Elsevier

Experimental Eye Research

Volume 81, Issue 4, October 2005, Pages 422-428
Experimental Eye Research

Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo

https://doi.org/10.1016/j.exer.2005.03.002Get rights and content

Abstract

The aim of the present study was to investigate the efficacy of fucoxanthin on endotoxin-induced uveitis (EIU) in rats. The effects of fucoxanthin on endotoxin-induced leucocyte and protein infiltration, nitric oxide (NO), prostaglandin (PG)-E2 and tumour necrosis factor (TNF)-alpha concentrations in rat aqueous humour, as well as on the cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein expression in a mouse macrophage cell line (RAW 264.7 cells) were studied. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS injection, either 0.1, 1 or 10 mg kg−1 of fucoxanthin was injected intravenously. The aqueous humour was collected 24 hr later from both eyes, and both the number of cells infiltrating into the aqueous humour and the aqueous humour protein concentration were measured. The levels of PGE2, NO and TNF-alpha were determined by enzyme-linked immunosorbent assay. The RAW 264.7 cells were pretreated with various concentrations of fucoxanthin for 24 hr and subsequently incubated with LPS for 24 hr. COX-2 and iNOS protein expression was analysed by the Western blotting method. Levels of PGE2, NO and TNF-alpha production were determined. Fucoxanthin suppressed the development of EIU in a dose-dependent fashion. Treatment with fucoxanthin resulted in a reduction in PGE2, NO and TNF-alpha concentrations in the aqueous humour. The expression of COX and iNOS protein in the fucoxanthin treated RAW264.7 cells decreased significantly compared to that the LPS group. It also significantly reduced the concentration of PGE2, NO and TNF-alpha production in the medium of cells. The present result indicate fucoxanthin suppresses the inflammation of EIU by blocking the iNOS and COX-2 protein expression and its anti-inflammatory effect on eye is comparable with the effect of predinisolone used in similar doses.

Introduction

Endotoxin-induced uveitis (EIU) is an acute anterior segment intraocular inflammation that can be induced by injection of lipopolysaccharide (LPS) (Bhattacherjee et al., 1983, Hikita et al., 1995, Suzuma et al., 1998). It has been suggested that this phenomenon could serve as a model for certain types of human uveitis such as those associated with seronegative arthritis, where a gram-negative bacteria may play a role in the pathogenesis (Rosenbaum et al., 1980). The inflammatory response in EIU is characterized by a breakdown of the blood-aqueous barrier and is cell-mediated with leakage of proteins into the anterior chamber of the eye and infiltration with a large number of inflammatory cells. Exposure to LPS stimulates cellular inflammatory responses, and releases factors such as cytokines (Hoekzema et al., 1991, Tracey and Cerami, 1994) and chemokines (Tuaillon et al., 2001).

Prostaglandin (PG)-E2 is a major cyclooxygenase (COX) product at inflammatory sites where it contributes to local blood flow increases, edema formation, and pain sensitization. We have previously reported that the PGE2 levels in the aqueous humour significantly increase in EIU, and therefore these data suggest that COX plays an important role in EIU (Shiratori et al., 2004). Also, one of the responses in the eye to LPS injection is the generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) (Goureau et al., 1995, McMenamin and Crewe, 1997). In addition, LPS and pro-inflammation cytokines induce iNOS in macrophages, neutrophils and endothelial cells, which can further contribute to the levels of nitric oxide in the eye during EIU (Mandai et al., 1994).

Carotenoids have been demonstrated to have beneficial effects on health. Fucoxanthin is a carotenoid that is found in common edible seaweed, such as Hijikia fusiformis, Undaria pinnatifida and Sargassum fulvellum. The chemical structures of fucoxanthin are shown in Fig. 1. Many biological functions of this compound have been studied, including cancer-preventing (Kotake-Nara et al., 2001), antimutagenic effects (Nishino, 1998). Recently, Ohgami et al. (2003) reported that carotenoid astaxanthin prevents the inflammation of EIU.

In the present study, we investigated the influence of fucoxanthin on EIU in rats. The anti-inflammatory potency of fucoxanthin in vivo was compared with that of prednisolone. In addition, we also investigated the in vitro concentrations of NO, PGE2, and tumour necrosis factor (TNF)-alpha and the expression of iNOS and COX-2 in RAW 264.7 cells after treatment with fucoxanthin in order to clarify the anti-inflammatory effect. To the best of our knowledge, there have been no reports on the effect of fucoxanthin on ocular inflammation.

Section snippets

Animals groups and EIU

Eight-week-old male Lewis rats were used. The rats weighed about 250 g. EIU was induced by injection into one footpad of 200 μg of LPS from Salmonella typhimurium (Sigma, St. Louis, MO) that had been diluted in 0.2 ml of saline.

The rats were injected intravenously with 0.1, 1 or 10 mg kg−1 fucoxanthin or 10 mg kg−1 prednisolone (Sigma, St. Louis, MO) in a 0.1% dimethyl sulfoxide solution (Sigma, St. Louis, MO) mixed with 0.1 ml phosphate-buffered saline (PBS). Fucoxanthin was isolated from brown algae

Effects on LPS-induced cellular infiltration in the aqueous humour

Cells were not detected in the aqueous humour of the control group. In the LPS group, the number of cells in the aqueous humour was 62.1±11.7×105 cells ml−1. Treatment with 0.1, 1 or 10 mg kg−1 of fucoxanthin showed reduced numbers of cells (0.1 mg kg−1; 27.2±5.5×105 cells ml−1, 1 mg kg−1; 18.3±6.5×105 cells ml−1, 10 mg kg−1; 12.8±3.5×105 cells ml−1). Statistically significant differences were observed (P<0.01) between the LPS and fucoxanthin groups. The effect of 10 mg kg−1 fucoxanthin on the number of cells in

Discussion

In the present study, we investigated the effect of fucoxanthin on EIU in rats. The results of the study indicate that fucoxanthin suppressed the development of EIU in a dose-dependent fashion. The anti-inflammatory activity of 10 mg kg−1 of fucoxanthin was comparable to that of 10 mg kg−1 of prednisolone. Clinical side effects of steroids were often reported, but no side effects of prednisolone were observed in this study.

Three types of NOS isoforms have been identified in cells. Endothelium NOS

Acknowledgements

This work was supported in part by a grant for Research on Sensory and Communicative Disorders, Ministry of Health, Labour and Welfare, Japan, and Uehara memorial foundation.

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