Skip to main content
SpringerLink
Log in

Inflammation and oxidative stress induced by lipid peroxidation metabolite 4-hydroxynonenal in human corneal epithelial cells

  • Basic Science
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Purpose

Oxidative stress is widely known to be a major contributor in the pathogenesis of dry eye disease (DED). 4-Hydroxynonenal (4-HNE), a well-known byproduct frequently measured as an indicator of oxidative stress-induced lipid peroxidation, has been shown to be elevated in both human and murine corneal DED samples. This study aims to investigate if 4-HNE is responsible for the oxidative stress in human corneal epithelial cells (HCECs) and explores the underlying mechanism by which it confers its effects.

Methods

SV40-immortalized HCECs were cultured in minimum essential media (MEM) with 1% penicillin/streptomycin and 10% fetal bovine serum. HCECs were exposed to media with or without 4-HNE and cell culture supernatants were collected at 4 and 24 h. Cellular reactive oxygen species (ROS) measurement was performed using a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay kit according to the manufacturer’s instructions. Protein levels of antioxidant enzymes copper/zinc superoxide dismutase 1 (SOD1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were analyzed by Western blot. NF-κB activation and expression of IL-6 and IL-8 were measured using an NF-κB p65 Total SimpleStep ELISA Kit and Proteome Profiler Human Cytokine Array Kit. Cell viability was evaluated by LDH cytotoxicity assay.

Results

Treatment with 4-HNE decreased cell viability of HCECs. Band intensities corresponding to levels of ROS production showed a significant increase in ROS generation after treatment with 4-HNE. 4-HNE decreased SOD1 levels and upregulated NQO1 expression in HCECs. A significant increase in activation of NF-κB and production of pro-inflammatory cytokines IL-6 and IL-8 was observed after treatment with 4-HNE. Exposure to N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the oxidative effects of 4-HNE on HCECs.

Conclusion

4-HNE induces oxidative stress in corneal epithelial cells by increasing levels of ROS generation and modifying the expression of antioxidant enzyme levels, decreasing cell viability of HCECs in vitro. This study demonstrates a potential pathway by which 4-HNE functions to confer its detrimental effects and provides a new therapeutic target for the treatment of DED.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DED:

dry eye disease

4-HNE:

4-hydroxynonenal

HCECs:

human corneal epithelial cells

ROS:

reactive oxygen species

MDA:

malondialdehyde

OSDs:

ocular surface diseases

DCFDA:

2′,7′-dichlorofluorescin diacetate

RFU:

fluorescence units

SOD1:

superoxide dismutase 1

NQO1:

NAD(P)H quinone dehydrogenase

References

  1. Craig JP, Nichols KK, Akpek EK, Caffery B, Dua HS, Joo CK, Liu Z, Nelson JD, Nichols JJ, Tsubota K, Stapleton F (2017) TFOS DEWS II definition and classification report. Ocul Surf 15(3):276–283. https://doi.org/10.1016/j.jtos.2017.05.008

    Article  PubMed  Google Scholar 

  2. Wakamatsu TH, Dogru M, Tsubota K (2008) Tearful relations: oxidative stress, inflammation and eye diseases. Arq Bras Oftalmol 71(6 Suppl):72–79

    Article  PubMed  Google Scholar 

  3. Schieber M, Chandel NS (2014) ROS function in redox signaling and oxidative stress. Curr Biol 24(10):R453–R462. https://doi.org/10.1016/j.cub.2014.03.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sacca SC, Cutolo CA, Ferrari D, Corazza P, Traverso CE (2018) The eye, oxidative damage and polyunsaturated fatty acids. Nutrients 10(6). https://doi.org/10.3390/nu10060668

  5. Shoham A, Hadziahmetovic M, Dunaief JL, Mydlarski MB, Schipper HM (2008) Oxidative stress in diseases of the human cornea. Free Radic Biol Med 45(8):1047–1055. https://doi.org/10.1016/j.freeradbiomed.2008.07.021

    Article  CAS  PubMed  Google Scholar 

  6. Nita M, Grzybowski A (2016) The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxidative Med Cell Longev 2016:3164734. https://doi.org/10.1155/2016/3164734

    Article  CAS  Google Scholar 

  7. Breitzig M, Bhimineni C, Lockey R, Kolliputi N (2016) 4-Hydroxy-2-nonenal: a critical target in oxidative stress? Am J Phys Cell Phys 311(4):C537–C543. https://doi.org/10.1152/ajpcell.00101.2016

    Article  Google Scholar 

  8. Seen S, Tong L (2018) Dry eye disease and oxidative stress. Acta Ophthalmol 96(4):e412–e420. https://doi.org/10.1111/aos.13526

    Article  CAS  PubMed  Google Scholar 

  9. Choi W, Lian C, Ying L, Kim GE, You IC, Park SH, Yoon KC (2016) Expression of lipid peroxidation markers in the tear film and ocular surface of patients with non-Sjogren syndrome: potential biomarkers for dry eye disease. Curr Eye Res 41(9):1143–1149. https://doi.org/10.3109/02713683.2015.1098707

    Article  CAS  PubMed  Google Scholar 

  10. Hessen M, Akpek EK (2014) Dry eye: an inflammatory ocular disease. J Ophthalmic Vis Res 9(2):240–250

    PubMed  PubMed Central  Google Scholar 

  11. Srivastava SK, Ramana KV (2009) Focus on molecules: nuclear factor-kappaB. Exp Eye Res 88(1):2–3. https://doi.org/10.1016/j.exer.2008.03.012

    Article  CAS  PubMed  Google Scholar 

  12. Lan W, Petznick A, Heryati S, Rifada M, Tong L (2012) Nuclear factor-kappaB: central regulator in ocular surface inflammation and diseases. Ocul Surf 10(3):137–148. https://doi.org/10.1016/j.jtos.2012.04.001

    Article  PubMed  Google Scholar 

  13. Yadav UC, Ramana KV (2013) Regulation of NF-kappaB-induced inflammatory signaling by lipid peroxidation-derived aldehydes. Oxidative Med Cell Longev 2013:690545. https://doi.org/10.1155/2013/690545

    Article  CAS  Google Scholar 

  14. Alves M, Calegari VC, Cunha DA, Saad MJ, Velloso LA, Rocha EM (2005) Increased expression of advanced glycation end-products and their receptor, and activation of nuclear factor kappa-B in lacrimal glands of diabetic rats. Diabetologia 48(12):2675–2681. https://doi.org/10.1007/s00125-005-0010-9

    Article  CAS  PubMed  Google Scholar 

  15. Nakamura S, Shibuya M, Nakashima H, Hisamura R, Masuda N, Imagawa T, Uehara M, Tsubota K (2007) Involvement of oxidative stress on corneal epithelial alterations in a blink-suppressed dry eye. Invest Ophthalmol Vis Sci 48(4):1552–1558. https://doi.org/10.1167/iovs.06-1027

    Article  PubMed  Google Scholar 

  16. Choi SI, Kim TI, Kim KS, Kim BY, Ahn SY, Cho HJ, Lee HK, Cho HS, Kim EK (2009) Decreased catalase expression and increased susceptibility to oxidative stress in primary cultured corneal fibroblasts from patients with granular corneal dystrophy type II. Am J Pathol 175(1):248–261. https://doi.org/10.2353/ajpath.2009.081001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wakamatsu TH, Dogru M, Ayako I, Takano Y, Matsumoto Y, Ibrahim OM, Okada N, Satake Y, Fukagawa K, Shimazaki J, Tsubota K, Fujishima H (2010) Evaluation of lipid oxidative stress status and inflammation in atopic ocular surface disease. Mol Vis 16:2465–2475

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Sano I, Kaidzu S, Tanito M, Hara K, Okuno T, Ohira A (2013) 4-Hydroxyhexenal- and 4-hydroxynonenal-modified proteins in pterygia. Oxidative Med Cell Longev 2013:602029. https://doi.org/10.1155/2013/602029

    Article  CAS  Google Scholar 

  19. Polak M, Zagorski Z (2004) Lipid peroxidation in diabetic retinopathy. Annales Universitatis Mariae Curie-Sklodowska Sectio D: Medicina 59(1):434–437

    Google Scholar 

  20. Niu L, Zhang S, Wu J, Chen L, Wang Y (2015) Upregulation of NLRP3 inflammasome in the tears and ocular surface of dry eye patients. PLoS One 10(5):e0126277. https://doi.org/10.1371/journal.pone.0126277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H (1995) An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 36(3):614–621

    CAS  PubMed  Google Scholar 

  22. Shah A, Farooq AV, Tiwari V, Kim MJ, Shukla D (2010) HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection. Mol Vis 16:2476–2486

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kawashima M, Kawakita T, Okada N, Ogawa Y, Murat D, Nakamura S, Nakashima H, Shimmura S, Shinmura K, Tsubota K (2010) Calorie restriction: a new therapeutic intervention for age-related dry eye disease in rats. Biochem Biophys Res Commun 397(4):724–728. https://doi.org/10.1016/j.bbrc.2010.06.018

    Article  CAS  PubMed  Google Scholar 

  24. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O (2012) Oxidative stress and antioxidant defense. World Allergy Organ J 5(1):9–19. https://doi.org/10.1097/WOX.0b013e3182439613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. He L, He T, Farrar S, Ji L, Liu T, Ma X (2017) Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell Physiol Biochem 44(2):532–553. https://doi.org/10.1159/000485089

    Article  PubMed  Google Scholar 

  26. Cejkova J, Ardan T, Simonova Z, Cejka C, Malec J, Dotrelova D, Brunova B (2008) Decreased expression of antioxidant enzymes in the conjunctival epithelium of dry eye (Sjogren’s syndrome) and its possible contribution to the development of ocular surface oxidative injuries. Histol Histopathol 23(12):1477–1483. https://doi.org/10.14670/HH-23.1477

    Article  CAS  PubMed  Google Scholar 

  27. Sykiotis GP, Bohmann D (2010) Stress-activated cap’n’collar transcription factors in aging and human disease. Sci Signal 3(112):re3. https://doi.org/10.1126/scisignal.3112re3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mittler R (2017) ROS are good. Trends Plant Sci 22(1):11–19. https://doi.org/10.1016/j.tplants.2016.08.002

    Article  CAS  PubMed  Google Scholar 

  29. Fang C, Gu L, Smerin D, Mao S, Xiong X (2017) The interrelation between reactive oxygen species and autophagy in neurological disorders. Oxidative Med Cell Longev 2017:8495160. https://doi.org/10.1155/2017/8495160

    Article  CAS  Google Scholar 

  30. Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15(6):1583–1606. https://doi.org/10.1089/ars.2011.3999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ross D, Siegel D (2017) Functions of NQO1 in cellular protection and CoQ10 metabolism and its potential role as a redox sensitive molecular switch. Front Physiol 8:595. https://doi.org/10.3389/fphys.2017.00595

    Article  PubMed  PubMed Central  Google Scholar 

  32. Asher G, Lotem J, Kama R, Sachs L, Shaul Y (2002) NQO1 stabilizes p53 through a distinct pathway. Proc Natl Acad Sci 99(5):3099–3104. https://doi.org/10.1073/pnas.052706799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu K, Jin B, Wu C, Yang J, Zhan X, Wang L, Shen X, Chen J, Chen H, Mao Z (2015) NQO1 stabilizes p53 in response to oncogene-induced senescence. Int J Biol Sci 11(7):762–771. https://doi.org/10.7150/ijbs.11978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Siegel D, Dehn DD, Bokatzian SS, Quinn K, Backos DS, Di Francesco A, Bernier M, Reisdorph N, de Cabo R, Ross D (2018) Redox modulation of NQO1. PLoS One 13(1):e0190717. https://doi.org/10.1371/journal.pone.0190717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yerramothu P, Vijay AK, Willcox MDP (2018) Inflammasomes, the eye and anti-inflammasome therapy. Eye (London, England) 32(3):491–505. https://doi.org/10.1038/eye.2017.241

    Article  CAS  Google Scholar 

  36. Yoon KC, Jeong IY, Park YG, Yang SY (2007) Interleukin-6 and tumor necrosis factor-alpha levels in tears of patients with dry eye syndrome. Cornea 26(4):431–437. https://doi.org/10.1097/ICO.0b013e31803dcda2

    Article  PubMed  Google Scholar 

  37. Wakefield D, Lloyd A (1992) The role of cytokines in the pathogenesis of inflammatory eye disease. Cytokine 4(1):1–5. https://doi.org/10.1016/1043-4666(92)90028-P

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. Deepak Shukla (University of Illinois, Chicago) for providing SV40-immortalized HCE cell line (RCB1834 HCE-T).

Funding

This work was supported by the Richard A. Perritt Charitable Foundation; Illinois Society for the prevention of Blindness; Science Foundation of Tianjin, China (No. 2018KJ054 to H.L.); and Natural Science Foundation of China (No. 81770890 to S.Z.Z.).

Author information

Author notes

  1. Hui Liu, Frank Gambino Jr and Crystal S. Algenio contributed equally to this work.

Authors and Affiliations

  1. Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin Medical University, Tianjin, 300384, China

    Hui Liu, Yichen Gao & Shaozhen Zhao

  2. Department of Ophthalmology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, IL, 60153, USA

    Hui Liu, Charles S. Bouchard & Ping Bu

  3. Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, IL, 60153, USA

    Frank Gambino Jr & Liang Qiao

  4. Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, IL, 60153, USA

    Crystal S. Algenio & Charles Wu

Authors
  1. Hui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Gambino Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Crystal S. Algenio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yichen Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles S. Bouchard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liang Qiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ping Bu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shaozhen Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.L. and F.G.J. designed the experiments and performed the study; H.L., F.G.J., and C.A. drafted the manuscript; C.W., Y.G., and C.A. coordinated the study and help performed some of experiments; C.B., L.Q., P.B., and S.Z.Z. conceived, designed experiment, contributed experimental materials, contributed to data interpretation, and manuscript editing.

Corresponding authors

Correspondence to Liang Qiao, Ping Bu or Shaozhen Zhao.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Research Office of Stritch School of Medicine, Loyola University. The HCE cell line (RCB1834 HCE-T) was kindly provided by Deepak Shukla, PhD (University of Illinois, Chicago, IL) in 2016. The HCE cell line was established by Dr. Kozaburo Hayashi (National eye Institute, Bethesda, MD) [21, 22]. This cell line has not been authenticated in our lab.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, H., Gambino, F., Algenio, C.S. et al. Inflammation and oxidative stress induced by lipid peroxidation metabolite 4-hydroxynonenal in human corneal epithelial cells. Graefes Arch Clin Exp Ophthalmol 258, 1717–1725 (2020). https://doi.org/10.1007/s00417-020-04647-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-020-04647-2

Keywords

Search

Navigation