Extra virgin olive oil and flaxseed oil have no preventive effects on DSS-induced acute ulcerative colitis

Extra virgin olive oil and flaxseed oil have no preventive effects on DSS-induced acute ulcerative colitis

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Extra-virgin olive oil and flaxseed oil have no preventive effects on dss-induced acute ulcerative colitis Roberto de Paula do Nascimento , Amanda Vieira Lima , Lila Missae Oyama , Ana Paula Ribeiro Paiotti , Leonardo Cardili , Carlos Augusto Real Martinez , Jose´ Aires Pereira , Milena Ferreira Silva , Ingrid Candido Garofolo , Vera Lucia Flor Silveira , Luciana Chagas Caperuto PII: DOI: Reference:

S0899-9007(20)30014-9 https://doi.org/10.1016/j.nut.2020.110731 NUT 110731

To appear in:

Nutrition

Received date: Accepted date:

31 March 2019 9 January 2020

Please cite this article as: Roberto de Paula do Nascimento , Amanda Vieira Lima , Lila Missae Oyama , Ana Paula Ribeiro Paiotti , Leonardo Cardili , Carlos Augusto Real Martinez , Jose´ Aires Pereira , Milena Ferreira Silva , Ingrid Candido Garofolo , Vera Lucia Flor Silveira , Luciana Chagas Caperuto , Extra-virgin olive oil and flaxseed oil have no preventive effects on dss-induced acute ulcerative colitis, Nutrition (2020), doi: https://doi.org/10.1016/j.nut.2020.110731

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1

HIGHLIGHTS 

EVOO and FO did not prevent the inflammatory outcome of experimental UC.



EVOO+FO caused an increase in the concentration of TNF-α in distal colon.



Photographic data of this study can be used for a precise evaluation of the DAI.

2 EXTRA-VIRGIN OLIVE OIL AND FLAXSEED OIL HAVE NO PREVENTIVE EFFECTS ON DSS-INDUCED ACUTE ULCERATIVE COLITIS

AUTHORS Roberto de Paula do NASCIMENTO (RPN) a - [email protected] Amanda Vieira LIMA (AVL) a - [email protected] Lila Missae OYAMA (LMO) b - [email protected] Ana Paula Ribeiro PAIOTTI (APRP) c - [email protected] Leonardo CARDILI (LC) d - [email protected] Carlos Augusto Real MARTINEZ (CARM) e - [email protected] José Aires PEREIRA (JAP) e - [email protected] Milena Ferreira SILVA (MFS) a - [email protected] Ingrid Candido GAROFOLO (ICG) a - [email protected] Vera Lucia Flor SILVEIRA (VLFS) a - [email protected] Luciana Chagas CAPERUTO (LCC) a - [email protected]

Institutional addresses a Laboratory of Metabolic Physiology, Universidade Federal de São Paulo - Diadema Campus, Professor Artur Riedel Street, 275, Diadema, São Paulo, Brazil. b Department of Physiology, Universidade Federal de São Paulo – São Paulo Campus, Botucatu Street, 862, São Paulo, São Paulo, Brazil.

3 c Laboratory of Molecular and Experimental Pathology/Laboratory of Hepatology Molecular Applied - Discipline of Gastroenterology, Universidade Federal de São Paulo – São Paulo Campus, Pedro de Toledo Street, 781, São Paulo, São Paulo, Brazil. d Department of Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM/UNIFESP), São Paulo, Brazil e Postgraduate Program in Health Sciences, Universidade São Francisco – Bragança Paulista Campus, São Francisco de Assis Avenue, 218, Bragança, São Paulo, Brazil

Corresponding author Luciana Chagas Caperuto - [email protected] Laboratory of Metabolic Physiology, Universidade Federal de São Paulo - Diadema Campus, Professor Artur Riedel Street, 275, Diadema, São Paulo, Brazil. +5511995810197

Running title Extra virgin olive oil and Flaxseed oil have no effects on DSS-induced colitis

Authors' contributions RPN – creation and development of the project, application in practice (experimental model, euthanasia, protein extraction, Western Blotting, Multiplex/ELISA), analysis of all steps, writing of the manuscript AVL – application in practice (experimental model and euthanasia) and analysis (histology) LMO - application in practice (Multiplex/ELISA) APRP – analysis (scores for histology)

4 LC - analysis (scores for histology) CRAM - application in practice (production of histological slides) JAP - application in practice (production of histological slides) MFS – application in practice (euthanasia), analysis (Disease Activity Index) ICG - application in practice (euthanasia), analysis (Disease Activity Index) VLFS - development of the project, application in practice (euthanasia), guidance of students involved, writing of the manuscript LCC - development of the project, application in practice (euthanasia), guidance of students involved, writing of the manuscript All authors read and approved the final manuscript

DECLARATIONS Original article This study was not previously published.

Declarations of interests None

Ethics approval This study was approved by the Ethics Committee on the Use of Animals (CEUA) of the Research Ethics Committee of the Universidade Federal de São Paulo (CEP/UNIFESP), number 7409170816.

5

Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, #2009/50041-0; #2017/02936-4) and in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001).

ABSTRACT The aim of this study was to evaluate the preventive effects of extra virgin olive oil (EVOO) and/or flaxseed oil (FO) on dextran sodium sulfate (DSS)-induced acute ulcerative colitis (UC) in female mice. Animals that received DSS presented: 1. elevated Disease Activity Index values; 2. increased colon weight/length ratio; 3. augmented leukocyte infiltration into the lamina propria and submucosa; and 4. increased production of TNF-α, IL-1β and IL-6 and greater iNOS expression in the distal colon. Individually or in combination, the oils were not able to reverse or mitigate any of the DSS-induced symptoms and/or damage. Additionally, the group of animals treated with DSS and supplemented with FO displayed increased spleen weight/body weight ratio, and the group that received a combination of EVOO and FO presented increased TNF-α levels, when compared to the respective control group. In conclusion, the consumption of large amounts of EVOO and FO as a treatment for and/or prevention against UC could potentially elicit unwanted adverse effects. Key words: inflammatory bowel disease; oleic acid; α-linolenic acid; phenolic compounds; inflammation; mucosa

ABBREVIATIONS ALA – Alfa-linolenic acid DAI – Disease activity index DSS – Dextran sodium sulfate

6 EVOO – Extra-virgin olive oil FO – Flaxseed oil HE – Hematoxilin-eosin IL – Interleucin iNOS – Inducible nitric oxide synthase MUFA – Monounsaturated fatty acid n-3 – Omega-3 n-6 – Omega-6 n-9 – Omega-9 NO – Nitric oxide OA – Oleic acid PUFA – Polyunsaturated fatty acid SO – Sunflower oil Th – T-helper TLR – Toll-like receptor TNBS – Trinitrobenzenesulfonic acid TNF – Tumor necrosis factor UC – Ulcerative colitis WB – Western Blotting

7

1. INTRODUCTION Ulcerative colitis (UC) is an inflammatory bowel disease of unknown etiology, and has a prevalence ranging from 5 to 550 individuals per 100,000 inhabitants [1]. Its greatest occurrence is in developed countries, such as those of Western Europe, the United States of America and Australia [1]. However, by 2025, it is predicted that the numbers of UC cases in Latin American and Asian countries will increase substantially [2] The disease primarily affects the colon and rectum and is characterized by an immune system mediated response, resulting in deformation of the intestinal mucosa, leukocyte infiltration, increased pro-inflammatory cytokine production (TNF-α, IL-1β and IL-6), and upregulated expression of genes and proteins, like inducible nitric oxide synthase (iNOS), which releases abundant amounts of nitric oxide (NO) [3]. UC is a non-transmissible chronic disease that presents acute episodes, which refer to the active phase of the disease [3]. Patients in this phase usually present severe abdominal pain, several episodes of diarrhea throughout the day and rectal bleeding. These symptoms, among others, can greatly impact the family, professional and social lives of the individual, and have a negative influence on their quality of life [4]. Currently, aminosalicylates, such as sulfasalazine and mesalazine, are employed for the induction or maintenance of the UC remission phase [5]. Other treatments for the disease include the use of corticosteroids, immunosuppressants and, more recently, therapies with monoclonal antibodies [6]. However, depending on the treatment, such therapies can cause a variety of adverse side-effects, including: diarrhea, headache, nausea, dyspepsia, fever, weakness, acute infections, hypersensitivity reactions, pancreatitis and/or liver toxicity [5-7]. Considering this, studies with humans and animals have correlated the consumption of some foods, rich in promising fatty acids and phenolic compounds, with improved outcomes in patients with UC, possibly with little or no adverse side-effects.

8 Inducing UC in mice, by administering Dextran Sodium Sulfate (DSS), has been useful in investigations with functional foods. In fact, this reagent is capable of recapitulating some of the clinical and histological manifestations of UC [8]. Among some of the foods previously evaluated using the murine DSS-induced UC model include: extra-virgin olive oil (EVOO) and flaxseed. EVOO is an oil rich in oleic acid (OA), omega-9 monounsaturated fatty acid (n-9 MUFA), and phenolic compounds, such as oleuropein, hydroxytyrosol and tyrosol [9], while flaxseed is a product rich in the omega-3 polyunsaturated fatty acid (n-3 PUFA), linolenic acid (ALA) and lignans [10,11] Sanchez-Fidalgo et al. [12, 13] reported satisfactory results with the utilization of EVOO or associated phenolic compounds in the prevention against acute DSS-induced UC in mice. These favorable results, however, represent the only two publications on the subject. On the other hand, studies with flaxseed show divergences among authors. Palla et al. [14] and Xu et al. [15] observed anti-inflammatory effects of the phenolic compounds of flaxseed, while Zarepoor et al. [10] found an increase in acute phase cytokines in the colon and plasma of animals with UC receiving flaxseed rich in ALA and lignans. Studies with flaxseed oil (FO) are lacking despite its potential benefits [16], which can be attributed to a higher ALA content [11]. Finally, the association of n-9 MUFA and n-3 PUFA looks promising for UC, since Reddy & Naidu [17] demonstrated that the combination of EVOO and fish oil showed greater efficacy, in comparison with only one or other, in the treatment of DSS-induced UC. Considering the potential benefits of these oils for UC, the present study was designed to investigate the protective effects previously observed with EVOO administration, and to gain insight into the effects of FO in a murine model of UC.

2. MATERIAL AND METHODS 2.1 Chemical analysis The oils used in this study were subjected to fatty acid and total phenolic content analysis. Gas chromatography was used for measuring the fatty acid levels. The method employed is based on

9 the “Ce 1a-13" methodology of the American Oil Chemists Society [18], which involves first submitting the samples to the Hartman and Lago’s [19] preparation of fatty acid methyl esters. Results were expressed as a percentage of the total of fatty acid content. The extraction and analysis of the total content of phenolic compounds was based on Kim et al. [20], using the Folin-Ciocalteu method. Results are presented as mg gallic acid/kg of oil, and values of less than 30 mg/kg were considered to be not detected (ND). The procedures were carried out by the Food Technology Institute (ITAL), in Campinas, Brazil. 2.2 Ethics, Animals and diets This study was approved by the Ethics Committee on the Use of Animals of the Research Ethics Committee at the Universidade Federal de São Paulo (number 7409170816). The animal protocol followed the National Institute of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), as well as the Brazilian guideline for care and use of animals in teaching or scientific research activities (DBCA). A total of 80 eight-to-nine weeks old female C57BL/6 mice were purchased from the Center for the Development of Experimental Models for Biology and Medicine at the Universidade Federal de São Paulo (UNIFESP, São Paulo, Brazil). Female mice were chosen because they are less likely to fight [21] and present fewer severe effects and reduced mortality in this DSS-induced UC model [22]. Mice were maintained two per cage, in a temperature (25 ± 2°C) and humidity (60-70%) controlled area with a 12 hour light/dark cycle and provided water ad libitum. During the acclimation period, 1 week before initiating the experimental protocol, animals received Nuvilab® feed. After acclimation, the mice were divided into four groups: control, fed with sunflower oil (SO), EVOO, FO and EVOO+FO. The diets were produced by supplementing the standard AIN-93M diet [23] with 10% of each oil, such that each specific diet contained 10% SO, 10% EVOO, 10% FO or 5% EVOO + 5% FO (Supplementary material 1) and stored at 20°C, as previously described [12, 17]. Approximately 10g of fresh diet was provided daily per cage.

10 2.3 Induction of acute UC After consuming each specific diet for 30 days, half of the animals from each group were subjected to DSS-induced UC. Acute UC was induced by providing the animals with a 3% solution of DSS for 5 days [12, 13]. Bottles with DSS were changed for new ones on the third day, in order to maintain drug efficacy [8]. This treatment period lasted 5 days, after which time the DSS was removed from the drinking water. The animals then received DSS-free water for an additional 4 days, and then euthanized. Water consumption was monitored from the start of DSS administration until the end of the experimental protocol. The disease activity index-DAI (percentage weight loss since day 30, bleeding and stool consistency) was recorded at days 30, 33, 35, 36, 38 and 39 after initiating the DSS treatment, and was based on Gommeaux et al. [24]. Bleeding and stool consistency analyses were evaluated by two researchers blind to the identification of the groups, using photographs taken of the stool and anus of the mice. Photographs were taken with a Nikon® SLR D3100 digital camera, with 14.2-megapixel resolution and a 18-55 mm lens. 2.4 Euthanasia and tissue measures All of the animals were anesthetized at day 40 of the experimental protocol with 60 mg/kg of Sodium Tiopenthal (Cristália, São Paulo, Brazil), administered intraperitoneal. Blood was collected by puncturing the orbital plexus, centrifuged at 2,600× g for 20 minutes and the serum was recovered. The colon was collected, cleaned with 0.9% saline solution, weighed and measured. The inflammatory index was based on the relationship between the colon weight and length [12, 13]. Approximately 1 cm of the distal colon was collected for histology experiments. Briefly, the distal colon was opened longitudinally, clamped with pins into pieces of cork and stored in 5% formaldehyde. Colon remains and serum were frozen in liquid nitrogen and stored at -80°C until analysis. The spleen was also removed from the animals and weighed.

11 2.5 Histopathology The distal colon was selected for the histopathological analysis, since is the portion of the intestine that is most affected by DSS-induced UC, and is considered to be a good representation of the disease [25]. Briefly, colon segments were removed from the 5% formaldehyde solution, washed with distilled water, dehydrated in successive increasing concentrations of alcohol, diaphanized in xylol, impregnated in liquid paraffin baths at 60ºC and included in paraffin blocks. Each block was submitted to microtomy (5μm) for the preparation of the slides for the histological study and stained with hematoxylin and eosin (HE). HE-stained sections were analyzed by assigning each sample two subjective scores (Supplementary material 2), generated by two specialized independent examiners, blind to the identification of the groups. The first score, modified from Dieleman et al. [26], evaluates and takes into consideration general aspects of the DSS model (leukocyte infiltration, inflammation extent and crypt damage), while the second one, modified from Stucchi et al. [27], considers a more detailed panorama (ulceration, mitotic activity, depletion of goblet cells, mononuclear infiltrate, granulocyte infiltrate, vascularization and edema). Each sample was analyzed at 100×, 200× and 400× magnification. 2.6 Protein extraction Protein extraction was achieved by placing portions of the colon in liquid nitrogen and simultaneously grinding it in a grail, so as to obtain homogenously powdered samples. Between 25-60 mg of this powder was dissolved in 100-400 μl of lysis buffer containing: 10 mM TrisHCl pH 7.5, 100 mM EDTA, 2 mM PMSF, 10 mM Na3VO4, 100 mM NaF, 10 mM NaPPi, protease inhibitor cocktail and 1% TX-100, using a pistil. The samples were then centrifuged at 15,000 × g at 4°C for 40 minutes, and the supernatant was collected for protein quantification by the Bradford assay.

12 2.7 Multiplex/ELISA The concentrations of TNF-α, IL-1β and IL-10 in the colon samples were measured using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay kit (Merck Millipore, Burlington, USA) and the concentration of IL-6 determined using a commercially available ELISA (R&D Systems, Minneapolis, USA). The results are presented as pg/mg. 2.8 Western Blotting A total of 25 μg of total protein per sample was denatured at 100ºC for 15 minutes and loaded onto 6.5% polyacrylamide gels (SDS-PAGE). The proteins were separated using a voltage of 15 to 20V. The proteins on the gel were then transferred to a nitrocellulose membrane, using a wet system, at 120V for 120 minutes. Membranes were incubated in blocking solution composed of 5% bovine serum albumin (BSA) in Tris buffered saline plus Tween 20 (TBST). The membranes were incubated overnight with anti-iNOS (Cell Signalling, Beverly, USA; rabbit, 1:1000) or anti-β-actin (Sigma Aldrich, Bellefonte, USA; mouse, 1:5000) in 5% BSA in TBST. Following incubation with the primary antibodies, the membranes were incubated with the corresponding secondary antibody (Cell signaling, Beverly, USA; rabbit, mouse, 1:3000). Membranes were then incubated with a chemiluminescent solution, and bands were detected using Uvitec Cambridge equipment and software (Warwickshire, UK). The density of the bands, corresponding to iNOS and β-actin, was analyzed using the ImageJ® software. iNOS content was normalized based on the band intensity of β-actin (iNOS/β-actin). 2.9 Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). Outliers were identified using the Grubbs test (5%) and discarded. A T-test with Welch’s correction (parametric data) or the Mann-Whitney test (non-parametric data) were utilized when comparing non-DSS and DSS groups (i.e. SO vs. SODSS; EVOO vs. EVODSS; FO vs. FODSS; EVOO+FO vs. EVOO+FODSS). Alternatively, one-way ANOVA followed by Tukey (parametric data) or

13 Kruskal-Wallis followed by Dunn (non-parametric data) were utilized when making comparisons among DSS groups (SODSS vs. EVOODSS vs. FODSS vs. EVOO+FODSS). Data were considered statistically different when p < 0.05. All statistical analyses were performed using GraphPad Prism 7.

3. RESULTS 3.1 Oils composition As shown in Table 1, the chemical analysis of EVOO and FO presented elevated levels of OA, n-9 MUFA (71%), and ALA, n-3 PUFA (57%), respectively. Moreover, these oils were also rich in phenolic compounds, with EVOO containing 244 and FO 100 mg/kg. In contrast, when compared to EVOO and FO, SO had greater amounts of omega-6 (n-6) PUFA, LA (42%), n-9 MUFA (44%) and undetectable levels of phenolic compounds (<30 mg/kg). 3.2 Characterization of DSS-induced acute UC model The animals that were provided water supplemented with 3% DSS displayed loose stools and/or diarrhea and bleeding (p<0.01) (data not shown), three days into the protocol. After five days, the body weight of the DSS mice was significantly reduced, when compared to controls (Figure 1A, p<0.05). At Day 36, the UC-induced animals presented high DAI values (p<0.001), which remained significantly elevated until the end of the experiment (Figure 1B, p<0.001). The DSSinduced UC group also displayed reduced colon length (Figures 1C, p<0.001) and an increased colon weight/length ratio (p<0.001) (Figures 1D, p<0.001). Additionally, the SODSS group presented a significantly increased (p<0.001) spleen weight/body weight (mg/g) ratio in comparison with the SO group (data not shown), and the histological analysis revealed high values for scores 1 and 2 (data not shown). Histological analyses of UC-induced animals was mainly characterized by the loss of epithelial architecture and crypt damage, extensive

14 Table 1 - Fatty acid and phenolic compound composition of SO, EVOO and FO. FATTY ACIDS COMPOSITION (SIMPLIFIED) (%) Fatty acids

SO

EVOO

FO

Saturated

9.22

15.21

8.26

Monounsaturated

44.32

72.13

16.47

Polyunsaturated

41.96

7.63

70.77

Omega-3

0.14

0.61

56.72

Omega-6

41.82

7.02

14.05

Total trans isomers

0.11

0

0

FATTY ACIDS COMPOSITION (DETAILED) (%) Fatty acids

SO

EVOO

FO

Palmitic C16:0

4.88

11.26

4.74

Palmitoleic C16:1 omega-7

0.08

0.79

0

Cis-10-heptadecanoic C17:1

0

0.10

0

Stearic C18:0

3.07

3.38

2.99

Oleic C18:3 omega-9

44.03

70.99

16.39

Trans linoleic C18:2 omega-6 t

0.11

0

0

Linoleic omega-6

41.82

7.02

14.05

Arachid C20:0

0.26

0.44

0.29

α-linolenic C18:3 omega-3 α

0.14

0.61

56.72

Cys-11-eicosenoic C20:1 omega-11

0.21

0.25

0.09

Behenic C22:0

0.75

0.12

0.16

Lignoceric C24: 0

0.27

0

0.08

TOTAL PHENOLIC COMPOUNDS (mg/kg) Methodology

SO

EVOO

FO

Folin-Ciocalteu (1965)

ND

243.8 ± 11.6

99.8 ± 1.3

Fatty acids: analysis by capillary gas chromatography. Factor used for fat to fatty acid conversion: 0.956. Total phenolic compounds: analysis by the Folin-Ciocalteu method, performed in triplicate. ND: not detected (values below 30 mg/kg of oil). SO: sunflower oil, EVOO: extra-virgin olive oil, FO: flaxseed oil.

15 ulceration, granulocyte and mononuclear cell infiltration in mucosa and submucosa and edema, most of which are shown in Figure 1E. Taken together, these characteristics also indicate that our experimental protocol, using DSS, produced a viable animal model for acute UC. Figures 2 and 3 provide the photographic representations of the scores used for the evaluation of bleeding and stool consistency. Figure 1 - Characterization of DSS-induced acute UC model.

Comparison among groups SO (n=7-8) and SODSS (n = 10-12) as a form of characterization of the DSS-induced acute UC induced model. T-test with Welch’s correction; *p<0.05; **p<0.01; ***p<0,.01 vs. SO. (A) Body weight loss (%) after DSS inclusion in water. (B) Disease activity index (DAI): score from 0 to 3. (C) Colon length (cm). (D) Colon weight/length (mg/cm). (E) Photographic representations of histological sections of HE-stained distal colon samples, 100× magnification. Color figure.

16 3.3 Effects of EVOO and FO in DSS-induced acute UC model In addition to the studies involving SO, the effects of EVOO and FO supplementation were also evaluated in the acute UC mouse model. The diet-related effects were assessed through the analysis of DAI values, tissue measurements, histopathology, as well as cytokine concentration and iNOS expression levels. Figure 2 - Photographic representation of the bleeding score.

(A) Score 0 – None. (B) Score 1 - Small spots of blood in stool; dry anal region. (C) Score 2 Large spots of blood in stool; blood appears through anal orifice. (D) Score 3 - Deep red stool; blood spreads largely around the anus (Based on Gommeaux et al. [24]). Color figure.

17 Figure 3 - Photographic representation of the score of stool consistency.

(A) Score 0 - Normal stools. (B) Score 1 - Soft pellets not adhering to the anus. (C) Score 2 Very soft pellets adhering to the anus. (D) Score 3 - Liquid stool on long streams; wet anus (Based on Gommeaux et al. [24]). Color figure.

3.3.1 DAI and tissue measures At Day 39, the FODSS group presented lower body weight in comparison with SODSS mice (Figure 4A, p<0.05). However, with regards to DAI, there were no observable differences among the DSS groups during the final days of the experiment. In fact, the DAI values for the EVOO+FODSS mice were only significantly different from the value of the SODSS group on Day 33 (Figure 4B, p<0.01). Additionally, there were no differences observed for the colon length nor the colon weight/length ratio, among all groups (Figures 4C and 4D, respectively).

18 Interestingly, the spleen weight/body weight ratio of the FODSS group increased significantly when compared to the other groups (Figure 4E, p<0.001). Figure 4 - Body weight, Disease Activity Index and tissue measurements

Comparison among DSS-induced UC groups (n = 10-12. (A) Body weight (g) after DSS inclusion in water; +p<0.05 vs. EVOODSS, #p<0.05 vs. SODSS. B. Disease Activity Index (DAI); ##p<0.01 vs. SODSS. C. Colon length (cm). D. Colon weight/length (mg/cm). E. Spleen weight/body weight (mg/g). Data were analyzed using one-way ANOVA followed by Tukey post-test, ###p<0.001 vs. SODSS; +++p<0.001 vs. EVOODSS; ===p<0.001 vs. EVOO+FODSS.

19 3.3.2 Histopathology With regards to histology, based on the totals of score 1 and 2, no significant differences were detected among the DSS-induced UC groups (Table 2). However, a separate analysis of the “leukocyte infiltration” category of score 1, revealed that the EVOO+FODSS group presented significantly increased levels of observable infiltration, when compared to the SODSS group (Table 2, p<0.05). Representative images of HE-stained distal colon from all of the DSSinduced UC groups are shown in Figure 5. Figure 5 - Photographic representations of HE-stained distal colon sections.

Comparison among DSS groups. 100× magnification. Color figure.

20 Table 2 - Results of scores 1 and 2 for histology.

(Modified from Dieleman et al. [26])

SCORE 1 SODSS

EVOODSS

FODSS

EVOO+FODSS

TOTAL

1.01±0.25

1.94±0.27

1.59±0.38

2.04±0.27

Leukocyte Infiltration

0.90±0.22

1.83±0.26

1.60±0.40

2.12±0.28 #

(Modified from Stucchi et

SCORE 2

al. [27])

SODSS

EVOODSS

FODSS

EVOO+FODSS

TOTAL

10.70±2.16

16.55±2.42

13.22±2.88

13.13±2.70

The analysis was performed by two independent examiners blind to the identification of the groups. Comparison among DSS groups (n = 8-12): one-way ANOVA followed by Tukey posttest. #p<0.05 vs. SODSS.

3.3.3 Cytokine concentrations and iNOS expression The levels of IL-1β and IL-6 were elevated in most of the DSS groups when compared to the non-DSS groups, while IL-10 levels were not perturbed by DSS treatment (data not shown). With regards to TNF-α, the EVOO+FODSS group had significantly higher levels of this cytokine, when compared to the SODSS group (Figure 6A, p<0.05). IL-6 concentrations were significantly elevated in FODSS group in comparison to EVOO+FODSS animals (Figure 6B). Concentrations of IL-1β were not different among all groups (Figure 6C). Finally, when comparing iNOS expression, animals treated with DSS versus untreated controls, there was a significant increase in the expression of this protein (data not shown); however, the same analysis failed to detect any significant changes among DSS groups (Figure 6D).

21 Figure 6 - Cytokine concentrations and iNOS expression.

C

O D +F O

EV O

O D

SS SO D

+F O O O EV

60

SS

0

SS

SS

50

D

SS FO

D

SS D O EV

O

SO

D

SS

0

100

EV O

1

=

FO D

IL-6 (pg/mg)

TNF-α (pg/mg)

2

B

150

#

SS

A

3

D iNOS/β-ACTIN

30 15 0

0.4

0.2

SS O D

SS EV O O +F

FO D

SS EV O O D

SS

D SS

EV

O

O

+F O

D SS FO

D SS O O EV

SO

D SS

0

SO D

IL-1β (pg/mg)

0.6

45

Comparison among DSS groups (n = 7-12). (A) TNF-α; #p<0.05 vs. SODSS. (B) IL-6; =p<0.05 vs. EVOO+FODSS. (C) IL-1β. (D) iNOS/β-actin. Data were analyzed using one-way ANOVA followed by Tukey post-test.

4. DISCUSSION Previous studies investigating the chemical composition of EVOO and FO revealed that these oils are rich in n-9 MUFA and OA [9], and n-3 PUFA and ALA [9], respectively, and that each contains up to 800 mg/kg of phenolic compounds [28, 29]. Moreover, these composition characteristics are also consistent with other studies utilizing these oils in UC models [12, 13, 30]. It has been suggested that these oils, which are rich in these chemical compounds, should be able to modulate the inflammatory response, thus providing a potential pro-health benefit.

22 However, the results of the present study failed to detect any potentially beneficial effects associated with EVOO or FO, individually or in combination, when evaluating the inflammatory response of female mice with DSS-induced acute UC. Animals started to present considerable loss of body weight after drinking the 3% DSS water, for 5-6 days. This loss reached a maximum of about 10% in relation to the weight at Day 30. Although our study uses a methodology similar to Sanchez-Fidalgo et al. [12, 13], there were some discrepancies in the results. For example, in their study, animals from the DSS groups reached a body weight loss of about 25% on the last day (Day 39 or 40). However, it should be pointed out that the acute DSS-induced UC model of Sánchez-Fidalgo et al. [12, 13] supplemented the diets of the animals with iron, which has the ability to aggravate experimental UC by increasing oxidative stress and neutrophil infiltration in the colon [31]. Thus, it is plausible that the increased weight loss and alterations of the DAI are also due to the effects of iron, and not only the DSS-induced UC model. In the present study, diets were not supplemented with iron, since in addition to the increased weight loss there is also an elevated risk of death. Indeed, the fact that no animals died during this study, indicates that employing female mice without iron supplementation produces an accurate model of acute UC. This is due, at least partially, to estradiol conferring partial protection in chemically induced UC models, by reducing myeloperoxidase activity [22]. Neither of the oils, individually or in combination, were able to prevent body weight loss or the UC associated increased DAI. Interestingly, the mean body weight of the FODSS group was significantly lower than the body weights of the other DSS groups at the end of the experiment. This result is in accordance with results obtained by Zarepoor et al. [10], who investigated acute UC prevention in C57BL/6 mice administered flaxseed, rich in lignans and ALA. On the other hand, it was later shown that isolated lignans, given through gavage, provided protective effects against chemically-induced UC in mice [15]. These discrepancies in the results demonstrate that the type and composition of the flaxseed, as well as the method utilized for administering the supplement all contribute to and influence these different responses. With regards to DAI, it is

23 important to emphasize that this is the first study to provide photographic reference material for the bleeding and stool consistency criteria, which will facilitate reproducibility of the analysis of DAI by other researchers. Especially, in studies evaluating DDS-induced UC in female C57BL/6 mice fed an AIN-93M diet. Previous studies have shown that individuals experiencing more severe UC phases present decreased distensibility and shortening of the colon [32, 33]. As shown in Figure 1, the DSSinduced UC mice had significantly reduced colon lengths, as well as an increased colon weight/length ratio, which is a marker for tissue edema [34] and inflammation [13]. Moreover, the colon length data resembled results reported by Jeengar et al. [35] and Wang et al. [36], which showed that the colon length can be reduced to as little as 6 cm, in C57BL/6 mice with acute DSS-induced UC. Furthermore, no differences in colon length were observed among the DSS groups, demonstrating that the EVOO and/or FO did not prevent inflammatory cell infiltration nor colonic mucosa deformation/destruction. The two scores employed for the histological evaluations are based on a large variety of properties and characteristics, with distinct criteria. Due to the subjectivity associated with histological analyses, two distinct complementary evaluations were employed when assessing the distal colon samples, thus strengthening the interpretation of the results. As expected, both scores provided similar results; however, a closer examination of the leukocyte infiltration category in score 1 showed that the EVOO+FODSS group presented increased leukocyte infiltration in comparison to the SODSS group. In general, the histological analysis results showed that inflammation was limited to the mucosa and submucosa, without presenting any evidence of transmural extension (muscular or serous). This is a common finding in studies using DSS for UC induction, and presents itself as one of the differences when compared to TNBS induction, which classically results in deeper lesions [37]. Acute DSS-induced UC has also been shown to promote other histological changes, including focal lesions in crypts, loss of goblet cells, infiltration of inflammatory cells at

24 lesioned sites and/or submucosal edema [38, 39]. The oils evaluated in this study did not prevent these histopathological alterations, thus further corroborating the DAI results. In the colon, cytokines are produced mainly by lymphocytes and monocytes/macrophages, and the histological analysis detected considerable amounts of these cell types in the epithelial mucosa of DSS treated animals. Since the inflammatory response is primarily mediated by the macrophages [38, 40], the observed increase in colon TNF-α, IL-1β and IL-6 levels in the DSSinduced UC groups was expected. The elevated TNF-α levels and augmented leukocyte infiltration detected in the EVOO+FODSS suggests that, in combination, these oils could actually exacerbate the cytokine-mediated inflammatory response. Another signaling pathway that has been shown to be perturbed in UC involves iNOS, which is also known to be classically increased during acute UC. iNOS produces nitric oxide (NO), via activation of the toll like receptor 4 (TLR4)/factor nuclear kappa B (Nf-kB) signaling pathway. In fact, activation of this signaling pathway, promotes the upregulation and activation of inflammatory factors, such as TNF-α and iNOS, resulting in the release of the free radical NO, and the vicious induction of the TLR4-mediated pathway [41]. Interestingly, DSS-induced UC animals administered an iNOS inhibitor or a nitric oxide sequestrant, showed a reduction or cessation of the typical inflammatory process associated with acute UC, indicating that these molecules are relevant to the pathogenesis of the disease [41]. In the present study, neither of the oils, individually or in combination, were able to significantly attenuate cytokine production or iNOS expression. Taken together, this would imply that the TLR4/Nf-kB pathway remained unaffected. It is plausible that the phenolic compounds, found in the oils, have antioxidant properties and could scavenge reactive oxygen species and free radicals, thus inhibiting the production and release of cytokines [42], however, there was no evidence that these oils functioned in this capacity. In the DSS treated animals there was an observable increase in spleen weight, a phenotypic characteristic described previously for this model of UC [36]. The enlargement of the spleen appears to be related to the inflammatory response occurring in the colon [8]. It is also possible

25 that this organ has increased number of Th17-positive CD4 cells, monocytes, myeloid suppressor cells, and a reduced number of regulatory T-positive CD4 cells in the DSS-induced UC model [43, 44]. Routinely, an increase in spleen weight/body weight of 60% was observed in mice administered DSS versus untreated controls; however, the FODSS group presented a further significant increase in this ratio when compared to the other DSS groups. It is unclear how FO provokes this further increase in acute UC, since our results provided evidence that FO supplementation had no major adverse effects on components of the inflammation response pathway. Future studies with this organ and the colon, are required to identify the molecular mechanisms behind these observations. The chemical compositions of EVOO and FO suggest that these oils may be beneficial in the treatment of acute UC. However, the results indicate otherwise, since EVOO and FO in combination increased TNF-α levels and FO was associated with increased spleen weight. Below, plausible explanations for why the specific diets failed to treat and/or prevent DSSinduced acute UC in mice. 1. 10% EVOO a. the oil utilized in our study may not be rich in important phenolic compounds typically found in EVOO. For example, Sanchez-Fidalgo et al. [13], showed that 3,4-dihydroxyphenylglycol, a phenolic present in EVOO, was ineffective in the prevention against DSS-induced acute UC, whereas hydroxytyrosol acetate had a wide-range of positive effects. Therefore, it is important to not only estimate the total amount of phenolic compounds, but also to identify the components of the oil, since EVOOs with higher concentrations of hydroxytyrosol, tyrosol and/or oleuropein have been shown to produce positive effects in DSS-induced UC models [9, 45, 46]; b. low n-6 PUFA content in the diet. This compound is an important component of the cell membrane, and just like n-3 PUFA and n-9 MUFA, plays a fundamental role

26 in inflammatory responses. Their presence in human and animal diets are necessary in adequate proportions [47]. 2. 10% FO a. n-3 PUFA present in FO may have negatively affected the results of our study. Zarepoor et al. [10] observed increased apoptosis of epithelial cells in DSS-induced UC mice fed flaxseed. n-3 PUFA appeared to prevent healing and exacerbate mucosal damage, which may account for the observed pro-apoptotic effects. These results, in association with other studies that found negative or inconclusive results using fish oil, which are known to be rich in n-3 PUFAs, in animals with experimental UC [48, 49] and in humans with UC [50, 51], lead us to believe that this fatty acid may not be suitable in every situation; b. the low conversion of n-3 PUFA, ALA, in EPA (from 5 to 10%) and DHA (less than 1%), which may impair FO-associated anti-inflammatory effects [47]; c. a low bioavailability of phenolic compounds in FO [52]; d. low n-6 PUFA content in the diet or insufficient proportions of fatty acids. According to recent studies with UC rodent models, diets with a 2:1 ratio of n-6:n-3 may present a more promising result than diets only rich in n-3 PUFA [53-55]. 3. 5% EVOO + 5% FO: this is the first study to investigate the effects of administering both oils, in an acute chemically induced UC model. The results indicated that this formulation may not be beneficial or even promising, since it increased the TNF-α concentrations and leukocyte infiltration in the colon. It is possible that different fatty acids and phenolic compounds, perhaps in less than ideal proportions, elicited the observed adverse effects in this experimental animal model. In order to verify this possibility more studies will need to be performed. Although our results indicate that EVOO and/or FO do not provide any beneficial effects in the prevention against or treatment of acute DSS-induced UC, a previous study, conducted by Jia et al. [48], showed that administering isolated fish oil with and without curcumin resulted in higher

27 mortality and mucosal damage in animals with acute DSS-induced UC, but during chronic UC induction, the therapies had positive effects, extenuating inflammation. Thus, additional studies assessing the effects of these oils at later stages of UC development or under chronic disease conditions need to be performed.

CONCLUSIONS Our data suggests that there are no beneficial effects associated with the consumption of EVOO and/or FO in an acute DSS-induced UC mouse model. The oils were not able to decrease or reverse some of the classical signs of inflammation, including: clinical and histological impairment, increased concentrations of proinflammatory cytokines and marked iNOS expression. In combination, EVOO and FO evoked an increased histological score for leukocyte infiltration and the concentration of TNF-α in the colon was significantly elevated, which could lead to more severe problems, if left untreated. Therefore, physicians and nutritionists should be cautious when recommending the consumption of large daily doses of EVOO and FO for the prevention and/or treatment of UC, at least until the effects of these oils are evaluated for longer periods of time or in chronic UC models.

ACKNOWLEDGMENTS This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, #2009/50041-0; #2017/02936-4) and in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001).

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35 Graphical Abstract