation between necrosis, apoptosis, inflammation, and tissue perfusion, we studied the time course of events by means of in vivo fluorescence microscopy in additional sets of saline-treated animals and animals with posttreatment of DPO at 2 and 4 hours after Gal-LPS exposure (n = 5 animals per time point and group each). These animals further served for sampling of blood and liver tissue for subsequent analysis (see below).Intravital Fluorescence MicroscopyFor in vivo analysis of the hepatic microcirculation 2, 4, and 6 hours after Gal-LPS exposure, ketamine/xylazine-anesthetized animals (75/25 mg/kg body weight i.p.) were placed in supine position on a heating pad for maintenance of body temperature at 36 to 37°C. Polyethylene catheters (PE 50, ID 0.28 mm; Smiths Medical International Ltd., Kent, UK) in the left carotid artery and jugular vein served for continuous monitoring of hemodynamics and injection of fluorescent dyes. After transverse laparotomy, the animals were positioned on their left side, and the left liver lobe was exteriorized and covered with a glass slide for intravital fluorescence microscopy. Using a Zeiss fluorescence microscope equipped with a 100 W mercury lamp and different filter sets for blue, green, and UV epi-illumination (Axiotech Vario; Zeiss, Jena, Germany), microscopic images were taken by water immersion objectives (×20/0.50W, ×40/0.8W; Zeiss), televised using a charge-coupled device video camera (FK 6990A-IQ; Pieper, Berlin, Germany), and recorded on videotape for subsequent off-line evaluation.9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google ScholarBlood perfusion within individual sinusoids was studied after tissue contrast enhancement by sodium fluorescein (2 μmol/kg body weight i.v.; Merck, Darmstadt, Germany) and blue light epi-illumination (450 to 490/>520 nm, excitation/emission wavelength).9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar In vivo labeling of leukocytes with rhodamine-6G (1 μmol/kg body weight i.v.; Merck) and green light epi-illumination (530 to 560/>580 nm) enabled quantitative analysis of intrahepatic leukocyte flow behavior.9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar For analysis of apoptotic cell death, in vivo staining of hepatocellular nuclei was achieved by intravenous injection of bisbenzimide (Hoechst 33342, 10 μmol/kg; Sigma-Aldrich) and UV epi-illumination (330 to 380/>415 nm).9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar, 21Schäfer T Scheuer C Roemer K Menger MD Vollmar B Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death.FASEB J. 2003; 17: 660-667Crossref PubMed Scopus (90) Google ScholarAssessment of hepatic microcirculatory parameters was performed off-line by frame-to-frame analysis of the videotaped images at magnifications of 424- and 823-fold, using a computer-assisted image analysis system with a 19-inch monitor (CapImage; Zeintl, Heidelberg, Germany). Within 10 acini and postsinusoidal venules per animal, microcirculatory analysis included the determination of sinusoidal perfusion, representing the number of perfused sinusoids in percentage of all sinusoids visible as well as the number of adherent leukocytes, located within postsinusoidal venules (given as cells/mm2 endothelial surface, calculated from diameter and length of vessel segment studied, assuming cylindrical geometry) and not moving during an observation period of 20 seconds.9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar Apoptotic cell death was analyzed within 10 lobules per animal by counting the number of cells that showed apoptosis-associated condensation, fragmentation, and crescent-shaped formation of chromatin (given as cells/mm2 observation field).9Eipel C Bordel R Nickels RM Menger MD Vollmar B Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.Am J Physiol. 2004; 286: G769-G776Google Scholar, 21Schäfer T Scheuer C Roemer K Menger MD Vollmar B Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death.FASEB J. 2003; 17: 660-667Crossref PubMed Scopus (90) Google ScholarSampling and AssaysAfter in vivo microscopy, animals were exsanguinated by puncture of the vena cava inferior for immediate separation of ethylenediaminetetraacetic acid plasma. Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and glutamate dehydrogenase activities were measured spectrophotometrically as indicators for hepatocellular disintegration and necrosis. Ethylenediaminetetraacetic acid plasma further served for analysis of tumor necrosis factor (TNF)-α and interleukin (IL)-6, using commercially available enzyme-linked immunosorbent assay kits in accordance with the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). Liver tissue was sampled for subsequent Western blot protein analysis, histology, and immunohistochemistry.Histology and ImmunohistochemistryLiver tissue was fixed in 4% phosphate-buffered formalin for 2 to 3 days and then embedded in paraffin. From the paraffin-embedded tissue blocks, 4-μm sections were cut and stained with hematoxylin and eosin (H&E). Necrosis was assessed by morphological features, such as vacuolization, swollen cytoplasm with disrupted cell and organelle membranes, as well as lytic nuclear changes.22Kerr JF Gobe GC Winterford CM Harmon BV Anatomical methods in cell death.Methods Cell Biol. 1995; 46: 1-27Crossref PubMed Scopus (470) Google Scholar The percentage of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared with the entire histological section.23Gujral JS Bucci TJ Farhood A Jaeschke H Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis?.Hepatology. 2001; 33: 397-405Crossref PubMed Scopus (339) Google Scholar To evaluate hepatocyte replication, mitotic figures were counted in 1000 hepatocytes (400-fold magnification) and given as mitotic index (number of mitotic figures per 1000 hepatocytes).24Eipel C Glanemann M Nuessler AK Menger MD Neuhaus P Vollmar B Ischemic preconditioning impairs liver regeneration in ex-tended reduced-size livers.Ann Surg. 2005; 241: 477-484Crossref PubMed Scopus (67) Google ScholarFor the immunohistochemical study of cleaved caspase-3, 4-μm sections of paraffin-embedded specimens were incubated overnight at 4°C with a rabbit polyclonal cleaved caspase-3 antibody (1:500, 9661 lot 15; Cell Signaling Technology, Frankfurt, Germany). This antibody detects endogenous levels of the large fragment (17/19 kd) of activated caspase-3 but not full-length caspase-3.25El-Gibaly AM Scheuer C Menger MD Vollmar B Improvement of rat liver graft quality by pifithrin-alpha-mediated inhibition of hepatocyte necrapoptosis.Hepatology. 2004; 39: 1553-1562Crossref PubMed Scopus (49) Google Scholar For the development of cleaved caspase-3, a biotinylated anti-rabbit immunoglobulin antibody was used as a secondary antibody for streptavidin-biotin complex peroxidase staining (1:20, horseradish peroxidase, P 0448; DakoCytomation, Hamburg, Germany). 3,3′-Diaminobenzidine (S 3000; DakoCytomation) was used as chromogen. The sections were counterstained with hemalaun. Cleaved caspase-3-positive hepatocytes were counted within 30 consecutive fields (×40 objective/0.65 numeric aperture) and are given as cells per mm2.Western Blot AnalysisFor Western blot analysis of protein levels of cleaved caspase-3, Bcl-XL, Bax, proliferating cell nuclear antigen (PCNA), and phosphorylated endothelial nitric-oxide synthase (phospho-eNOSSer1177), liver tissue was homogenized in lysis buffer (10 mmol/L Tris, pH 7.5, 10 mmol/L NaCl, 0.1 mmol/L ethylenediaminetetraacetic acid, 0.5% Triton X-100, 0.02% NaN3, and 0.2 mmol/L phenylmethyl sulfonyl fluoride), incubated for 30 minutes on ice, and centrifuged for 15 minutes at 10,000 × g. The supernatant was saved as whole protein fraction. Before use, the buffer received a protease inhibitor cocktail (1:100 v/v; Sigma-Aldrich). Protein concentrations were determined using the bicinchoninic acid protein assay (Sigma-Aldrich) with bovine serum albumin as standard. Sixty μg of protein/lane (cleaved caspase-3 and phospho-eNOS) or 20 μg of protein/lane (Bcl-XL, Bax, PCNA) were separated discontinuously on sodium dodecyl sulfate polyacrylamide gels (12%) and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Eschborn, Germany). After blockade of nonspecific binding sites, membranes were incubated for 2 hours at room temperature with a rabbit polyclonal anti-cleaved caspase 3 (Asp 175, 1:1000; Cell Signaling Technology), a mouse monoclonal anti-Bcl-XL (1:1000; BD PharMingen, Heidelberg, Germany), a mouse monoclonal anti-Bax (1:250; BD PharMingen), a rabbit polyclonal anti-PCNA (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA), or a rabbit polyclonal anti-phospho-eNOS (1:500; Cell Signaling Technology), followed by secondary peroxidase-linked goat anti-rabbit or anti-mouse antibodies (cleaved caspase-3, 1:2000; PCNA, 1:8000; and phospho-eNOS, 1:2500; Cell Signaling Technology; BcL-XL, 1:10,000; and Bax, 1:20,000; Sigma-Aldrich). Protein expression was visualized by means of luminol-enhanced chemiluminescence (ECL Plus; Amersham Pharmacia Biotech, Freiburg, Germany) and exposure of the membrane to a blue light-sensitive autoradiography film (Kodak BioMax Light Film; Kodak-Industrie, Chalon-sur-Saone, France). Signals were densitometrically assessed (Quantity One, Gel Doc XR; Bio-Rad Laboratories GmbH, Munich, Germany) and normalized to the β-actin or β-tubulin (for phospho-eNOS only) signals (mouse monoclonal anti-β-actin antibody, 1:20,000; Sigma-Aldrich; or rabbit polyclonal anti-β-tubulin antibody, 1:500; Santa Cruz Biotechnology).Statistical AnalysisAll data are expressed as means ± SEM. After proving the assumption of normality and equal variance across groups, differences between groups were assessed using analysis of variance followed by the appropriate post hoc comparison test. Statistical significance was set at P < 0.05. Statistics were performed using the software package SigmaStat (Jandel Corp., San Rafael, CA).ResultsDPO Attenuates Intrahepatic Inflammation and Systemic Cytokine Concentrations after Gal-LPS ChallengeAnalysis of hepatic microcirculation by fluorescence microscopy is given in Figure 1. In the Gal-LPS-treated mice, in vivo microscopy of the livers revealed characteristic features of acute injury, including severe sinusoidal perfusion failure of up to 40% and increased white blood cell accumulation with ∼250 adherent leukocytes per mm2 endothelial surface of postsinusoidal venules. DPO-pretreated and, in particular, DPO-posttreated animals exhibited an improvement of hepatic microvascular perfusion by ∼17% and a 50% reduction of inflammatory leukocyte adherence, respectively (Figure 1).Gal-LPS exposure was characterized by high concentrations of IL-6 when compared with control animals without liver injury. Pretreatment and, in particular, posttreatment with DPO was capable of significantly reducing systemic IL-6 concentrations (Table 1). At 6 hours after Gal-LPS exposure, TNF-α levels were already well less than 100 pg/ml and did not differ between the four groups studied (Table 1).Table 1Systemic Concentrations of TNF-α and IL-6 (pg/ml), Tissue Necrosis (%), and Transaminase Activities (U/L) in Animals That Were Injected with Gal (720 mg/kg Body Weight i.p.) and LPS (10 μg/kg Body Weight i.p.) for Induction of Acute Liver Injury and Either Pretreated (24 Hours before Gal-LPS Exposure) or Posttreated (30 Minutes after Gal-LPS Exposure) with Darbepoetin (DPOpre and DPOpost; 10 μg/kg i.v.)Gal-LPSControlDPOpreDPOpostTNF-α40 ± 358 ± 446 ± 341 ± 9IL-6302 ± 604869 ± 228*P < 0.05 versus control.2734 ± 630*P < 0.05 versus control.1648 ± 934†P < 0.05 versus Gal-LPS.Necrosis0.7 ± 0.550 ± 10*P < 0.05 versus control.11 ± 7*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.18 ± 7*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.ALT33 ± 4267 ± 51*P < 0.05 versus control.92 ± 24*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.145 ± 76*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.AST140 ± 13453 ± 13*P < 0.05 versus control.249 ± 51*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.267 ± 91*P < 0.05 versus control.†P < 0.05 versus Gal-LPS.LDH1249 ± 2663468 ± 295*P < 0.05 versus control.1627 ± 329†P < 0.05 versus Gal-LPS.1439 ± 667†P < 0.05 versus Gal-LPS.GLDH15 ± 259 ± 11*P < 0.05 versus control.33 ± 6†P < 0.05 versus Gal-LPS.33 ± 12†P < 0.05 versus Gal-LPS.Control animals received isotonic saline only (control). For further information, please see Materials and Methods. Values are given as means ± SEM; analysis of variance and post hoc comparison test.ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; GLDH, glutamate dehydrogenase.* P < 0.05 versus control.† P < 0.05 versus Gal-LPS. Open table in a new tab DPO Ameliorates Apoptotic Cell Death and Caspase-3 Cleavage after Gal-LPS ChallengeAnalysis of apoptotic cell death is given in Figure 2, Figure 3, including in vivo microscopy of hepatocellular apoptosis as well as immunohistochemistry and Western immunoblot for cleaved caspase-3. Compared with sham control animals, Gal-LPS induced a notable increase of hepatocellular apoptosis, as indicated by the ∼12-fold rise in the number of bisbenzimide-stained hepatocytes with nuclear chromatin condensation and fragmentation (Figure 2, left). In support of these in vivo data, immunohistochemistry (Figure 2, right) and Western immunoblot (Figure 3) revealed an intense activation of the effector caspase-3 in Gal-LPS-exposed liver tissue. Not only pretreatment but also posttreatment of DPO remarkably ameliorated the extent of apoptotic cell death to almost sham control values (Figure 2, Figure 3).Figure 2Representative images with quantitative analysis of apoptotic hepatocytes in animals that were injected with Gal (720 mg/kg body weight i.p.) and LPS (10 μg/kg body weight i.p.) for induction of acute liver injury and either pretreated (24 hours before Gal-LPS exposure) or posttreated (30 minutes after Gal-LPS exposure) with DPO (DPOpre and DPOpost; 10 μg/kg i.v.). Endotoxic controls were treated with saline only (Gal-LPS). Sham-operated animals without liver injury served as controls (control). Hepatocellular apoptosis was assessed by either fluorescence microscopy and bisbenzimide staining (left) or immunohistochemistry for cleaved caspase-3 (right). Note the individual hepatocytes with condensation, fragmentation, and/or margination of nuclear chromatin as characteristic signs of apoptotic cell death on Gal-LPS exposure and the marked reduction of cell apoptosis in the images of Gal-LPS/DPOpreanimals, being also representative for those found in Gal-LPS/DPOpost animals. Values are given as means ± SEM; analysis of variance and post hoc comparison test; *P < 0.05 versus control; #P < 0.05 versus Gal-LPS. Original magnifications: ×823 (left); ×1600 (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Representative Western blot and densitometric analysis of cleaved caspase-3 protein levels in livers of animals that were injected with Gal (720 mg/kg body weight i.p.) and LPS (10 μg/kg body weight i.p.) for induction of acute liver injury and either pretreated (24 hours before Gal-LPS exposure) or posttreated (30 minutes after Gal-LPS exposure) with DPO (DPOpre and DPOpost; 10 μg/kg i.v.). Endotoxic controls were treated with saline only (Gal-LPS). Sham-operated animals without liver injury served as controls (control). β-Actin served as loading control. Values are given as means ± SEM; analysis of variance and post hoc comparison test; *P < 0.05 versus control; #P < 0.05 versus Gal-LPS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DPO Reduces Hepatic Morphological Damage and Liver Enzyme Release after Gal-LPS ChallengeH&E histopathology of Gal-LPS-exposed liver exhibited disruption of the general architecture, microvascular disintegration, as well as tissue apoptosis and necrosis (Figure 4). Morphological criteria, such as vacuolization, swollen cytoplasm with disrupted cell and organelle membranes, as well as lytic nuclear changes served to determine necrosis and revealed a substantial amount of necrosis with a fraction of 50% positive fields on Gal-LPS exposure (Table 1). In line with histopathology, liver injury caused a threefold to eightfold rise in transaminase levels when compared with saline exposed controls (Table 1). Sections obtained from DPO pre- and posttreated animals appeared different, often exhibiting grossly retained general architecture and lacking evidence of major morphological injury (Figure 4). Occasional sections mostly of the posttreated animals exhibited minor to moderate injury (Table 1). Accordingly, the fraction of observation fields encompassing necrotic tissue amounted to only 10 and 18% on DPO pre- and posttreatment, respectively. Concomitantly, transaminase levels were found significantly reduced in DPO pre- an
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