, Volume 27, Issue 5, pp 1057–1068 | Cite as

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia

  • Hsiao-Ling Chen
  • Chih-Ching Yen
  • Shih-Ming Wang
  • Tung-Chou Tsai
  • Zi-Lun Lai
  • Jheng-Yue Sun
  • Willei Lin
  • Wu-Huei Hsu
  • Chuan-Mu ChenEmail author


This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO2 = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.


Hyperoxic lung injury Lung fibrosis Aerosol therapy Bovine lactoferrin Oxygen toxicity 



The authors would like to thank Prof. Jiung-Wang Liao for his help with the pathology analysis and our colleagues (Drs. Yu-Tang Tung, and Cheng-Wei Lai) in the Molecular Embryology & DNA Methylation Laboratory for their help with discussions and technical issues. We also would like to thank the Biostatic Center of China Medical University for assistance with the statistical analysis. This research was supported by grant NSC-95-2313-B-005-012 from the National Science Council, grant COA-97-6.2.1-U1(9) from the Council of Agriculture, and the Ministry of Education, Taiwan, Republic of China, under the aiming top university plan (ATU-101-S-0508).

Supplementary material

10534_2014_9750_MOESM1_ESM.tif (408 kb)
Supplemental Figure S1. Image of the ultrasonic nebulizer system to aerosolize bLF for the murine model of hyperoxia-induced lung injury and measurement of its efficiency for nebulization of bLF. An ultrasonic nebulizer was connected to the oxygen delivery system to aerosolize the bLF protein drug. The efficiency of aerosolized bLF in this system was measured. A total of 3.6 mg bLF dissolved in 70 mL PBS was delivered by this aerosol system and approximately 78 % of bLF protein was detected in the cage after 10 h of aerosolization. (TIFF 407 kb)


  1. Actor JK, Hwang SA, Kruzel ML (2009) Lactoferrin as a natural immune modulator. Curr Pharm Des 15:1956–1973PubMedCentralPubMedCrossRefGoogle Scholar
  2. Ando K, Hasegawa K, Shindo K, Furusawa T, Fujino T, Kikugawa K, Nakano H, Takeuchi O, Akira S, Akiyama T, Gohda J, Inoue J, Hayakawa M (2010) Human lactoferrin activates NF-kappaB through the toll-like receptor 4 pathway while it interferes with the lipopolysaccharide-stimulated TLR4 signaling. FEBS J 277:2051–2066PubMedCrossRefGoogle Scholar
  3. Asikainen TM, White CW (2005) Antioxidant therapy for lung injury. In: Notter RH, Finkelstein JN, Holm BA (eds) Lung injury: mechanisms, pathophsiology, and therapy. Tayler & Francis Group Press, NY, pp 665–703Google Scholar
  4. Bagby GC Jr, Bennett RM (1982) Feedback regulation of granulopoiesis: polymerization of lactoferrin abrogates its ability to inhibit CSA production. Blood 60:108–112PubMedGoogle Scholar
  5. Brock JH (2002) The physiology of lactoferrin. Biochem Cell Biol 80:1–6PubMedCrossRefGoogle Scholar
  6. Chang LY, Subramaniam M, Yoder BA, Day BJ, Ellison MC, Sunday ME, Crapo JD (2003) A catalytic antioxidant attenuates alveolar structural remodeling in bronchopulmonary dysplasia. Am J Respir Crit Care Med 167:57–64PubMedCrossRefGoogle Scholar
  7. Chen CM, Chen HL, Hsiau TH, Hsiau AH, Shi H, Brock GJ, Wei SH, Caldwell CW, Yan PS, Huang TH (2003) Methylation target array for rapid analysis of CpG island hypermethylation in multiple tissue genomes. Am J Pathol 163:37–45PubMedCentralPubMedCrossRefGoogle Scholar
  8. Chen HL, Lai YW, Yen CC, Lin YY, Lu CY, Yang SH, Tsai TC, Lin YJ, Lin CW, Chen CM (2004) Production of recombinant porcine lactoferrin exhibiting antibacterial activity in methylotrophic yeast, Pichia pastoris. J Mol Microbiol Biotechnol 8:141–149PubMedCrossRefGoogle Scholar
  9. Chen HL, Wang LC, Chang CH, Yen CC, Cheng WT, Wu SC, Hung CM, Kuo MF, Chen CM (2008a) Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonatal mice from a lethal challenge with enterovirus type 71. Vaccine 26:891–898PubMedCrossRefGoogle Scholar
  10. Chen YJ, Wu CY, Shen JL, Chu SY, Chen CK, Chang YT, Chen CM (2008b) Psoriasis independently associated with hyperleptinemia contributing to metabolic syndrome. Arch Dermatol 144:1571–1575PubMedGoogle Scholar
  11. Chen HL, Lai YW, Chen CS, Chu TW, Lin W, Yen CC, Lin MF, Tu MY, Chen CM (2010) Probiotic lactobacillus casei expressing human lactoferrin elevates antibacterial activity in the gastrointestinal tract. Biometals 23:543–554PubMedCrossRefGoogle Scholar
  12. Chen JY, Chen HL, Cheng JC, Lin HJ, Tung YT, Lin CF, Chen CM (2012) A Chinese herbal medicine, Gexia-Zhuyu Tang (GZT), prevents dimethylnitrosamine- induced liver fibrosis through inhibition of hepatic stellate cells proliferation. J Ethnopharmacol 142:811–818PubMedCrossRefGoogle Scholar
  13. Curran CS, Demick KP, Mansfield JM (2006) Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell Immunol 242:23–30PubMedCrossRefGoogle Scholar
  14. El-Loly MM, Mahfouz MB (2011) Lactoferrin in relation to biological functions and applications: a review. Int J Dairy Sci 6:79–111CrossRefGoogle Scholar
  15. Ghio AJ, Carter JD, Richards JH, Richer LD, Grissom CK, Elstad MR (2003) Iron and iron-related proteins in the lower respiratory tract of patients with acute respiratory distress syndrome. Crit Care Med 31:395–400PubMedCrossRefGoogle Scholar
  16. Griffiths CE, Cumberbatch M, Tucker SC, Dearman RJ, Andrew S, Headon DR, Kimber I (2001) Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans. Br J Dermatol 144:715–725PubMedCrossRefGoogle Scholar
  17. Guillen C, McInnes IB, Vaughan D, Speekenbrink AB, Brock JH (2000) The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis. Arthritis Rheum 43:2073–2080PubMedCrossRefGoogle Scholar
  18. Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I (2002) Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 220:83–95PubMedCrossRefGoogle Scholar
  19. Holm BA, Chess PR, Notter RH (2005) Cell and animal models of lung injury. In: Notter RH, Finkelstein JN, Holm BA (eds) Lung injury: mechanisms, pathophsiology, and therapy. Tayler & Francis Group Press, NY, pp 366–370Google Scholar
  20. Hung CM, Wu SC, Yen CC, Lin MF, Lai YW, Tung YT, Chen HL, Chen CM (2010a) Porcine lactoferrin as feedstuff additive elevates avian immunity and potentiates vaccination. Biometals 23:579–587PubMedCrossRefGoogle Scholar
  21. Hung CM, Yeh CC, Chen HL, Lai CW, Kuo MF, Yeh MH, Lin W, Tu MY, Cheng HC, Chen CM (2010b) Porcine lactoferrin administration enhances peripheral lymphocyte proliferation and assists infectious bursal disease vaccination in native chickens. Vaccine 28:2895–2902PubMedCrossRefGoogle Scholar
  22. Ioannou YA, Chen FW (1996) Quantitation of DNA fragmentation in apoptosis. Nucl Acids Res 24:992–993PubMedCentralPubMedCrossRefGoogle Scholar
  23. Jiang D, Liang J, Li Y, Noble PW (2006) The role of Toll-like receptors in non-infectious lung injury. Cell Res 16:693–701PubMedCrossRefGoogle Scholar
  24. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8:e1000412PubMedCentralPubMedCrossRefGoogle Scholar
  25. Kruzel ML, Harari Y, Mailman D, Actor JK, Zimecki M (2002) Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol 130:25–31PubMedCentralPubMedCrossRefGoogle Scholar
  26. Kruzel ML, Actor JK, Boldogh I, Zimecki M (2007) Lactoferrin in health and disease. Postepy Hig Med Dosw (Online) 61:261–267Google Scholar
  27. Lagan AL, Melley DD, Evans TW, Quinlan GJ (2008) Pathogenesis of the systemic inflammatory syndrome and acute lung injury: role of iron mobilization and decompartmentalization. Am J Physiol Lung Cell Mol Physiol 294:161–174CrossRefGoogle Scholar
  28. Matute-Bello G, Frevert CW, Martin TR (2008) Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol 295:L379–L399PubMedCentralPubMedCrossRefGoogle Scholar
  29. Montuschi P, Ciabattoni G, Paredi P, Pantelidis P, du Bois RM, Kharitonov SA, Barnes PJ (1998) 8-isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am J Respir Crit Care Med 158:1524–1527PubMedCrossRefGoogle Scholar
  30. Montuschi P, Collins JV, Ciabattoni G, Lazzeri N, Corradi M, Kharitonov SA, Barnes PJ (2000) Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 162:1175–1177PubMedCrossRefGoogle Scholar
  31. Nakao K, Imoto I, Gabazza EC, Yamauchi K, Yamazaki N, Taguchi Y, Shibata T, Takaji S, Ikemura N, Misaki M (1997) Gastric juice levels of lactoferrin and Helicobacter pylori infection. Scand J Gastroenterol 32:530–534PubMedCrossRefGoogle Scholar
  32. Paesano R, Pietropaoli M, Gessani S, Valenti P (2009) The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia. Biochimie 91:44–51PubMedCrossRefGoogle Scholar
  33. Paesano R, Berlutti F, Pietropaoli M, Goolsbee W, Pacifici E, Valenti P (2010a) Lactoferrin efficacy versus ferrous sulfate in curing iron disorders in pregnant and non-pregnant women. Int J Immunopathol Pharmacol 23:577–587PubMedGoogle Scholar
  34. Paesano R, Berlutti F, Pietropaoli M, Pantanella F, Pacifici E, Goolsbee W, Valenti P (2010b) Lactoferrin efficacy versus ferrous sulfate in curing iron deficiency and iron deficiency anemia in pregnant women. Biometals 23:411–417PubMedCrossRefGoogle Scholar
  35. Paesano R, Pietropaoli M, Berlutti F, Valenti P (2012) Bovine lactoferrin in preventing preterm delivery associated with sterile inflammation. Biochem Cell Biol 90:468–475PubMedCrossRefGoogle Scholar
  36. Puddu P, Latorre D, Carollo M, Catizone A, Ricci G, Valenti P, Gessani S (2011) Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells. PLoS One 6:e22504PubMedCentralPubMedCrossRefGoogle Scholar
  37. Simonson SG, Welty-Wolf KE, Huang YC, Taylor DE, Kantrow SP, Carraway MS, Crapo JD, Piantadosi CA (1997) Aerosolized manganese sod decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry. J Appl Physiol 83:550–558Google Scholar
  38. Sinclair SE, Altemeier WA, Matute-Bello G, Chi EY (2004) Augmented lung injury due to interaction between hyperoxia and mechanical ventilation. Crit Care Med 32:2496–2501PubMedCrossRefGoogle Scholar
  39. Sreedhara A, Flengsrud R, Langsrud T, Kaul P, Prakash V, Vegarud GE (2010) Structural characteristic, pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins. Biometals 23:1159–1170PubMedCrossRefGoogle Scholar
  40. Tung YT, Chen HL, Lai CW, Shen CJ, Lai YW, Chen CM (2011) Curcumin reduces pulmonary tumorigenesis in vascular endothelial growth factor (VEGF)-overexpressing transgenic mice. Mol Nutr Food Res 55:1036–1043PubMedCrossRefGoogle Scholar
  41. Ward PP, Paz E, Conneely OM (2005) Multifunctional roles of lactoferrin: a critical overview. Cell Mol Life Sci 62:2540–2548PubMedCrossRefGoogle Scholar
  42. Ware LB, Matthay MA (2000) The acute respiratory distress syndrome. N Engl J Med 342:1334–1349PubMedCrossRefGoogle Scholar
  43. Wen ST, Chen W, Chen HL, Lai CW, Yen CC, Lee KH, Wu SC, Chen CM (2013) Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury. PLoS One 8:e75383PubMedCentralPubMedCrossRefGoogle Scholar
  44. Xiang M, Fan J, Fan J (2010) Association of Toll-like receptor signaling and reactive oxygen species: a potential therapeutic target for posttrauma acute lung injury. Mediators Inflamm 2010:916425PubMedCentralPubMedCrossRefGoogle Scholar
  45. Yang F, Coalson JJ, Bobb HH, Carter JD, Banu J, Ghio AJ (1999) Resistance of hypotransferrinemic mice to hyperoxia-induced lung injury. Am J Physiol 277:L1214–L1223PubMedGoogle Scholar
  46. Yen CC, Lin CY, Chong KY, Tsai TC, Shen CJ, Lin MF, Su CY, Chen HL, Chen CM (2009) Lactoferrin as a natural regimen for selective decontamination of the digestive tract: recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonates from pathogenic challenge in the gastrointestinal tract. J Infect Dis 199:590–598PubMedCrossRefGoogle Scholar
  47. Yen CC, Lai YW, Chen HL, Lai CW, Lin CY, Chen W, Kuan YP, Hsu WH, Chen CM (2011a) Aerosolized human extracellular superoxide dismutase prevents hyperoxia-induced lung injury. PLoS One 6:e26870PubMedCentralPubMedCrossRefGoogle Scholar
  48. Yen CC, Shen CJ, Hsu WH, Chang YH, Lin HT, Chen HL, Chen CM (2011b) Lactoferrin: an iron-binding antimicrobial protein against Escherichia coli infection. Biometals 24:585–594PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Hsiao-Ling Chen
    • 1
  • Chih-Ching Yen
    • 2
    • 3
    • 4
  • Shih-Ming Wang
    • 2
  • Tung-Chou Tsai
    • 2
  • Zi-Lun Lai
    • 2
  • Jheng-Yue Sun
    • 2
  • Willei Lin
    • 5
  • Wu-Huei Hsu
    • 3
  • Chuan-Mu Chen
    • 2
    • 6
    Email author
  1. 1.Department of BioresourcesDa-Yeh UniversityChanghwaTaiwan
  2. 2.Department of Life Sciences, Agricultural Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan
  3. 3.Department of Internal MedicineChina Medical University HospitalTaichungTaiwan
  4. 4.College of Health CareChina Medical UniversityTaichungTaiwan
  5. 5.Program of Technology ManagementFun Jen Catholic UniversityHsinchuangTaiwan
  6. 6.Rong Hsing Research Center for Translational Medicine, iEGG CenterNational Chung Hsing UniversityTaichungTaiwan

Personalised recommendations