**2. Results**

#### *2.1. Fucoidan Diet Rescued Psoriasis Symptoms on m-Traf3ip2 Mouse Faces and Reduced Scratching*

Psoriasis is a chronic immunological inflammatory disease of the skin characterized by dry silvery scales with eruptions. In this study, the severity of symptoms on the faces of *m-Traf3ip2* mice was graded by phenotype using a scoring system based on clinical Psoriasis Area and Severity Index (PASI) and an ethological method as a scratch test. For this study, 1% fucoidan or 1% cellulose was administered by adding it to the AIN-93G diet, which is a normal diet. *m-Traf3ip2* mice facial skin showed significant di fferences in symptoms between the fucoidan and normal diet groups. *m-Traf3ip2* mice which were on the normal diet showed severe symptoms of psoriasis on their faces, whereas the mice on the fucoidan diet showed improved facial symptoms after 21 days and tended to enter remission in the following weeks (Figure 1A). The normal diet group exhibited increased scratching within a certain time frame, whereas the fucoidan diet group showed significantly decreased facial scratching from 1 week until after 56 days (Figure 1B). In the normal diet group, the mice with the highest PASI scores showed the most serious symptoms and the most severe scratch test results. On the other hand, the symptoms of the fucoidan diet mice group decreased from 21 days until after 63 days, and were significantly lower than in those of the control group (Figure 1C). Histological sections of the facial skin of mice that had high PASI scores showed thickening of both the epithelium and keratin layer of the epidermis by hematoxylin-eosin (HE) staining (Figure 1D).

**Figure 1.** Effects of fucoidan diet on *m-Traf3ip2* mouse faces by PASI score analysis and scratching actions. (**A**) Aspects of *m-Traf3ip2* typical mouse face over time. (a), (b), (c) Sections are normal diet mice and (d), (e), (f) sections are fucoidan diet mice. (**B**) Scratching behavior of individual mice. Closed triangle shows normal diet group and closed circle shows fucoidan diet group. PASI test scored 5 persons and each value. The calculated values for the in vitro experiments are mean ± SD (fucoidan diet group *n* = 14 and normal diet group *n* = 9). (**C**) The severity of the psoriasis-like skin condition. (**D**) Histological analysis by HE staining of faces. Normal diet mice and fucoidan diet mice epidermal sections are shown on left and right, respectively. Bars show 0.2 mm. Asterisk (\*) shows significant difference (*p* < 0.05).

These results suggested that fucoidan improved psoriasis symptoms on the faces of *m-Traf3ip2* mice.

#### *2.2. Fucoidan Drastically Changed Microbiota in the Small Intestines of m-Traf3ip2 Mice*

Dietary fiber is a prebiotic, which is useful in the intestinal environment [11,12]. In the present study, 16S rRNA from intestinal microorganisms in fecal samples was analyzed using next-generation genome sequences. In phylum analysis, fecal microflora in the fucoidan diet group showed significantly increasing relative abundance of the *Bacteroidetes* and *Proteobacteria* phyla during each time course (i.e., 6, 28, and 56 days) compared to mice fed the normal diet, whereas the relative abundance of the *Firmicutes* and *TM7* phyla significantly decreased in the fucoidan diet group after 6 days and remained at a low level until the end of the experiment (Figure 2A) compared to the fecal microorganisms of the normal diet fed mice. Table 1 shows the changes in phylum levels in fecal microflora of *m-Traf3ip2* fucoidan diet and normal diet mice over time. The relative abundance of *Deferribacteres* and *Actinobacteria* phyla, which were the lowest of all the phyla, were higher in the fucoidan diet group than in the normal diet group. The differences between the fucoidan and normal diet samples were then analyzed by taxonomy, class, order, family, and genus. After 56 days, levels of fecal microbiota belonging to the *Bacteroidaceae* and *Paraprevotellaceae* families in the *Bacteroidetes* phylum in fucoidan diet group were significantly higher than those of normal diet group. On the other hand, the fecal microbiota of fucoidan diet group in the family of *F16* in the phylum of *TM-7,* and those in the family of *Odoribacteraceae* in the phylum *Bacteroidetes*, showed decreased levels of the same kinds of microflora after 56 days (Figure 2B,C) compared to the microbiota of normal diet mice group. In the genus analysis, fecal microflora of fucoidan diet group showed significantly increased relative abundance of unclassified *Paraprevotellaceae* genera and *Bacteroides* genera in the family of *Bacteroidetes* compared to fecal microflora of the normal diet group (Figure 2D,E).


**Table 1.** Changes in phylum levels in fecal microbiota of *m-Traf3ip2* fucoidan diet group and normal diet group. Significant difference between fucoidan and normal diets. (*p* < 0.01 (c), *p* < 0.05 (d)). Significant difference from before to after same diet administration. (*p* < 0.01 (a), *p* < 0.05 (b)).

Microorganisms of the *Desulfovibrionaceae* family were found at slightly higher levels in the fecal microbiota of the fucoidan diet group compared to that of the normal diet group. The fecal microbiota of fucoidan diet group showed significantly lower populations of the genera *Prevotella* and *Odoribacter*, as well as of an unclassified *S24-7* family in the *Bacteroidetes* phylum, than in the normal diet group after 56 days. In the *Firmicutes* phylum, the genera of *Coprococcus*, unclassified members of the *Ruminococcaceae* family, and unclassified members of the order *Clostridiales* were lower in fecal microbiota of the fucoidan diet group compared to those of the normal diet group. The normal diet group showed significant increase in the fecal microbiota of *F16* family compared to those of the fucoidan diet group.

These results suggested that the fucoidan diet altered the taxonomic compositions of many microbiota in *m-Traf3ip2* mice 6 days after feeding, and thereafter, the microbiota conditions were continuously maintained.

**Figure 2.** *Cont*.

**Figure 2.** Fecal microbiota analysis. 16S rRNA genome-wide screening analysis. Time dependence of fecal microbiota analyses were performed on the phylum (**A**), family (**B**), (**C**), and genus (**D**), (**E**), levels of normal diet group (left) and fucoidan diet group (right). White bar shows normal diet group and black bar shows fucoidan group. The calculated values for the *in vitro* experiments are means ± SD (*n* =5).

#### *2.3. Fucoidan Diet Promoted Fecal Mucin Production*

Mucin, which is produced by mucous/goblet cells, is a major component of mucosal fluid. Alterations in gastrointestinal mucin induced by dietary fiber may affect nutrient bioavailability, cytoprotection of the mucosa, or other aspects of gastrointestinal function [20,21]. In this study, mucin production in feces by mucous goblet cells was measured. Fluorescence labeling was used to measure terminal N-acetylgalactosamine in mucin components in feces. In *m-Traf3ip2* mice, the fucoidan diet group showed increased mucin production, especially from 21 days after the diet had begun, whereas the normal diet group showed little change (Figure 3A). Similarly, wild-type, not mutated gene of *Traf3ip2* BALB/c mice, fed a fucoidan diet showed significantly increasing mucin secretion compared to the group of a normal diet mice (Figure 3B). These results suggested that fucoidan promoted mucin production and created a better intestinal environment.

**Figure 3.** Quantification of mouse fecal mucin. Volume concentrations of (**A**) *m-Traf3ip2* and (**B**) wild type mouse in fecal mucin. White bar shows normal diet group and black bar shows fucoidan group from fecal. The calculated values for the in vitro experiments are mean ± SD (*n* = 5). Asterisk (\*) shows significant difference (*p* < 0.05).

#### *2.4. Production of IgA in Feces and Cecum*

Secreted of IgA in the gu<sup>t</sup> mucosal layer plays important roles in intestinal immune function, e.g., prevention of bacterial and viral invasion. IgA suppresses the expression of bad bacteria and maintains a healthy balance of gu<sup>t</sup> microbiota. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate IgA expression in feces and cecum of *m-Traf3ip2* mice fed on fucoidan with or without diet for 63 days. The assay showed that secreted IgA was not present, or was present at levels below detection, in fecal samples from both groups. It was reported that the cecum produces IgA [22]. Our study detected total IgA in the cecum of both groups in the samples obtained at 63 days. Total IgA volume was obviously higher in the ceca of the fucoidan fed mice group compared to those of the normal diet mice group (Figure 4A). Wild type mice fed a fucoidan diet also showed higher total IgA compared to the normal diet group of wild type mice (Figure 4B).

**Figure 4.** Quantification of total IgA in cecal contents of fucoidan diet group and normal diet group. Volume concentrations of (**A**) *m-Traf3ip2* and (**B**) wild type mouse total IgA were quantified from 63 days and 31 days, respectively, in cecum contents by ELISA. White bar shows normal diet group and black bar shows fucoidan diet group from cecum. The absorbance of reaction products was determined at 450 nm, and the production was quantified. The calculated values for the in vitro experiments are expressed as mean ± SD (*n* = 5). Asterisk (\*) shows significant difference (*p* < 0.05).

This result indicated that fucoidan promotes the secretion of IgA in the cecum and improves the intestinal immunity.
