Abstract
Purpose
This study assessed the anti-inflammatory effect and mechanism of action of hinokitiol in human corneal epithelial (HCE) cells.
Methods
HCE cells were incubated with different concentrations of hinokitiol or dimethylsulfoxide (DMSO), which served as a vehicle control. Cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assay. After polyriboinosinic:polyribocytidylic acid (poly(I:C)) stimulus, cells with or without hinokitiol were evaluated for the mRNA and protein levels of interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-1β (IL-1β) using real-time PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear and cytoplasmic levels of nuclear factor kappa B (NF-κB) p65 protein and an inhibitor of NF-κB α (IκBα) were evaluated using western blotting.
Results
There were no significant differences among the treatment concentrations of hinokitiol compared with cells incubated in medium only. Incubating with 100 μM hinokitiol significantly decreased the mRNA levels of IL-8 to 58.77±10.41% (P<0.01), IL-6 to 64.64±12.71% (P<0.01), and IL-1β to 54.19±8.10% (P<0.01) compared with cells stimulated with poly(I:C) alone. The protein levels of IL-8, IL-6, and IL-1β had similar trend. Further analysis revealed that hinokitiol maintained the levels of IκBα and significantly reduced NF-κB p65 subunit translocation to the nucleus which significantly inhibiting the activation of the NF-κB signal pathway.
Conclusion
Hinokitiol showed a significant protective effect against ocular surface inflammation through inhibiting the NF-κB pathway, which may indicate the possibility to relieve the ocular surface inflammation of dry eye syndrome (DES).
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Acknowledgements
We thank Ying-Gang Yan, PhD, Yu Wu, PhD, Bin Zhang, PhD (Department of Neurobiology in Zhejiang University) for technical assistance in sample testing and statistical analysis. This study was supported by grant 81070756 from the Natural Science Foundation of China, by grant 2012BAI08B01 from the National Twelfth Five-Year Plan for Science and Technology Support of China, by grant NCET-11-0161 from the Program for New-Century Excellent Talents in Universities of China, by the Zhejiang Provincial Program for Cultivation of High-level Innovative Health Talents, by grant 2012C13023-2 from the Specialized Key Science and Technology Foundation of Zhejiang Provincial Science and Technology Department, and by grant 2011ZDA014 from the Zhejiang Provincial Key Project of Medicine and Health.
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Ye, J., Xu, YF., Lou, LX. et al. Anti-inflammatory effects of hinokitiol on human corneal epithelial cells: an in vitro study. Eye 29, 964–971 (2015). https://doi.org/10.1038/eye.2015.62
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DOI: https://doi.org/10.1038/eye.2015.62
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