Anti-inflammatory effect of fucoxanthin on dextran sulfate sodium-induced colitis in mice

Abstract The anti-inflammatory activities of fucoxanthin, a marine carotenoid derived from the macroalgae and microalgae, have been demonstrated in the previous studies. However, the effect of fucoxanthin on ulcerative colitis (UC), an inflammatory bowel disease, was still unclear. In this study, we evaluated the in vivo anti-inflammatory effect of fucoxanthin on dextran sulfate sodium(DSS)-induced colitis in mice. Fucoxanthin at the doses of 50 and 100 mg/kg/day significantly protected against DSS-induced gradual loss of body weight, exhibited inhibitory effects on the DSS-induced increase of disease activity index and colon shortening. Moreover, fucoxanthin treatment resulted in a marked amelioration of the histological damage in the colon, and reduced the colonic PGE2 levels in colitic mice. In addition, the DSS-induced overexpressions of inflammation-related molecules including COX‐2 and NF-κB were significantly decreased in fucoxanthin-treated mice. These finding suggested that the use of fucoxanthin provides a new and attractive alternative to control UC. Graphical Abstract


Introduction
As a marine carotenoid derived from the macroalgae and microalgae (e.g. Undaria pinnatiida, Laminaria japonica, Cylindrotheca closterium, and Phaeodactylum tricornutum), fucoxanthin has been paid great attention due to its significant pharmacological activities (Zhang et al. 2015). In particular, the results of in vitro and in vivo studies have demonstrated the anti-inflammatory activities of fucoxanthin. Using an lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophage culture system, fucoxanthin has been repeatedly confirmed to exhibit an anti-inflammatory activity (Shiratori et al. 2005;Heo et al. 2012;Islam et al. 2013). Moreover, fucoxanthin also served as a negative feedback regulator of neuroinflammation in amyloid-b42-induced (Pangestuti et al. 2013) and LPS-induced (Zhao et al. 2017) BV2 microglia cells. These data suggested fucoxanthin might be ultimately proved useful in the control of inflammatory diseases. Accordingly, fucoxanthin has been reported to dose-dependently suppress the development of endotoxin-induced uveitis in rats (Shiratori et al. 2005), ameliorate the state of low-level inflammation in high-fat-diet-induced obesity in mice (Tan and Hou 2014), significantly attenuate carrageenan/kaolin-induced experimental arthritis in rats (Gong et al. 2014), as well as inhibit the inflammation response in carrageenaninduced paw edema model (Choi et al. 2016). And the in vivo anti-inflammatory potency of fucoxanthin was believed to be compared with that of prednisolone (Shiratori et al. 2005).
Excessive or prolonged inflammation is proved harmful, and is a hallmark of a variety of disorders, including ulcerative colitis (UC), an inflammatory bowel disease (Wang et al. 2016). Hwang et al. (2016) have demonstrated the anti-inflammatory effect of fucoxanthin in an LPS-induced intestinal epithelial Caco-2 cell damage, an in vitro model of inflammatory bowel disease. Here, we evaluated the in vivo anti-inflammatory effect of fucoxanthin on dextran sulfate sodium(DSS)-induced colitis in mice.

Fucoxanthin prevented the development of DSS-induced colitis in mice
As shown in Figure S1, fucoxanthin at the doses of 50 and 100 mgÁkg À1 Áday À1 (chosen based on a preliminary experiment) significantly protected against DSS-induced gradual loss of body weight, starting on day 2 after the initiation of DSS challenge, and lasted up to day 7 (p < 0.01). On day 7, fucoxanthin treatment was found to exhibit inhibitory effects on the DSS-induced disease activity index (DAI) increase in a dosedependent manner (p < 0.05; p < 0.01) and improve the colon shortening induced by DSS (p < 0.05). Compared to the normal group, there were no abnormal changes in the body weight and colons of mice in the solely fucoxanthin group. And the protective effects exerted by fucoxanthin on the colitis severity were comparable to those obtained with salazosulfapyridine (SASP), which is a first-line anti-inflammatory medication for active UC managements. However, many side effects associated with SASP, including fever, abdominal pain, diarrhea, rash, cramps and kidney failure, render its use undesirable (Wang et al. 2016). But the safety of fucoxanthin and its metabolite has been demonstrated to be excellent (Zhang et al. 2015), which suggests that the use of fucoxanthin provides a relatively nontoxic and attractive alternative to control UC.

Fucoxanthin relieved colonic inflammation in mice with DSS-induced colitis
Histological analysis showed that, compared with the DSS control group, mice in the fucoxanthin-treated and SASP-treated groups exhibited relieved inflammatory conditions in the colon, as indicated by decreased inflammatory cell infiltration, wellpreserved colonic structure, and improved edema ( Figure S2a and b).Treatment of low-or high-dose fucoxanthin, or 500 mgÁkg À1 Áday À1 SASP, resulted in a significant reduction in the histological scores, when compared with the DSS control group (p < 0.05; p < 0.01).
Prostaglandin(PG)E 2 , as an important inflammatory mediator, has been known to be upregulated proportionately to the severity of mucosal inflammation in UC, thus be considered as a possible inflammation marker in UC patients (Shiratori et al. 2005;Kim et al. 2010). As shown in Figure S2c, fucoxanthin alone treatment at 100 mgÁkg À1 Áday À1 did not affect the colonic PGE 2 level, compared to the normal control group, importantly, the increase of colonic PGE 2 production induced by DSS challenge was significantly prevented by the presence of 50 or 100 mgÁkg À1 Áday À1 fucoxanthin, or 500 mgÁkg À1 Áday À1 SASP (p < 0.01). The inhibitory effect of fucoxanthin on the enhanced production of PGE 2 under inflammatory conditions has been reported in LPS-activated murine RAW 264.7 macrophage (Shiratori et al. 2005;Kim et al. 2010;Heo et al. 2012), LPS-induced BV2 microglia cells (Zhao et al. 2017), and in the aqueous humour of rat with endotoxin-induced uveitis (Shiratori et al. 2005). In line with these previous studies, here we provide an additional evidence for the reduction in PGE 2 production in response to fucoxanthin using DSS-induced colitis model.

Fucoxanthin downregulated the DSS-induced expression of COX-2 and NF-jB in colon tissues
Development of UC was accompanied by the increased expression of cyclooxygenase (COX)-2, an inducible enzyme contributing to the PGE 2 production. And there are increasing evidences on the role of COX-2 in mediating the barrier dysfunction and the prolonged epithelial secretion observed in murine colonic inflammation (Sklyarov et al. 2011). As shown in Figure S3a, the increase in COX-2 mRNA expression in colon tissues induced by DSS was attenuated by 33%, 60% and 59%, respectively, after low-, high-dose fucoxanthin and SASP administration (p < 0.05; p < 0.01). Immunofluorescence staining ( Figure S3c) showed a significantly positive protein expression of COX-2 in colon, especially in ulcer surface area, from the DSS control mice. However, the DSS-induced expression of COX-2 was markedly repressed by fucoxanthin.
In UC patients, nuclear factor (NF)-jB as a transcription factor mediating transcriptional activation of numerous inflammatory genes was found to be significantly activated and strongly affect the course of mucosal inflammation. Accordingly, blockade of NF-jB activation has been considered as a therapeutic strategy for UC (Liu et al. 2018). Figure S3b showed that fucoxanthin dose-dependently reduced the upregulated expression of NF-jBp65 mRNA in mice with DSS-induced colitis (p < 0.05; p < 0.01). As shown in Figure S3c, co-staining NF-jBp65 of with 4,6-diamidino-2phenylindole (DAPI), the latter of which was used to define nuclei, showed that DSS treatment caused an increase of expression levels and a cytoplasmic to nuclear translocation of NF-jBp65, which could be reversed by fucoxanthin treatment. There was no marked change in the expressions of COX-2 and NF-jB in the fucoxanthin 100 mg/ kg group, compared to the normal control group.

Conclusions
This study demonstrated the anti-inflammatory effect of fucoxanthin administration in DSS-induced colitis model, suggesting that the use of fucoxanthin provides an attractive alternative to control UC. It is worth noting that, in addition to it's antiinflammatory activity, fucoxanthin is considered as a potential natural antioxidant (Zhang et al. 2015). And targeting oxidatives tress, a key pathogenic factor in the inflamed colonic tissue, might provide an exciting approach to combat inflammationassociated damage in UC (Jena et al. 2012). Therefore, it merits further elucidation as to whether the antioxidant property of fucoxanthin also underlies its protective effect on the experimental colitis.

Disclosure statement
No potential conflict of interest was reported by the authors.