Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation

Abstract

Purpose: Ultraviolet B (UVB) irradiation activates nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in the cornea, resulting in inflammatory responses and malondialdehyde (MDA) accumulation. This study aims to determine the effect of zerumbone, a potent NF-κB inhibitor and inflammation modulators, on UVB-induced corneal damages in a mouse model.

Methods: Fifty female imprinting control region (ICR) mice were randomly divided into five groups. The mice were anaesthetized with their ocular surfaces exposed to UVB light (0.72J/cm(2)/daily), followed by daily dietary zerumbone supplements at 0, 1, 10, and 100 mg/kg of bodyweight. Mice without zerumbone supplements were used as treatment controls and mice without UVB irradiation as blank controls. Corneal surface damages were graded according to smoothness, opacity, and the extent of lissamine green staining. Histopathological changes were also examined, along with the expression of NF-κB, iNOS, and tumor necrosis factor-α (TNF-α). MDA accumulation and the levels of two antioxidant enzymes, glutathione (GSH) and GSH reductase (GR) were also examined.

Results: UVB irradiation caused significant damages to cornea, including sustained inflammation, apparent corneal ulcer, and severe epithelial exfoliation, leading to thinning of corneal epithelial layer, and infiltration of polymorphonuclear leukocytes. NF-κB expression was highly activated with nuclear translocation. The expression of iNOS and TNF-α were increased. MDA accumulation was also increased in both the corneal epithelial layer and the stroma. With dietary zerumbone, corneal damages were ameliorated in a dose-dependent manner. NF-κB activation and its nuclear translocation were blocked with decreased expression of iNOS and TNF-α. Infiltration of polymorphonuclear leukocytes was also blocked by dietary zerumbone. Besides, MDA accumulation was reduced with concomitant increase of GSH and GR levels.

Conclusions: Dietary zerumbone prevents UVB-induced corneal damages by inhibition of NF-κB, iNOS, and TNF-α, with concomitant reduction of MDA accumulation and increase of GSH and GR levels in the mouse model. Results of this study suggest that dietary zerumbone may be used as a prophylactic agent against UVB-induced photokeratitis.

Figure 1

Chemical structure of zerumbone and experimental protocol for dietary zerumbone supplementation after UVB irradiation to the mouse cornea. A: The chemical structure of zerumbone. B: Daily UVB light exposure (indicated by arrows) was performed from Day 1 to Day7, with dietary zerumbone given at 1, 10, and 100 mg/kg of bodyweight, respectively, from Day 0 until Day 8. No zerumbone was given to the UVB group or the blank control group.

Figure 2

Comparison and scoring of corneal smoothness, opacity, and lissamine green staining among UVB (UVB exposure without dietary zerumbone), UVB/ZER (UVB exposure with dietary zerumbone at 1, 10, and 100 mg/kg of bodyweight, respectively), and blank control (no UVB exposure and no dietary zerumbone) groups. All scale bars: 1.25 mm.

Figure 3

UVB-induced corneal damages, central corneal epithelial thickness, NF-κB and iNOS expression, and MDA accumulation among study groups. A-E: Hematoxylin-Eosin (HE) stain of corneas showed much thinner corneal epithelial layer with UVB exposure and the reverse effect with dietary zerumbone. U: The central corneal epithelial thickness was significantly reversed with dietary zerumbone, starting at 10 mg/kg. F-T: Immunohistochemical staining showed evident inhibition of NF-κB and iNOS expression with dietary zerumbone supplementation at 100 mg/kg and 10 mg/kg, respectively. NF-κB nuclear translocation in the UVB-exposed cornea in (F) was evidently reversed in the cornea with dietary zerumbone at 100 mg/kg in (I). MDA accumulation was reduced with dietary zerumbone supplementation at 100 mg/kg. All scale bars in E, J, O, and T are equal to 20μm.

Figure 4

Anti-inflammatory effects of dietary zerumbone. A-E: Infiltration of polymorphonuclear leukocytes following UVB exposure, which was evidently blocked by dietary zerumbone. F: TNF-α expression was inhibited by dietary zerumbone. Scale bar in E is equal to 20 μm.

Figure 5

Diagrammatic illustration of the effects of dietary zerumbone against UVB-induced photokeratitis as shown in this study.