The cultivation of SPI requires intense sunlight, high temperatures and low rainfall, nutrients, and a pH between 9 and 10. Initially, the inocula received from Antenna (Geneve, Switzerland) were cultivated in the authors’ laboratory at the Federal University of Ceara, Brazil, utilizing Zarrouk media. After filtration, the material was weighed to determine the wet biomass and submitted to desiccation for 5 hours (50℃). The dried material was weighed to determine the dried biomass and the production per square meter (8 to 10 g/day/m²).

C57BL/6 male mice aged 6-8 weeks, weighing 20-25 g, were divided randomly into five groups, with six mice/groups, as follows: healthy-mice not exposed to DSS and treated with saline only; DSS+Vei–mice exposed to 3% DSS and treated with saline by gavage; DSS+50, 100 or 250 mg/kg/day–mice exposed to DSS 3% and treated by gavage with different concentrations of SPI. Colitis was induced by 3% DSS (MP Biomedicals, Illkirch, France, Molecular weight: 36,000-50,000 kDa) added continuously to the drinking water for six consecutive days. Saline and SPI were given orally by gavage for five days. After this period, the mice were euthanized with an intraperitoneal injection of 2, 2, 2-tribromoethanol followed by a cervical dislocation, and the colon samples were collected for immunological and histological assays. The results are representative of two independent experiments. The extract was obtained by macerating SPI dried biomass with saline to obtain the target concentrations.

All animals were housed in specific pathogen-free and standard-controlled environmental conditions at a constant temperature (25℃) on a 12 hours light/dark cycle and provided with food and water ad libitum in the animal housing facility of the Federal University of Triângulo Mineiro, Brazil. The Institutional Animal Care and Use Committee of Federal University of Triângulo Mineiro approved this study under protocol 388, and all procedures were performed in accordance with the international ethics guidelines. All mice were monitored for their general wellbeing before, during, and after the experiments, as described previously.¹⁷

The mice were euthanized on day six, and the colon was removed for further analysis. The colon length was evaluated immediately, and the samples were divided into two smaller sections: 1) immersed into PBS/10% formaldehyde for paraffin embedding; and 2) immersed in a solution containing phosphate-buffered saline (PBS), 2% Nonidet P-40 and protease inhibitors (Complete^®, Roche Pharmaceuticals, Mannheim, Germany) for myeloperoxidase (MPO), nitric oxide (NO) and cytokine/chemokine quantification. The dry weights of all sections were determined before storage.

All mice were evaluated daily for their food and water intake, bodyweight variation, and clinical signs of disease. The clinical, overall clinical, post mortem, and accumulated scores were assessed to obtain a disease score for every mouse, as described previously.¹⁸ Briefly, each sign presented by the animals (wet anus, bleeding stools, stools consistency, piloerection, and hypoactivity) corresponded to one point, and the sum of points for each mouse was defined as the overall clinical score. For weight variation, mice that lost 5 to 10% of their body weight in 24 hours received one point. Weight losses higher than 10% received two points. The percentage of weight variation was calculated as follows: the weight of each mouse was determined at the beginning of the study and was considered to be 100%. The weight variation was calculated for each animal during the experimental period based on the daily weight gain or loss and compared to their weight on day 0. The post mortem score was calculated based on the macroscopical alterations of the colon (consistency of stool and bleeding). One point was attributed to each alteration if these alterations were not diffused. Regarding the diffused alterations, two points were attributed to each alteration observed. The accumulated score was calculated based on the clinical score on the day of euthanasia and the macroscopic score. The sum of the signs or macroscopic disturbances for each mouse was represented graphically as a bar chart.

Colon sections stored in PBS containing the protease inhibitors were kept at 4℃ and disrupted mechanically using TissueRuptor II (Qiagen, Germantown, MD, USA) until visual homogenization was achieved. The samples were then centrifuged at 1,000 xg, and the supernatants were stored at -80℃ until the MPO, NO, and cytokine quantification.

The activity of MPO was determined as previously described.¹⁹ Briefly, a tetramethylbenzidine substrate (BD OptEIA™, San Diego, CA, USA) was added to colon homogenate samples in 96-well plates at 37℃. The reaction was stopped, and readings were performed in a spectrophotometer (Perkin Elmer Cetus, CA, USA) at 450 nm. The results were normalized to the dry weight of each colon section and are expressed as the optical density per gram of tissue (nm/g of tissue).

For the nitric oxide measurements, the nitrite accumulated in the colon homogenates was measured as an indicator of NO production using a Griess reaction.²⁰ The tissue homogenate was mixed 1:1 with Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl-ethylenediamine-hydrogen chloride) and incubated at room temperature for 10 min. The absorbance was measured at 540 nm in a 96-well plate reader (Perkin Elmer Cetus). The amount of nitrite in the samples was calculated based on the absorbance of a serial dilution of the sodium nitrite standard curve. The results were normalized to the dry weight of each colon section and are expressed as picograms per milliliter per gram of tissue (pg/mL/g).

To assess the microscopic damage induced by DSS in mice treated with or without SPI, the colon sections were cut longitudinally, washed with PBS, fixed in 10% buffered formalin for 24 hours, and then processed for paraffin embedding followed by microtome sectioning. Tissue sections (5 μm) were obtained and stained with hematoxylin and eosin. For histopathological analysis, the mucosa, submucosa, muscle layers, and serosa were evaluated. These colon sections were also assessed for the presence of edema, inflammatory infiltrate, and epithelial abnormalities.

Images were captured using a digital video camera (Axion camicC5, Zeiss, Berlin, Germany) coupled to an optical microscope (Nikon Eclipse 50i, Nikon, Melville, NY, USA). Semi-quantitative analyses of infiltrating edema and epithelial abnormalities were also performed and classified according to the level of tissue involvement as follows: mild (>25%), moderate (25-50%), or severe (>50%). A trained pathologist who was blinded to the treatment performed the histopathological analysis.

Cytokines IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17 (BD Biosciences, San Jose, CA, USA) and IL-27 (Invitrogen eBioscience, Vienna, Austria) were quantified in the colon homogenates by ELISA according to the manufacturer’s instructions. The results were normalized to the dry weight of each intestinal section and are expressed as nanograms per milliliter per gram of tissue (ng/mL/g of tissue).

The number of animals per group (n:6) was estimated based on previous publications from the authors’ group using C57BL/6 mice exposed to DSS. The sample size was determined by assessing the feasibility of minimally detectable effect sizes. All results are representative of two independent experiments. The normal distribution and homogeneous variance were tested for all the variables. Parametric tests were used when the distribution was considered normal, and the variance was homogeneous: unpaired Student’s t-test or one-way analysis of variance followed by Tukey’s post hoc test. The following nonparametric tests were used in cases of non-Gaussian distribution of data: Mann-Whitney test or Kruskal-Wallis test accompanied by a Dunn’s post hoc test. The results are expressed as mean±SD. The differences observed were considered significant when p<0.05. Statistical analysis was performed using GraphPad Prism, version 7.0 (GraphPad Software, La Jolla, CA, USA).

One of the most frequent clinical manifestations observed in DSS-induced inflammation is weight loss. In general, this clinical sign results from the lesions induced by DSS in the colon, which leads to the lower absorption of nutrients and weight loss. Therefore, the influence of an SPI extract on weight variations was assessed in mice exposed to DSS, as described in SUBJECTS AND METHODS. Treatment using the SPI extract resulted in reduced weight loss, particularly when administered at the highest concentration (black circle) (Fig. 1A), compared to the mice treated with the lowest concentration of SPI (50 mg/kg depicted as a clear triangle) or the vehicle (inverted black triangle).

The effects of SPI extract on the clinical outcome and macroscopic features of the colon were assessed. Treatment with the SPI extract reduced the disease worsening in a dose-dependent manner compared to their control counterparts (Fig. 1B, E). A significant inhibitory effect on the presentation of clinical signs of disease was observed in the mice treated with SPI at 100 mg/kg compared to the untreated mice (Fig. 1C). The beneficial effects of SPI were further evidenced by the postmortem and accumulated scores, particularly when SPI was given at 250 mg/kg (Fig. 1D, E; respectively). No difference in the colon length of mice exposed to DSS was observed, regardless of the treatment used (Fig. 1F). Overall, SPI had a positive impact on the clinical outcomes and macroscopic features, particularly when administered at the highest concentration.

After evaluating the effect of SPI extract on clinical outcomes, this study examined whether the clinical amelioration is related to the modulation of NO and MPO locally in the colon. Regardless of the treatment used, there were no significant differences in the production of MPO and NO in the tissue homogenates of mice exposed to DSS (Fig. 2A, B; respectively).

Because of the beneficial effects of SPI on the clinical outcomes, this study examined the role of SPI on the intestinal architecture. Untreated mice exposed to DSS showed moderate edema (arrow), moderate mononuclear cell infiltration (arrowhead), and intense destroyed crypts (asterisk) (Fig. 3A). In contrast, the mice treated with 50, 100, and 250 mg/kg of SPI extract (Fig. 3B-D; respectively) showed mild edema (arrow), mild mononuclear cell infiltration (arrowhead), and mild destroyed crypts (asterisk). These results suggest that regardless of the concentration of SPI used, the treatment attenuated inflammation, edema, and epithelial abnormalities of the colon compared to the untreated mice exposed to DSS.

Because the therapy using SPI improved the clinical outcomes and reduced the severity of DSS-induced injuries in the intestine, this study examined whether these ameliorations were related to the local modulation of cytokines. Mice treated with SPI, regardless of the concentration used, showed the reduced production of inflammatory cytokines, such as TNF-α and IFN-γ (Fig. 4A, B; respectively). Despite the suppression of IFN-γ production with SPI at 50 and 250 mg/kg, mice treated with SPI at 100 mg/kg produced higher levels of this cytokine, but the difference was not significant. A similar trend was observed for the quantification of TNF-α (Fig. 4A) and IL-10 (Fig. 4F). Surprisingly, the mice treated with SPI, particularly at 50 and 250 mg/kg, showed a decrease in the production of IL-10 compared to those with intestinal inflammation and treated with vehicle only (Fig. 4F). On the other hand, there were no differences in the production of IL-4 (Fig. 4C), IL-6 (Fig. 4D), IL-17 (Fig. 4E), and IL-27 (Fig. 4G). Overall, these results highlight the therapeutic potential of SPI extract to treat DSS-induced intestinal inflammation.