Skip to main content

Antioxidants inhibit the inflammatory and apoptotic processes in an intermittent hypoxia model of sleep apnea

Abstract

Background

Sleep apnea causes intermittent hypoxia (IH). We aimed to investigate the proteins related to oxidative stress, inflammation and apoptosis in liver tissue subjected to IH as a simulation of sleep apnea in conjunction with the administration of either melatonin (MEL, 200 μL/kg) or N-acetylcysteine (NAC, 10 mg/kg).

Methods

Seventy-two adult male Balb-C mice were divided: simulation of IH (SIH), SIH + MEL, SIH + NAC, IH, IH + MEL and IH + NAC. The animals were subjected to simulations of sleep apnea for 8 h a day for 35 days. The data were analyzed with ANOVA and Tukey tests with the significance set at p < 0.05.

Results

In IH, there was a significant increase in oxidative stress and expression of HIF-1a. In addition, we observed increase in the activation levels of NF-kB. This increase may be responsible for the increased expression of TNF-alpha and iNOS as well as the significant increase of VEGF signaling and expression of caspase-3 and caspase-6, which suggests an increase in apoptosis. In the groups treated with antioxidants, the analysis showed that the enzyme activity and protein levels were similar to those of the non-simulated group.

Conclusions

Thus, we show that IH causes liver inflammation and apoptosis, which may be protected with either MEL or NAC.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

ALP:

Alkaline phosphatase

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

CAT:

Catalase

EDTA:

Ethylenediaminetetraacetic acid

H2O2 :

Hydrogen peroxide

HE:

Hematoxylin and eosin

HEPES:

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HIF-1α:

Hypoxia-inducible factor 1 alpha

IH:

Intermittent hypoxia

IH + MEL:

Intermittent hypoxia and treatment with melatonin

IH + NAC:

Intermittent hypoxia and treatment with N-acetylcysteine

iNOS:

Inducible NO synthase

KCl:

Potassium chloride

MEL:

Melatonin

NAC:

N-Acetylcysteine

NaCl:

Sodium chloride

NASH:

Nonalcoholic steatohepatitis

NF-kB:

Nuclear factor kappa B

NO:

Nitric oxide

O •-2 :

Superoxide anion radical

OSA:

Obstructive sleep apnea syndrome

PVDF:

Polyvinylidene difluoride

SIH:

Simulation of intermittent hypoxia

SIH + MEL:

Simulation of intermittent hypoxia and treatment with melatonin

SIH + NAC:

Simulation of intermittent hypoxia and treatment with N-acetylcysteine

SOD:

Superoxide dismutase

TGF-β:

Transforming growth factor beta

TNF-alpha:

Tumor necrosis factor

VEGF:

Vascular endothelial growth factor

XO:

Xanthine oxidase

References

  1. 1.

    Dempsey JA, Veasey SC, Morgan BJ, O’Donnell CP. Pathophysiology of sleep apnea. Physiol Rev. 2010;90:47–112.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  2. 2.

    Zhou W, Li S, Wan N, Zhang Z, Guo R, Chen B. Effects of various degrees of oxidative stress induced by intermittent hypoxia in rat myocardial tissues. Respirology. 2012;17(5):821–9.

    PubMed  Article  Google Scholar 

  3. 3.

    Lavie L. Obstructive sleep apnoea syndrome—an oxidative stress disorder. Sleep Med Rev. 2003;7:35–51.

    PubMed  Article  Google Scholar 

  4. 4.

    Lavie L. Sleep-disordered breathing and cerebrovascular disease: a mechanistic approach. Neurol Clin. 2005;23:1059–75.

    PubMed  Article  Google Scholar 

  5. 5.

    Suzuki YJ, Jain V, Park AM, Day RM. Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases. Free Radic Biol Med. 2006;40:1683–92.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  6. 6.

    Lavie L. Oxidative stress—a unifying paradigm in obstructive sleep apnea and comorbidities. Prog Cardiovasc Dis. 2009;51:303–12.

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Aalto TK, Raivio KO. Nucleotide depletion due to reactive oxygen metabolites in endothelial cells: effects of antioxidants and 3-aminobenzamide. Pediatr Res. 1993;34:572–6.

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Szabo C, Zingarelli B, Salzman AL. Role of poly-ADP ribosyltransferase activation in the vascular contractile and energetic failure elicited by exogenous and endogenous nitric oxide and peroxynitrite. Circ Res. 1996;78:1051–63.

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Zhang J, Dawson VL, Dawson TM, Snyder SH. Nitric oxide activation of poly (ADP-ribose) synthetase in neurotoxicity. Science. 1994;263:687–9.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Singh H, Pollock R, Uhanova J, Kryger M, Hawkins K, Minuk GY. Symptoms of obstructive sleep apnea in patients with nonalcoholic fatty liver disease. Dig Dis Sci. 2005;50:2338–43.

    PubMed  Article  Google Scholar 

  11. 11.

    Tanne F, Gagnadoux F, Chazouilleres O, Fleury B, Wendum D, Lasnier E, Lebeau B, Poupon R, Serfaty L. Chronic liver injury during obstructive sleep apnea. Hepatology. 2005;41:1290–6.

    PubMed  Article  Google Scholar 

  12. 12.

    Tatsumi K, Saibara T. Effects of obstructive sleep apnea syndrome on hepatic steatosis and nonalcoholic steatohepatitis. Hepatol Res. 2005;33:100–4.

    PubMed  Article  Google Scholar 

  13. 13.

    Jouet P, Sabate JM, Maillard D, Msika S, Mechler C, Ledoux S, Harnois F, Coffin B. Relationship between obstructive sleep apnea and liver abnormalities in morbidly obese patients: a prospective study. Obes Surg. 2007;17:478–85.

    PubMed  Article  Google Scholar 

  14. 14.

    Kallwitz ER, Herdegen J, Madura J, Jakate S, Cotler SJ. Liver enzymes and histology in obese patients with obstructive sleep apnea. J Clin Gastroenterol. 2007;41:918–21.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Zamora-Valdes D, Mendez-Sanchez N. Experimental evidence of obstructive sleep apnea syndrome as a second hit accomplice in nonalcoholic steatohepatitis pathogenesis. Ann Hepatol. 2007;6:281–3.

    PubMed  Google Scholar 

  16. 16.

    Kheirandish-Gozal L, Sans Capdevila O, Kheirandish E, Gozal D. Elevated serum aminotransferase levels in children at risk for obstructive sleep apnea. Chest. 2008;133:92–9.

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Norman D, Bardwell WA, Arosemena F, Nelesen R, Mills PJ, Loredo JS, Lavine JE, Dimsdale JE. Serum aminotransferase levels are associated with markers of hypoxia in patients with obstructive sleep apnea. Sleep. 2008;31:121–6.

    PubMed Central  PubMed  Google Scholar 

  18. 18.

    Byrne CD, Olufadi R, Bruce KD, Cagampang FR, Ahmed MH. Metabolic disturbances in non-alcoholic fatty liver disease. Clin Sci (Lond). 2009;116:539–64.

    CAS  Article  Google Scholar 

  19. 19.

    Savransky V, Nanayakkara A, Vivero A, Li J, Bevans S, Smith PL, Torbenson MS, Polotsky VY. Chronic intermittent hypoxia predisposes to liver injury. Hepatology. 2007;45:1007–13.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Savransky V, Reinke C, Jun J, Bevans-Fonti S, Nanayakkara A, Li J, Myers AC, Torbenson MS, Polotsky VY. Chronic intermittent hypoxia and acetaminophen induce synergistic liver injury in mice. Exp Physiol. 2009;94:228–39.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  21. 21.

    Ryan S, Taylor CT, McNicholas WT. Systemic inflammation: a key factor in the pathogenesis of cardiovascular complications in obstructive sleep apnoea syndrome? Thorax. 2009;64:631–6.

    CAS  PubMed  Google Scholar 

  22. 22.

    Htoo AK, Greenberg H, Tongia S, Chen G, Henderson T, Wilson D, Liu SF. Activation of nuclear factor kappaB in obstructive sleep apnea: a pathway leading to systemic inflammation. Sleep Breath. 2006;10:43–50.

    PubMed  Article  Google Scholar 

  23. 23.

    Greenberg H, Ye X, Wilson D, Htoo AK, Hendersen T, Liu SF. Chronic intermittent hypoxia activates nuclear factor-kappaB in cardiovascular tissues in vivo. Biochem Biophys Res Commun. 2006;343:591–6.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Selmi C, Montano N, Furlan R, Keen CL, Gershwin ME. Inflammation and oxidative stress in obstructive sleep apnea syndrome. Exp Biol Med (Maywood). 2007;232:1409–13.

    CAS  Article  Google Scholar 

  25. 25.

    Yamauchi M, Tamaki S, Tomoda K, Yoshikawa M, Fukuoka A, Makinodan K, Koyama N, Suzuki T, Kimura H. Evidence for activation of nuclear factor kappaB in obstructive sleep apnea. Sleep Breath. 2006;10:189–93.

    PubMed  Article  Google Scholar 

  26. 26.

    Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, et al. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 1998;394:485–90.

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Ziment I. Acetylcysteine: a drug that is much more than a mucokinetic. Biomed Pharmacother. 1988;42:513–9.

    CAS  PubMed  Google Scholar 

  28. 28.

    Rosa DP, Martinez D, Picada JN, Semedo JG, Marroni NP. Hepatic oxidative stress in an animal model of sleep apnoea: effects of different duration of exposure. Comp Hepatol. 2011;10:1.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  29. 29.

    Savransky V, Bevans S, Nanayakkara A, Li J, Smith PL, Torbenson MS, Polotsky VY. Chronic intermittent hypoxia causes hepatitis in a mouse model of diet-induced fatty liver. Am J Physiol Gastrointest Liver Physiol. 2007;293:G871–7.

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Halliwell B. Free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. Br J Exp Pathol. 1989;70:737–57.

    CAS  PubMed Central  PubMed  Google Scholar 

  31. 31.

    Martinez D, Fiori CZ, Baronio D, Carissimi A, Kaminski RS, Kim LJ, Rosa DP, Bos A. Brown adipose tissue: is it affected by intermittent hypoxia? Lipids Health Dis. 2010;9:121.

    PubMed Central  PubMed  Article  Google Scholar 

  32. 32.

    Guven A, Yavuz O, Cam M, Ercan F, Bukan N, Comunoglu C, Gokce F. Effects of melatonin on streptozotocin-induced diabetic liver injury in rats. Acta Histochem. 2006;108:85–93.

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Vercelino R, Tieppo J, Dias AS, Marroni CA, Garcia E, Meurer L, Picada JN, Marroni NP. N-Acetylcysteine effects on genotoxic and oxidative stress parameters in cirrhotic rats with hepatopulmonary syndrome. Basic Clin Pharmacol Toxicol. 2008;102:370–6.

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    [Anon]. AVMA updates its euthanasia guidelines. Vet Rec. 2007;161:502–502.

  35. 35.

    [Anon]. AVMA releases updated euthanasia guidelines. JAVMA. 2007, 231:827-827.

  36. 36.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302–10.

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170–5.

    CAS  PubMed  Google Scholar 

  39. 39.

    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979;76:4350–4.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  41. 41.

    Park AM, Suzuki YJ. Effects of intermittent hypoxia on oxidative stress-induced myocardial damage in mice. J Appl Physiol. 2007;102:1806–14.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Dutta A, Ray K, Singh VK, Vats P, Singh SN, Singh SB. l-carnitine supplementation attenuates intermittent hypoxia-induced oxidative stress and delays muscle fatigue in rats. Exp Physiol. 2008;93:1139–46.

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Bertuglia S, Reiter RJ. Melatonin reduces microvascular damage and insulin resistance in hamsters due to chronic intermittent hypoxia. J Pineal Res. 2009;46:307–13.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Lavie L, Hefetz A, Luboshitzky R, Lavie P. Plasma levels of nitric oxide and l-arginine in sleep apnea patients: effects of nCPAP treatment. J Mol Neurosci. 2003;21:57–63.

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Sohn HY, Krotz F, Gloe T, Keller M, Theisen K, Klauss V, Pohl U. Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. Cardiovasc Res. 2003;58:638–46.

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Jordan W, Cohrs S, Degner D, Meier A, Rodenbeck A, Mayer G, Pilz J, Ruther E, Kornhuber J, Bleich S. Evaluation of oxidative stress measurements in obstructive sleep apnea syndrome. J Neural Transm. 2006;113:239–54.

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Phillips SA, Olson EB, Lombard JH, Morgan BJ. Chronic intermittent hypoxia alters NE reactivity and mechanics of skeletal muscle resistance arteries. J Appl Physiol. 2006;100:1117–23.

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Shpirer I, Copel L, Broide E, Elizur A. Continuous positive airway pressure improves sleep apnea associated fatty liver. Lung. 2010;188(4):301–7.

    PubMed  Article  Google Scholar 

  49. 49.

    Carmiel-Haggai M, Cederbaum AI, Nieto N. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats. FASEB J. 2005;19:136–8.

    CAS  PubMed  Google Scholar 

  50. 50.

    Ludwig J, McGill DB, Lindor KD. Review: nonalcoholic steatohepatitis. J Gastroenterol Hepatol. 1997;12:398–403.

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–5.

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, Ratcliffe PJ. Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts. J Biol Chem. 2009;284:16767–75.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  53. 53.

    Semenza GL. Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology (Bethesda). 2009;24:97–106.

    CAS  Article  Google Scholar 

  54. 54.

    Yuan G, Khan SA, Luo W, Nanduri J, Semenza GL, Prabhakar NR. Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia. J Cell Physiol. 2011;226:2925–33.

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Nath B, Levin I, Csak T, Petrasek J, Mueller C, Kodys K, Catalano D, Mandrekar P, Szabo G. Hepatocyte-specific hypoxia-inducible factor-1alpha is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice. Hepatology. 2011;53:1526–37.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  56. 56.

    Blouin CC, Page EL, Soucy GM, Richard DE. Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1alpha. Blood. 2004;103:1124–30.

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Peyssonnaux C, Cejudo-Martin P, Doedens A, Zinkernagel AS, Johnson RS, Nizet V. Cutting edge: essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis. J Immunol. 2007;178:7516–9.

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J. 2008;412:477–84.

    PubMed Central  PubMed  Article  Google Scholar 

  59. 59.

    Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, Johnson RS, Haddad GG, Karin M. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature. 2008;453:807–11.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  60. 60.

    Beraza N, Malato Y, Vander Borght S, Liedtke C, Wasmuth HE, Dreano M, de Vos R, Roskams T, Trautwein C. Pharmacological IKK2 inhibition blocks liver steatosis and initiation of non-alcoholic steatohepatitis. Gut. 2008;57:655–63.

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 1999;18:6853–66.

    CAS  PubMed  Article  Google Scholar 

  62. 62.

    Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25:581–611.

    CAS  PubMed  Article  Google Scholar 

  63. 63.

    Minet E, Michel G, Remacle J, Michiels C. Role of HIF-1 as a transcription factor involved in embryonic development, cancer progression and apoptosis (review). Int J Mol Med. 2000;5:253–9.

    CAS  PubMed  Google Scholar 

  64. 64.

    Zhu H, Zeng L, Zhu D, Yuan Y. The role of TGF-beta 1 in mice hepatic fibrosis by Schistosomiasis Japonica. J Tongji Med Univ. 2000;20(320–321):329.

    Google Scholar 

  65. 65.

    Jeong WI, Do SH, Yun HS, Song BJ, Kim SJ, Kwak WJ, Yoo SE, Park HY, Jeong KS. Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver. Liver Int. 2004;24:658–68.

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Hampl V, Cornfield DN, Cowan NJ, Archer SL. Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells. Eur Respir J. 1995;8:515–22.

    CAS  PubMed  Google Scholar 

  67. 67.

    Feelisch M, Fernandez BO, Bryan NS, Garcia-Saura MF, Bauer S, Whitlock DR, Ford PC, Janero DR, Rodriguez J, Ashrafian H. Tissue processing of nitrite in hypoxia: an intricate interplay of nitric oxide-generating and -scavenging systems. J Biol Chem. 2008;283:33927–34.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  68. 68.

    Semenza GL. Surviving ischemia: adaptive responses mediated by hypoxia-inducible factor 1. J Clin Invest. 2000;106:809–12.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  69. 69.

    Moritz W, Meier F, Stroka DM, Giuliani M, Kugelmeier P, Nett PC, Lehmann R, Candinas D, Gassmann M, Weber M. Apoptosis in hypoxic human pancreatic islets correlates with HIF-1alpha expression. FASEB J. 2002;16:745–7.

    CAS  PubMed  Google Scholar 

  70. 70.

    Akakura N, Kobayashi M, Horiuchi I, Suzuki A, Wang J, Chen J, Niizeki H, Kawamura K, Hosokawa M, Asaka M. Constitutive expression of hypoxia-inducible factor-1alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation. Cancer Res. 2001;61:6548–54.

    CAS  PubMed  Google Scholar 

  71. 71.

    Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol. 2004;57:1009–14.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

Download references

Acknowledgments

Fundo de Investimento à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE-HCPA), Brazil.

Conflict of interest

No conflicts of interest in this work.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Darlan Pase da Rosa.

Additional information

Responsible Editor: John Di Battista.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

da Rosa, D.P., Forgiarini, L.F., e Silva, M.B. et al. Antioxidants inhibit the inflammatory and apoptotic processes in an intermittent hypoxia model of sleep apnea. Inflamm. Res. 64, 21–29 (2015). https://doi.org/10.1007/s00011-014-0778-5

Download citation

Keywords

  • Sleep apnea
  • Intermittent hypoxia
  • Liver