**3. Results**

DNBS challenge in rats induced a substantial decrease in the body weight starting by day 2 following the enema, in contrast to the sham group which showed body weight gain. During the same period DNBS group experienced diarrhea and also rectal bleeding was observed. AphaMax® (20–100 mg/Kg for 14 day) dose-dependently prevented the drop in the animal weight and improved the diarrheal status and bleeding in DNBS treated rats (Table 2).


**Table 2.** Effects of Aphamax® on body weight change, stool consistency, rectal bleeding 2- and 7- days after colitis-induction.

Percent of body weight change compared to the original body weight (taken as 100%), consistency of the stool rectal bleeding scored following Cooper et al. [22] method in sham rats or in 20, 50, and 100 mg/kg Aphamax®- treated rats with or without colitis induction. Data are means ± S.E.M. *n* = 5 animals/ each group. \* *p* < 0.05 *versus* Sham group; § *p* < 0.05 *versus* 2,4 dinitrobenzensulfonic acid (DNBS) group. #### *3.1. AphaMax*® *E*ff*ects on Macroscopic Changes in Colon of Colitis Animals*

Macroscopic evaluation demonstrated that in the sham group, the distal colon showed no epithelial damage differently from colitis group in which intense mucosal damage, increased wall thickness, hyperemia, ulceration, edema and necrosis were observed associated with high macroscopic score (Figure 1A). DNBS rats revealed also colon shortening accompanied by an increased in the ratio colon weight/length, a marker of tissue inflammation, (13.27 ± 0.46 cm vs. 9.75 ± 0.41 cm colon length in Sham group and in DNBS group respectively *p* < 0.05; 1330.01 ± 120.3 mg vs. 3167.10 ± 443.21 mg colon weight in Sham group and in DNBS group respectively *p* < 0.05) (Figure 1B). AphaMax® (20, 50, or 100 mg/kg) treatment decreased the DNBS-induced macroscopic changes by improving the inflammation symptoms in colon tissues such as mucosal injury, size of ulcer area and also a reduction of the colon weight/length ratio, (11.25 ± 0.45; 12.16 ± 0.65 and 12.91 ± 0.47 cm in colon length in DNBS + AphaMax® 20, 50, or 100 mg/kg respectively ; 2447.03 ± 147.51 mg, 2141.66 ± 80.01 mg, 2095.01 ± 122.03 mg colon weight in in DNBS + AphaMax® 20, 50, or 100 mg/kg respectively, Figure 1B). Diffuse adhesions of the colon with other organs, typically observed during the acute phase of colitis, were unremarkable and only sporadically observed in AphaMax®-treated animals at all doses tested. AphaMax® *perse*,atthehighestdoseused,100mg/Kg,hadnoeffectonshamanimals(Figure1A,B).

**Figure 1.** Effects of Aphamax® on DNBS-induced macroscopic colonic damage. (**A**) Macroscopic damage score based on Appleyard and Wallace method and (**B**) Colon weight/Length ratio in sham rats or in Aphamax®-treated rats with or without colitis induction. Data are means ± S.E.M. *n* = 5 animals /group. \* *p* < 0.05 *versus* Sham animals; § *p* < 0.05 *versus* 2,4 dinitrobenzensulfonic acid (DNBS) animals.

#### *3.2. AphaMax*® *E*ff*ects on Histopathological Changes in Inflamed Colon Tissue*

Histological analyses showed in the sham group a colon tissue with normal mucosa, submucosa, crypts, muscularis, lamina propria, and serosa, without inflammatory cell infiltration and necrosis. A score of the histological changes in colitis-induced rats was calculated, as previously described [18–21] (Table 2). In the DNBS group, the colon showed serious histological changes with high scores of microscopic damages, characterized by large ulcers with necrosis, transmural inflammation, disrupted crypts and massive infiltration into lamina propria and submucosa of inflammatory cells, consisting of lymphocytes, macrophages, and neutrophils. A net distinction of the colon layers from one another was not allowed AphaMax® (20–100 mg/Kg) treatment induced a progressive reduction of inflammatory cell infiltration and decreased the DNBS-mediated colonic damages leading to an improvement in the microscopic damage score; however no one of the concentrations tested completely resolved the histological changes (*p* < 0.05 compared to sham group) (Figure 2A,B).

**Figure 2.** Effects of Aphamax® on DNBS-induced histological damage. (**A**) Photomicrographs of the colon stained with H&E from (**a**) Sham animals, (**b**) Sham rats treated with Aphamax® (100 mg/kg), (**c**) DNBS animals showing colonic damage and a diffuse infiltration of inflammatory cells in mucosa and submucosa or (**d**) DNBS rats treated with Aphamax® (20 mg/kg), (**e**) or with Aphamax® (50 mg/kg), showing progressive reduction of submucosal infiltration of inflammatory cells in submucosal layer and (**f**) DNBS rats treated with Aphamax® (100 mg/kg) showing few inflammatory cells close to the mucosal layer. (Scale bar = 100 μm, magnification 20×, red arrows = colonic damage, black arrows = inflammatory infiltrate)) (**B**) Microscopic damage scored with Hunter method and (**C**) activity of colonic myeloperoxidase in sham rats or Aphamax® treated rats with or without colitis induction. Data are means ± S.E.M. *n* = 5 animals for each group. \* *p* < 0.05 *versus* Sham group; § *p* < 0.05 *versus* 2,4 dinitrobenzensulfonic acid (DNBS) group.

#### *3.3. AphaMax* ® *E*ff*ects on MPO and Cytokine Levels*

To explore the possible action mechanism of AphaMax ® extract, we examined, at first, the myeloperoxidase levels, index of a massive infiltration of neutrophil into the inflamed tissue. In DNBS animals, the MPO activity was significantly increased compared to the sham group. MPO activity was reduced by AphaMax ® treatment in a dose-dependent manner, confirming the histological observation showing a lower infiltration of leukocytes in the colonic tissue (Figure 3C). Moreover, we evaluated the production of proinflammatory cytokines, highly involved in IBD, such as IL-1β and IL-6. In accordance with the immune cell infiltration detected in the histological examination of the DNBS colon, increased levels of IL-1β, IL-6 in comparison to sham animals were observed. Moreover, the reduction in the histological score was correlated with a significant reduction of the levels of IL-1β and IL-6 in AphaMax ®-treated animals, compared with DNBS group, being however still significantly higher compared to sham group (*p* < 0.05) (Figure 3A).

**Figure 3.** Effects of Aphamax ® on pro-inflammatory mediator levels ( **A**) Levels of IL-1β, IL-6 levels; (**B**) NF-kB, ( **C**) COX-2 and ( **D**) iNOS mRNA expression. Data are means ± S.E.M. *n* = 5 animals for each group. \* *p* < 0.05 *versus* Sham group; § *p* < 0.05 *versus* 2,4 dinitrobenzensulfonic acid (DNBS) group.

#### *3.4. AphaMax* ® *E*ff*ect on NF-*κ*B p65, COX-2 and iNOS mRNA Expression*

We assessed the expression of the activated NF-κB p65 subunit, which plays a crucial proinflammatory role during the pathogenesis of IBD. The colon from the DNBS group showed an extensive increase in the mRNA expression of NF-κB p65. NF-κB p65 subunit mRNA expression was significant decreased in AphaMax ®-treated animals suggesting that a down-regulation of these proinflammatory factors could be implicated in AphaMax ® beneficial protective e ffects (Figure 3B).

As already mentioned, the beneficial e ffects of blue-green algae could be partly ascribed to the ability of their components to selectively inhibit key enzymes in inflammatory disease as COX-2 and iNOS [32]. Thus, we analyzed mRNA expression of both enzymes in the colonic tissues from the di fferent animal groups. DNBS group showed an increased expression of both enzyme compared to control group, which was reduced after AphaMax® treatment in dose-dependent manner, (*p* < 0.05 compared to DNBS and to sham group) (Figure 3C,D).

#### *3.5. AphaMax*® *E*ff*ect on Nitrite and ROS Production*

Lastly, to investigate possible antioxidant proprieties of AphaMax® we evaluated if the treatment could affect the tissue NO and ROS generation.

DNBS induced a significant increase of the levels of nitrites and ROS in the colon of DNBS group compared with sham group (*p* < 0.05). AphaMax® at the different doses tested induced a moderate (*p* < 0.05 compared to sham groups) but significant down-regulation in the production of both nitrites and ROS in colitis rats (*p* < 0.05 compared to DNBS group) (Figure 4A,B).

**Figure 4.** Effects of Aphamax® on ROS and nitrite levels. (**A**) ROS levels measured using DCFH-DA, a peroxide/redox-sensitive fluorescent method. (**B**) nitrite levels detected using Griess assay. Data are means ± S.E.M. *n* = 5 for each group. \* *p* < 0.05 *versus* Sham group; § *p* < 0.05 *versus* 2,4 dinitrobenzensulfonic acid (DNBS) group.
