June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Histatin-1 attenuates the production nitric oxide by inhibiting MAPK signaling in LPS-induced RAW264.7 macrophages
Author Affiliations & Notes
  • Sang Min Lee
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Kyung-No Son
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Dhara Shah
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Marwan Ali
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Arun Balasubramaniam
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Vinay Kumar Aakalu
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   Sang Min Lee, None; Kyung-No Son, None; Dhara Shah, None; Marwan Ali, None; Arun Balasubramaniam, None; Vinay Aakalu, Horizon Pharma (C), University of Illinois at Chicago (P), ViSo Therapeutics Inc. (I)
  • Footnotes
    Support  NEI/NIH K08EY024339, R01EY029409, Department of Defense: W81XWH1710122, VA I01BX004080
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1243. doi:
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      Sang Min Lee, Kyung-No Son, Dhara Shah, Marwan Ali, Arun Balasubramaniam, Vinay Kumar Aakalu; Histatin-1 attenuates the production nitric oxide by inhibiting MAPK signaling in LPS-induced RAW264.7 macrophages. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1243.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Nitric oxide (NO) is a free radical which plays an important role in immune and inflammatory responses as an important intercellular messenger. In addition, NO has an important role in inflammatory responses in the ocular surface. Histatin peptides are well established antimicrobial and wound healing agents. These peptides are important in multiple biological systems, playing roles in responses to the environment and immunomodulation. Given the importance of macrophages in responses to environmental triggers and pathogens, we investigated the effect of histatin-1 (H1) on LPS-induced inflammatory responses and the underlying molecular mechanisms in RAW264.7 macrophages.

Methods : RAW264.7 mouse macrophages were stimulated by adding 10 ng/mL of lipopolysaccharide (LPS) with or without H1, dose-dependently. The production of NO were determined by Griess assay (Sigma, St. Louis, MO) in RAW264.7 seeded in 96-well plates. Expression of inducible nitric oxide synthase (iNOS) was measured by Western blot analysis. Activation of downstream signaling molecules were detected by Western blotting using phosphorylation specific MAPKs antibodies.

Results : Griess assay testing demonstrated that treatment with H1 inhibited significantly the LPS stimulated NO production in RAW264.7 cells, in a dose dependent manner. iNOS expression was detected in LPS treated RAW264.7 cells, and pre-treatment of H1 inhibited activation. Macrophages did not exhibit cytotoxic response to up to 100 µM of H1 treatment. Phosphorylation of p38 and JNK1/2 MAPKs were inhibited after pre-treatment with H1; however, pro-survival ERK1/2 MAPK protein was not inhibited by H1 in LPS stimulated RAW264.7 cells.

Conclusions : These results demonstrate that H1 can inhibit LPS induced inflammatory mediator production via suppressing activation of MAPK signaling pathways in macrophages.

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1. Effects of H1 on nitric oxide production in murine macrophages.
RAW264.7 cells were pre-treated indicated concentrations of H1 for 2 h then co-treated with LPS (10ng/mL) for 16 h. The culture supernatants were subsequently isolated and analyzed for nitrite levels. The values for nitrite are mean±S.D. from three independent experiments. **P 〈 0.001 compared to treatment with LPS alone.

Figure 1. Effects of H1 on nitric oxide production in murine macrophages.
RAW264.7 cells were pre-treated indicated concentrations of H1 for 2 h then co-treated with LPS (10ng/mL) for 16 h. The culture supernatants were subsequently isolated and analyzed for nitrite levels. The values for nitrite are mean±S.D. from three independent experiments. **P 〈 0.001 compared to treatment with LPS alone.

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