Amelioration of endotoxin-induced uveitis treated with the sea urchin pigment echinochrome in rats

Abstract

Purpose: Echinochrome is a pigment present in the shells and spines of sea urchins. It has been reported to have several biologic protective effects, including in experimental models of myocardial ischemia/reperfusion injury, for which the proposed mechanisms are scavenging reactive oxygen species (ROS) and chelating iron. Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation that is induced by the injection of lipopolysaccharide (LPS). In this study, the therapeutic effect of echinochrome was examined in uveitis using the EIU model.

Methods: EIU was induced in Lewis rats via 200 μg subcutaneous injections of LPS from Escherichia coli. Echinochrome was administered intravenously in 10, 1, or 0.1 mg/kg doses suspended in PBS (controls were injected with PBS only). Twenty-four hours after LPS injection, the number of infiltrating cells and the protein concentration in aqueous humor were determined. Aqueous tumor necrosis factor α (TNF-α) concentration was quantified with enzyme-linked immunosorbent assay, eyes were stained with nuclear factor (NF) κB antibodies, and ROS production was determined by dihydroethidium staining in fresh frozen samples.

Results: The number of inflammatory aqueous cells and protein levels were lower in the groups treated with 10 and 1 mg/kg of echinochrome than in the untreated LPS group (p<0.01). Treatment with 10 and 1 mg/kg of echinochrome significantly reduced TNF-α concentrations in aqueous humor (p<0.01). The numbers of NFκB-positive cells and ROS signals were also reduced by echinochrome administration (p<0.05).

Conclusions: Echinochrome ameliorated intraocular inflammation caused by EIU by reducing ROS production, thereby also decreasing the expression of NFκB and TNF-α. As a natural pigment, echinochrome may therefore be a promising candidate for the safe treatment of intraocular inflammation. The use of sea urchin shells and spines in health foods and medical products is thus both economically and environmentally meaningful.

Figure 1

The chemical structure of echinochrome is shown. Echinochrome has two main isomers.

Figure 2

Effects of echinochrome (10, 1, and 0.1 mg/kg) on A: cellular infiltration and B: protein concentration in aqueous humor collected 24 h after lipopolysaccharide (LPS) treatment are shown. Data are presented as mean±standard error of the mean (SEM; n=4). **Groups treated with echinochrome 10 and 1 mg/kg have significantly less infiltrating cells and protein concentrations than untreated endotoxin-induced uveitis (EIU) controls (p<0.01).

Figure 3

Effects of echinochrome (10, 1, and 0.1 mg/kg) on tumor necrosis factor-α (TNF-α) in aqueous humor collected 24 h after lipopolysaccharide treatment are shown. Data are presented as mean ± standard error of the mean (SEM; n=4). ** TNF-α concentration is significantly lower in echinochrome (10 and 1 mg/kg) treated groups than untreated endotoxin-induced uveitis (EIU) rats (p<0.01).

Figure 4

Effect of echinochrome on nuclear factor κB (NFκB) p65 (red) activation in the iris/ciliary body 3 h after lipopolysaccharide injection is shown. Dual-immunofluorescence labeling showed NFκB colocalization (yellow) in nuclei (green). A: Control naïve animals were not injected with lipopolysaccharide (LPS); an inactive NFκB signal was observed only in the cytoplasm area of cells and no nuclear colocalization of NFκB was detected. B: The group of endotoxin-induced uveitis (EIU) rats treated with echinochrome showed reduced NFκB colocalization compared to C: untreated EIU rats. D: Quantitative analysis of NFκB-positive cells in the iris/ciliary body (ICB). Data are shown as mean±standard error of mean (SEM; n=4). *Amount of NFκB nuclear colocalization was significantly lower in echinochrome treated group than untreated EIU controls (p<0.05).

Figure 5

Reactive oxygen species (ROS) signal expression 24 h after lipopolysaccharide injection is shown. ROS signal (red) was weak in the A: naïve eyes. B: ROS signal was reduced by echinochrome treatment compared with C: iris/ciliary body (ICB) tissue of untreated endotoxin induced uveitis (EIU) rats, in which ROS signal was strongly expressed. D: Mean gray values of the corneal epithelium in dihydroethidium (DHE)-stained slides of the ICB area were evaluated. Mean gray values were significantly lower in the ICB of rats treated with echinochrome than in the eyes of untreated EIU controls (p<0.05). Data are shown as mean ± standard error of the mean (SEM; n=4). *Mean gray value of echinochrome treated eyes was significantly lower than untreated EIU controls (p<0.05).