Advertisement

Molecular Medicine

, Volume 13, Issue 9–10, pp 509–517 | Cite as

Erythropoietin Protects the Intestine Against Ischemia/Reperfusion Injury in Rats

  • Ensari Guneli
  • Zahide Cavdar
  • Huray Islekel
  • Sulen Sarioglu
  • Serhat Erbayraktar
  • Muge Kiray
  • Selman Sokmen
  • Osman Yilmaz
  • Necati Gokmen
Research Article

Abstract

Previous studies have shown that erythropoietin (EPO) has protective effects against ischemia/reperfusion (I/R) injury in several tissues. The aim of this study was to determine whether EPO could prevent intestinal tissue injury induced by I/R. Wistar rats were subjected to intestinal ischemia (30 min) and reperfusion (60 min). A single dose of EPO (5000 U/kg) was administered intraperitoneally at two different time points: either at five minutes before the onset of ischemia or at the onset of reperfusion. At the end of the reperfusion period, jejunum was removed for examinations. Myeloperoxidase (MPO), malondialdehyde (MDA), and antioxidant defense system were assessed by biochemical analyses. Histological evaluation was performed according to the Chiu scoring method. Endothelial nitric oxide synthase (eNOS) was demonstrated by immunohistochemistry. Apoptotic cells were determined by TUNEL staining. Compared with the sham, I/R caused intestinal tissue injury (Chiu score, 3 ± 0.36 vs 0.4 ± 0.24, P < 0.01) and was accompanied by increases in MDA levels (0.747 ± 0.076 vs 0.492 ± 0.033, P < 0.05), MPO activity (10.51 ±1.87 vs 4.3 ± 0.45, P < 0.05), intensity of eNOS immunolabelling (3 ±0.4 vs 1.3 ±0.33, P < 0.05), the number of TUNEL-positive cells (20.4 ± 2.6 vs 4.6 ± 1.2, P < 0.001), and a decrease in catalase activity (16.83 ±2.6 vs 43.15 ±4.7, P < 0.01). Compared with the vehicle-treated I/R, EPO improved tissue injury; decreased the intensity of eNOS immunolabelling (1.6 ± 0.24 vs 3 ± 0.4, P < 0.05), the number of TUNEL-positive cells (9.2 ±2.7 vs 20.4 ± 2.6, P < 0.01), and the high histological scores (1 ± 0.51 vs3± 0.36, P < 0.01), and increased catalase activity (42.85 ± 6 vs 16.83 ± 2.6, P < 0.01) when given before ischemia, while it was found to have decreased the levels of MDA (0.483 ± 0.025 vs 0.747 ± 0.076, P < 0.05) and MPO activity (3.86 ± 0.76 vs 10.51 ± 1.87, P < 0.05), intensity of eNOS immunolabelling (1.4 ± 0.24 vs 3 ± 0.4, P < 0.01), the number of TUNEL-positive cells (9.1 ±3 vs 20.4 ± 2.6, P < 0.01), and the number of high histological scores (1.16 ± 0.4 vs 3 ± 0.36, P < 0.05) when given at the onset of reperfusion. These results demonstrate that EPO protects against intestinal I/R injury in rats by reducing oxidative stress and apoptosis. We attributed this beneficial effect to the antioxidative properties of EPO.

Notes

Acknowledgments

This research was supported by grants from Dokuz Eylul University Research Foundation (Project 2005. KB. SAG.031).

References

  1. 1.
    Granger DN, Korthuis RJ. (1995) Physiologic mechanisms of postischemic tissue injury. Annu. Rev. Physiol. 57:311–32.CrossRefGoogle Scholar
  2. 2.
    Brandt, JL. (2003) Mesenteric vascular disease. Current Diagnosis and Treatment in Gastroenterology. New York: Section 1.9.Google Scholar
  3. 3.
    Mallick IH, Yang W, Winslet WC, Seifalian AM. (2004) Ischemia—Reperfusion Injury of the Intestine and Protective Strategies Against Injury. Dig. Dis. Sci. 49:1359–77.CrossRefGoogle Scholar
  4. 4.
    Erbayraktar S et al. (2006) Carbamylated erythropoietin reduces radiosurgically-induced brain injury. Mol. Med. 12:74–80.CrossRefGoogle Scholar
  5. 5.
    Siren AL, Ehrenreich H. (2001) Erythropoietin— a novel concept for neuroprotection. Eur. Arch. Psychiatry Clin. Neurosci. 25:179–84.Google Scholar
  6. 6.
    Celik M et al. (2002) Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc. Natl. Acad. Sci. 99:2258–63.CrossRefGoogle Scholar
  7. 7.
    Erbayraktar S et al. (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuro-protective activity in vivo. Proc. Natl. Acad. Sci. 100:6741–6746.CrossRefGoogle Scholar
  8. 8.
    Erbayraktar S, Yilmaz O, Gokmen N, Brines M. (2003) Erythropoietin is a multifunctional tissue-protective cytokine. Curr. Hematol. Rep. 2:465–470.PubMedGoogle Scholar
  9. 9.
    Contaldo C et al. (2007) Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice. Am. J. Physiol. Heart. Circ. Physiol. 293:274–283.CrossRefGoogle Scholar
  10. 10.
    Brines M et al. (2004) Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc. Natl. Acad. Sci. 101:14907–12.CrossRefGoogle Scholar
  11. 11.
    Juul SE et al. (1999) Why is erythropoietin present in human milk? Studies of erythropoietin receptors on enterocytes of human and rat neonates. Pediatr. Res. 46: 263–8.CrossRefGoogle Scholar
  12. 12.
    Cuzzocrea S et al. (2004) Erythropoietin reduces the development of experimental inflammatory bowel disease. J. Pharmacol. Exp. Ther. 311:1272–80.CrossRefGoogle Scholar
  13. 13.
    Ledbetter DJ, Juul SE. (2000) Erythropoietin and the incidence of necrotizing enterocolitis in infants with very low birth weight. J. Pediatr. Surg. 35:178–82.CrossRefGoogle Scholar
  14. 14.
    Fatouros M et al. (1999) Alterations in body weight, breaking strength, and wound healing in wistar rats treated pre- and postoperatively with erythropoietin or granulocyte macrophage-colony stimulating factor: Evidence of a previously unknown anabolic effect of erythropoietin? J. Lab. Clin. Med. 133:253–9.CrossRefGoogle Scholar
  15. 15.
    Lipsic E et al. (2004) Timing of erythropoietin treatment for cardioprotection in ischemia-reperfusion. J. Cardiovasc. Pharmacol. 44:473–9.CrossRefGoogle Scholar
  16. 16.
    Sharples EL et al. (2004) Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion. J. Am. Soc. Nephrol. 15:2115–24.CrossRefGoogle Scholar
  17. 17.
    Solaroglu A, Dede FS, Okutan E, Bayrak A, Haberal A, Kilinc K. (2004) A single dose of erythropoietin attenuates lipid peroxidation in experimental liver ischemia—reperfusion injury in the rat fetus. J. Matern. Fetal. Neonatal. Med. 16:231–4.CrossRefGoogle Scholar
  18. 18.
    Wu H et al. (2006) Pretreatment with recombined human erythropoietin attenuates ischemia— reperfusion-induced lung injury in rats. Eur. J. Cardiothorac. Surg. 29:902–7.CrossRefGoogle Scholar
  19. 19.
    Villa P et al. (2003) Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J. Exp. Med. 198:971–5.CrossRefGoogle Scholar
  20. 20.
    Junk AK et al. (2002) Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc. Natl. Acad. Sci. 99:10659–64.CrossRefGoogle Scholar
  21. 21.
    Tatum VL, Changchit C, Chow CK. (1990) Measurement of malonaldehyde by HPLC with fluorescence detection. Lipids 25:226–9.CrossRefGoogle Scholar
  22. 22.
    Wiechelman KJ, Braun RD, Fitzpatrick JD. (1988) Investigation of the bicinchoninic acid protein 517 assay: identification of the groups responsible for color formation. Anal. Biochem. 175:231–7.CrossRefGoogle Scholar
  23. 23.
    Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. (1970) Intestinal mucosal lesion in low-flow states. I. A morphologic, hemodynamic and metabolic reappraisal. Arch. Surg. 101:478.CrossRefGoogle Scholar
  24. 24.
    Park PO, Haglund U, Bulkley GB, Falt K. (1990) The sequence of development of intestinal tissue injury following strangulation ischemia and reperfusion. Surgery. 107:574.PubMedGoogle Scholar
  25. 25.
    Zimmerman BJ, Granger DN. (1994) Mechanisms of reperfusion injury. Am. J. Med. Sci. 307:284–92.CrossRefGoogle Scholar
  26. 26.
    Kettle AJ, Winterbourn CC. (1997) Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox. Rep. 3:3–15.CrossRefGoogle Scholar
  27. 27.
    Liu X et al. (2006) Mechanism of the cardioprotection of rhEPO pretreatment on suppressing the inflammatory response in ischemia-reperfusion. Life Sci. 78:2255–64.CrossRefGoogle Scholar
  28. 28.
    Chattonadhyay T, Das C, Bandyopadhyay D, Datta AG. (2000) Protective effect of erythropoietin on the oxidative damage of erythrocyte membrane by hydroxyl radical. Biochem. Pharmacol. 59:419–25.CrossRefGoogle Scholar
  29. 29.
    Chakraborty M, Ghosal J, Biswas T, Data AG. (1988) Effect of erythropoietin on membrane lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase of rat RBC. Biochem. Med. Metab. Biol. 40:8–18.CrossRefGoogle Scholar
  30. 30.
    Akisu M, Kullahcioglu GF, Baka M, Husseyinov A, Kultursay N. (2001a) The role of recombinant human erythropoietin in lipid peroxidation and platelet-activating factor generation in a rat model of necrotizing enterocolitis. Eur. J. Pediatr. Surg. 11:167–72.CrossRefGoogle Scholar
  31. 31.
    Akisu M, Tuzun S, Arslanoglu S, Yalaz M, Kultursay N. (2001b) Effect of recombinant human erythropoietin administration on lipid peroxidation and antioxidant enzyme(s) activities in preterm infants. Acta. Med. Okayama. 55:357–62.PubMedGoogle Scholar
  32. 32.
    Cross CE, Halliwell B, Allen A. (1984) Antioxidant protection: A function of tracheobronchial and gastrointestinal mucus. Lancet. 1:1328–30.CrossRefGoogle Scholar
  33. 33.
    Digicaylioglu M, Lipton SA. (2001) Erythropoietin-mediated neuroprotection involves crosstalk between Jak2 and NF-κB signaling cascades. Nature. 412:641–7.CrossRefGoogle Scholar
  34. 34.
    Genc S, Akhisaroglu M, Kuralay F, Genc K. (2002) Erythropoietin restores glutathione peroxidase activity in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced neurotoxicity in C57BL mice and stimulates murine astroglial glutathione peroxidase production in vitro. Neurosci. Lett. 321:73–76.CrossRefGoogle Scholar
  35. 35.
    Kumral A et al. (2005) Protective effects of erythropoietin against ethanol-induced apoptotic neurodegeneration and oxidative stress in the developing C57BL/6 mouse brain. Brain Res. Dev. Brain Res. 160:146–56.CrossRefGoogle Scholar
  36. 36.
    Sakanaka M et al. (1998) In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc. Natl. Acad. Sci. 95:4635–40.CrossRefGoogle Scholar
  37. 37.
    Turi S, Nemeth I, Varga I, Bodrogi T, Matkovics B. (1992) The effect of erythropoietin on the cellular defense mechanism of red blood cells in children with chronic renal failure. Pediatr. Nephrol. 6:536–41.CrossRefGoogle Scholar
  38. 38.
    Kerr JF, Wyllie AH, Currie AR. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26: 239–57.CrossRefGoogle Scholar
  39. 39.
    Ikeda H et al. (1998) Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium. Gut. 42:530–7.CrossRefGoogle Scholar
  40. 40.
    Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY. (1998) Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am. J. Physiol. 274:270–6.Google Scholar
  41. 41.
    Parsa CJ et al. (2004) Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts. J. Biol. Chem. 279:20655–62.CrossRefGoogle Scholar
  42. 42.
    Brines M and Cerami A. (2006) Discovering erythropoietin’s extra-hematopoietic functions: biology and clinical promise. Kidney Int. 70:246–50.CrossRefGoogle Scholar
  43. 43.
    Bai J, Cederbaum AI. (2001) Mitochondrial catalase and oxidative injury. Biol. Signals Recept. 10: 189–99.CrossRefGoogle Scholar
  44. 44.
    Buttke TM, Sandstrom PA. (1994) Oxidative stress as a mediator of apoptosis. Immunol. Today. 15:7–10.CrossRefGoogle Scholar
  45. 45.
    Curtin JF, Donovan M, Cotter TG. (2002) Regulation and measurement of oxidative stress in apoptosis. J. Immunol. Methods. 265:49–72.CrossRefGoogle Scholar
  46. 46.
    Kojima M et al. (2003) Effects of antioxidative agents on apoptosis induced by ischaemia-reperfusion in rat intestinal mucosa. Aliment Pharmacol. Ther. 1:139–45.CrossRefGoogle Scholar
  47. 47.
    Roviezzo F et al. (2007) Protective role of PI3-kinase-Akt-eNOS signaling pathway in intestinal injury associated with splanchnic artery occlusion shock. Br. J. Pharmacol. 151:377–83.CrossRefGoogle Scholar
  48. 48.
    Munzel T, Daiber A, Ullrich V, Mulsch A. (2005) Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase. Arterioscl. Thromb. Vasc. Biol. 25:1551–7.CrossRefGoogle Scholar
  49. 49.
    Rui T et al. (2005) Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1. Cardiovasc. Res. 65:719–27.CrossRefGoogle Scholar
  50. 50.
    Lopez Ongil SL, Saura M, Lamas S, Rodriguez Puyol M, Rodriguez Puyol D. (1996) Recombinant human erythropoietin does not regulate the expression of endothelin-1 and constitutive nitric oxide synthase in vascular endothelial cells. Exp. Nephrol. 4:37–42.PubMedGoogle Scholar
  51. 51.
    Banerjee D, Rodriguez M, Nag M, Adamson JW. (2000) Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity. Kidney Int. 57:1895–904CrossRefGoogle Scholar
  52. 52.
    Wang XQ, Vaziri ND. (1999) Erythropoietin depresses nitric oxide synthase expression by human endothelial cells. Hypertension. 33:894–9CrossRefGoogle Scholar
  53. 53.
    Calapai MC et al. (2000) Erythropoietin protects against brain ischemia injury by inhibition of nitric oxide formation, Eur. J. Pharmacol. 401:349–356.CrossRefGoogle Scholar
  54. 54.
    Li H, Wallerath T, Munzel T, Förestermann U. (2002) Regulation of endothelial-type NO synthase expression in pathophysiology and in response to drugs. Nitric Oxide. 7:149–64.CrossRefGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2007

Authors and Affiliations

  • Ensari Guneli
    • 1
  • Zahide Cavdar
    • 2
  • Huray Islekel
    • 2
  • Sulen Sarioglu
    • 3
  • Serhat Erbayraktar
    • 4
  • Muge Kiray
    • 5
  • Selman Sokmen
    • 6
  • Osman Yilmaz
    • 1
  • Necati Gokmen
    • 7
  1. 1.Department of Laboratory Animal Sciences, Health Sciences InstituteDokuz Eylul, UniversityIzmirTurkey
  2. 2.Department of Biochemistry, Health Sciences InstituteDokuz Eylül UniversityIzmirTurkey
  3. 3.Department of Pathology, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey
  4. 4.Department of Neurosurgery, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey
  5. 5.Department of Histology and Embryology, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey
  6. 6.Department of Surgery, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey
  7. 7.Department of Anesthesiology and Reanimation, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey

Personalised recommendations