Polysaccharides derived from Morinda citrifolia Linn reduce inflammatory markers during experimental colitis

Journal Pre-proof Polysaccharides derived from Morinda citrifolia Linn reduce inflammatory markers during experimental colitis Jalles Arruda Batista, Diva de Aguiar Magalhães, Stefany Guimarães Sousa, Jayro dos Santos Ferreira, Cynthia Maria Carvalho Pereira, José Victor do Nascimento Lima, Ieda Figueira de Albuquerque, Nayonara Lanara Sousa Dutra Bezerra, Carlos Eduardo da Silva Monteiro, Alvaro Xavier Franco, David Di Lenardo, Lorena Almeida Oliveira, Judith Pessoa de Andrade Feitosa, Regina Célia Monteiro de Paula, Jefferson Soares de Oliveira, Daniel Fernando Pereira Vasconcelos, Pedro Marcos Gomes Soares, André Luiz dos Reis Barbosa PII:

S0378-8741(19)31800-8

DOI:

https://doi.org/10.1016/j.jep.2019.112303

Reference:

JEP 112303

To appear in:

Journal of Ethnopharmacology

Received Date: 5 May 2019 Revised Date:

10 October 2019

Accepted Date: 11 October 2019

Please cite this article as: Batista, J.A., Magalhães, D.d.A., Sousa, Stefany.Guimarã., Ferreira, J.d.S., Pereira, C.M.C., Victor do Nascimento Lima, José., Albuquerque, I.F.d., Dutra Bezerra, N.L.S., Monteiro, C.E.d.S., Franco, A.X., Lenardo, D.D., Oliveira, L.A., Feitosa, J.P.d.A., de Paula, Regina.Cé.Monteiro., Oliveira, J.S.d., Vasconcelos, D.F.P., Soares, P.M.G., Barbosa, André.Luiz.dos.Reis., Polysaccharides derived from Morinda citrifolia Linn reduce inflammatory markers during experimental colitis, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112303. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Polysaccharides derived from Morinda citrifolia Linn reduce inflammatory markers during experimental colitis Jalles Arruda Batistaa, Diva de Aguiar Magalhãesa, Stefany Guimarães Sousaa, Jayro dos Santos Ferreiraa, Cynthia Maria Carvalho Pereiraa, José Victor do Nascimento Limaa, Ieda Figueira de Albuquerquea, Nayonara Lanara Sousa Dutra Bezerraa, Carlos Eduardo da Silva Monteirob, Alvaro Xavier Francob, David Di Lenardoc, Lorena Almeida Oliveirad, Judith Pessoa de Andrade Feitosad, Regina Célia Monteiro de Paulad, Jefferson Soares de Oliveirae, Daniel Fernando Pereira Vasconcelosc, Pedro Marcos Gomes Soaresb, André Luiz dos Reis Barbosaa*. a

Laboratory of Experimental Physiopharmacology, LAFFEX, Post-graduation program in Biotechnology - Federal University of Piauí. Parnaiba, Brazil

b

Laboratory of Physiopharmacology Study of Gastrointestinal Tract, LEFFAG, Federal University of Ceará. Fortaleza, Brazil

c

Laboratory of Analysis and Histological Processing, LAPHIS, Department of Biomedicine – Federal University of Piauí. Parnaíba, Brazil d

Laboratory of Proteins and Carbohydrates of Marine Algae, Department of Biochemistry and Molecular Biology, Federal University of Ceará. Fortaleza, Brazil

e

Laboratory of Biochemistry and Microorganisms and Plant Biology, Department of Biomedicine, Federal University of Piauí. Parnaíba, Brazil

*Author to whom correspondence should be addressed; BIOTEC/LAFFEX/UFPI, Av. São Sebastião, nº 2819, CEP 64202-020, Parnaíba, PI, Brazil. E-Mail: [email protected] Tel.: +55-86-998199358/ +55-86-33225340; Fax: +55-86-33235406

ABSTRACT Ethnopharmacological relevance: There are many reports of pharmacological activities of extracts and fractions of different vegetable-derived products in the scientific literature and in folk medicine. Ethnopharmacological use of these products by various communities continues to be extensively explored, and they account for more than half of all medications used worldwide. Polysaccharides (PLS) extracted from plants such as Morinda Citrifolia Linn present therapeutic potential in treatment of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC). Aim of the study: To evaluate the anti-inflammatory action of Noni PLS against the intestinal damage in UC induced by acetic acid in mice. Materials and methods: In acetic acid-induced colitis, the mice were treated intraperitoneally (ip) with PLS (0.1, 0.3, and 3.0 mg/kg) or subcutaneously (sc) with dexamethasone (2.0 mg/kg) 30 min before euthanasia to determine the best dose of PLS with an anti-inflammatory effect in the course of UC. The colonic tissue samples were collected for macroscopic, wet weight, microscopic and biochemical (myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), nitrate/nitrite (NO3/NO2), cytokines, and cyclooxygenase (COX-2)) analyses. Results: Treatment with PLS reduced the intestinal damage induced by acetic acid as it reduced macroscopic and microscopic scores and the wet weight of the colon. In addition, MPO activity and levels of GSH, MDA, NO3/NO2, pro-inflammatory cytokines, and COX-2 expression reduced. Conclusions: This study suggests that PLS exhibits anti-inflammatory action against intestinal damage by reducing inflammatory cell infiltration, oxidative stress, proinflammatory action of cytokines, and COX-2 expression in the inflamed colon. PLS shows therapeutic potential against inflammatory disorders like UC.

Keywords: Colitis, Morinda Citrifolia Linn., Acetic acid, Anti-inflammatory, Antioxidant.

1. Introduction Plants have always represented a source of biomolecules for drug development (Yilmazer et al., 2016) and continue to provide new drugs for the pharmaceutical industry. As a result, natural products and derivatives account for more than 50% of all drugs in clinical use worldwide (Krishnaiah et al., 2011; Gurib-Fakim, 2006). Plant macromolecules have several therapeutic effects (Phosrithong and Nuchtavorn, 2016; Remila et al., 2015), and the ethnopharmacological application and efficacy, safety, and therapeutic potential of medicinal plants by many communities continues to be explored (Ningthoujam et al., 2012; Gurib-Fakim, 2006). Polysaccharides (PLS) extracted from plants can be considered a group of heterogeneous and complex macromolecules present in plant extracellular tissues (Vasconcelos et al., 2015; Patel, 2012; Rodrigues et al., 2012) used in food, biomedical, pharmaceutical, and cosmetic production (Cunha et al., 2009). When isolated from medicinal plants, PLS are studied because of the large spectrum of activities in vitro and in vivo and because of their relatively low toxicity (Inngjerdingen et al., 2005). Morinda citrifolia Linn (Noni PLS), known by many names such as Noni or Indian, Mengkudu in Malaysia, painkiller bush in the Caribbean, or cheese fruit in Australia, belongs to the Rubiaceae family and is a native tropical shrub of Asia and Polynesia (Wang et al., 2002). This plant has been used in traditional medicine for more than 2,000 years and is cultivated and utilized by Polynesians because of its antibacterial, antiviral, antifungal, antitumor, anthelmintic, analgesic, anti-inflammatory, hypotensive, immunostimulatory, antimicrobial, and anticancer properties (Wang et al., 2002; Bui et al., 2006). In folk medicine, every part of Noni (fruits, leaves, bark, trunk, and roots) can be used for therapeutic purposes (Pandy et al., 2017). To explore the medicinal properties of Noni, we used Noni PLS extracted from Noni juice. This PLS occurs as two fractions with different molecular weights. Fraction I consists of homogalacturonan and rhamnogalacturonan (1.71 × 105 g/mol), and fraction II contains arabinogalactan type I and arabinogalactan-type II (2.57 × 104 g/mol) (Sousa et al., 2018; supplementary data). These residues are important for reversing inflammatory conditions (Sousa et al., 2018). Noni PLS can be used against inflammatory disorders such as inflammatory bowel disease (IBD). One type of IBD is ulcerative colitis (UC) that affects the colon and rectum through formation of ulcers in the intestinal mucosa and submucosa (Conrad

et al., 2014; Hindryckx et al., 2016). This disease has an unknown etiology, but evidence shows that it can result from a deregulated immune response (Cetinkaya et al., 2005; Hill et al., 2010), characterized by increased production of pro-inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6), and by elevated inflammatory infiltration with increased production of reactive oxygen species (ROS) in the colon mucosa (Damiani et al., 2007; RobertsThomson et al., 2014). Symptoms often related to UC include fecal urgency, rectal bleeding, abdominal pain,

diarrhea,

fever,

fatigue,

and

weight

loss.

Eventually,

extra-intestinal

manifestations and various complications such as abscesses, fistulas, and colorectal cancer can occur (Escoté, 2018). In addition, active UC is associated with high rates of incapacity of working, increase in absenteeism, and reduction in labor productivity (Gibson et al, 2014). Currently, standard therapy for UC is the use of aminosalicylates, corticosteroids, antibiotics, and TNF-α antagonists. However, curative therapy is not yet available, and drug treatment can often be flawed and/or associated with adverse effects (Sobczak et al., 2014; Duijvestein et al., 2018). The development of innovative and effective drugs from natural sources for UC may be an interesting alternative for reducing adverse effects and improving treatment. Considering the biological effects of Morinda citrifolia L., this study aimed to evaluate the anti-inflammatory action of Noni PLS against intestinal damage in UC induced by acetic acid in mice.

2. Material and Methods

2.1. Extraction and characterization of noni PLS The fruits of Morinda citrifolia L. were harvested in a locality of the city of Fortaleza in the State of Ceará, located at 14 meters of altitude (Latitude: 3º 43 '6' 'South, Longitude: 38º 32' 36 '' West). PLS extraction was performed according to Sousa et al., (2018). Afterwards, the fruits were peeled and processed using a Mondial Juicer centrifuge for the acquisition of Noni puree, which was lyophilized to analyze its dry mass. Then, distilled water was added every 5 g of puree and the suspension was kept under magnetic stirring at room temperature for 1 hour. Subsequently, the suspension was centrifuged (6000 rpm, 20 min, 25°C) and the residue was discarded.

The supernatant was filtered and lyophilized, then the dried dried residue was dissolved in distilled water. PLS was precipitated, slowly and under stirring in 96% commercial ethyl alcohol in the ratio of 1: 4 (volume of Noni solution/volume of alcohol) (Bui, Bacic and Pettolino, 2006). After precipitation, the suspension containing the polysaccharide was centrifuged (8000 rpm; 30 min; 10 °C) and the supernatant was discarded. Finally, the precipitate was dissolved in water and lyophilized, being denominated as noni polysaccharide. The characterization of the molecular and structural arrangement of PLS was performed by Size exclusion chromatography, Infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, according to the protocol described by Sousa et al., (2018).

2.2. Materials and chemicals

Acetic acid was obtained from Sigma-Aldrich, Brazil. All drugs were dissolved in 0.9% saline or phosphate-buffered saline (PBS) (0.02M, pH: 7.0). All other chemicals and reagents were analytical grade and obtained from standard commercial suppliers.

2.3. Animals Male Swiss mice (25-30g) from the Federal University of Piauí (UFPI) were used, where they were allocated at a temperature of 25 ± 2ºC under a light/dark cycle of 12/12 h. These animals were deprived of food for 16 hours prior to the experimental trials, but with free access to water. All treatments and procedures performed were conducted according to the principles currently established by the Laboratory Animal Care and Use Guide (National Institute of Health, Bethesda, MD, USA) and approved by the UFPI Ethics Committee on Animal Experimentation (number protocol: 001/18). 2.4. Induction of colitis Colitis was induced according to the method described by Guazelli et al., (2013) with modifications. The animals were fasted for 16 hours and for the induction of colitis were anesthetized intraperitoneally (i.p.) with ketamine (80 mg/kg) and xylazine (10 mg/kg) and placed in left lateral decubitus position. Colitis was induced by

administration (200 µL) of acetic acid (6%) dissolved in saline solution in each colon of the mouse rectally via a polyethylene catheter 3 centimeter (cm) from the anal margin. Immediately afterwards, the animals were upright upside down for 30s and relocated to the boxes with free access to food and water. After 18 hours of induction of colitis, the animals were euthanized with a lethal dose of ketamine (240 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.) and samples of colonic tissue were removed for analysis of the inflammatory parameters.

2.5. Experimental Protocols

2.5.1. Anti-inflammatory effect of noni PLS on the assessment of acetic acid-induced colitis

The animals were divided into six groups (n = 6/group), in which group 1 received intracolon saline solution (0.9%, 200 µL); group 2 received only 6% intracolon acetic acid (200 µL); groups 3, 4 and 5 received intracolon acetic acid (6%, 200 µL) and were treated 30 minutes before euthanasia with noni PLS at doses 0.1, 0.3 and 3.0 mg/kg, i.p respectively, in order to determine the best PLS dose of noni with an antiinflammatory effect in the course of UC; and the positive control group 6 received intracolon acetic acid (6%, 200 µL) and the reference drug, dexamethasone (Dexa; 2.0 mg/kg, s.c.), was given subcutaneously (s.c.) 30 minutes prior to euthanasia. Afterwards, all animals were euthanized after 18 hours of induction of colitis and colon tissue samples were collected for macroscopic, wet weight, microscopic and biochemical analyzes. 2.5.2. Macroscopic lesion scores of the colon evaluation The colon was removed in which it was opened longitudinally and washed with physiological solution. Subsequently, macroscopic lesion scores were established using specific criteria (Morris et al., 1989) with modifications. The established criteria were: no damage (score 0); localized hyperemia but no ulcers (score 1); linear ulcers without significant inflammation (score 2); linear ulcers with inflammation in one place (score 3); two or more sites of ulceration and inflammation (score 4); a site of inflammation > 1 cm along the length of the colon (score 5); local inflammation > 2 cm

along the length of the colon, with quantification increased to 1 for each additional centimeter (score 6-10). 2.5.3. Colon wet weight The distal segments of the colon were sectioned, measuring 5 cm, to determine the of the humid weight of the colon. The results were expressed as percent weight gain of the colon (g) of the animals with colitis compared to a normal control group, without colitis and in relation to the animals treated with different doses of the noni PLS. 2.5.4. Histological analysis In the evaluation of the histological parameters, tissue samples from the colon were fixed in 10% formalin solution for a period of 24 hours, in which they were then transferred to 70% alcohol solution. Subsequently, the intestinal segment was immersed in paraffin, sectioned, deparaffinized, stained with hematoxylin and eosin and analyzed under a microscope (blind analysis). The evaluation had as reference criteria the following parameters: loss of mucosal architecture (0-3 score), cell infiltration (score 03), muscle thickening (score of 0-3), crypt abscess (score of 0 -1) and depletion of goblet cells (score of 0-1) (Appleyard e Wallace, 1995). The evaluation was performed by a reference histopathologist. 2.6. Biochemical assays 2.6.1. Myeloperoxidase Activity (MPO) The extent of neutrophil accumulation in colon tissue was measured indirectly by the enzymatic levels of MPO (Bradley, 1982). 50 to 100 mg of tissue were homogenized in 1 ml of potassium buffer with 0.5% hexadecyltrimethylammonium bromide per 50 mg of tissue. The homogenate was centrifuged at 40,000 x g for 7 min at 4 °C and the supernatant analyzed by spectrophotometry to determine the MPO activity at 450 nm. The results were expressed as the MPO units per milligram of tissue of the colon. 2.6.2. Glutathione (GSH) assay

The colon samples were homogenized in 0.02M EDTA (1 mL/100mg tissue). Subsequently, 400 µL of homogenate were mixed with 300 µL of distilled water and 80 µL of trichloroacetic acid (50%, w/v) and centrifuged at 3000 rpm for 15 minute. Then, 400 ml of supernatant was mixed with 800 ml of Tris buffer (0.4M, pH: 8.9) and 20 ml of 0.01M DTNB was added, and the samples were subsequently stirred for a period of 3 minute. A reading spectrophotometer with absorbance adjustment measured at 412nm was used. The results are expressed as µg of GSH/g tissue (Sedlak e Lindsay, 1968). 2.6.3. Dosage of Malondialdehyde (MDA) Colon intestinal tissue samples were homogenized in 1.15% KCl (1 mL/100 mg tissue). 250 ml of the homogenate was added in 1.5 ml of H 3 PO 4 (1%) and 0.5 ml of an aqueous solution of aqueous thiobarbituric acid (0.6). The tubes were heated for 45 minutes in a boiling water bath and the reaction mixture was then cooled in an ice-water bath, followed by the addition of 2ml of n-butanol. The contents were mixed for 1min. with a vortex mixer, centrifuged and the absorbance of the organic layer was measured at 520 and 535nm. The results were expressed as mmol of MDA/g of tissue (Mihara e Uchiyama, 1978). 2.6.4. Determination of NO3 (nitrate) and NO2 (nitrite) on colon mucosa For the determination of colonic concentrations of nitrate and nitrite the Griess method was used (Green et al., 1982). The processed samples of animal colonic tissue were incubated in a microplate with nitrate reductase (16 µL per well) for 12hours for the conversion of NO3 (nitrate) to NO2 (nitrite). Nitric oxide (NO) production was determined by measuring nitrite concentrations in an ELISA plate reader at 540nm. The results were expressed as micromoles (µM) of nitrite. 2.6.5. Cytokines assay Colon samples were homogenized in sterile saline and levels of IL-1β interleukin and tumor necrosis factor alpha (TNF-α) in the colon samples were determined by the Enzyme-Linked Immunoabsorbent Assay (ELISA) kit according to the recommendations of the manufacturer (Brito et al., 2013). The results were expressed in picograms per milliliter of homogenate (pg/ml). 2.6.6. Western blotting for COX-2

Segments of the distal colon were homogenized in a solution consisting of 0.2% octylphenyl ether Nonaetilenoglicol (Sigma, St. Louis, MO), 2mM EDTA, TritonX 100 a 1% 1X and proteinase inhibitors (Sigma, St. Louis, MO). The BCA assay (Sigma, Saint Louis, MO) (Smith et al., 1985) was used to measure protein concentrations with bovine albumin (1 mg/ml) as standard for a spectrophotometer length of 570 nm. Protein samples (25 µL) were loaded onto 12% polyacrylamide gels -SDS and electrophoresis performed for 90 min (Laemmli, 1970). The protein bands were transferred to PVDF membranes, washed for 5 min with Tris buffered saline (TBS) and placed in a blocking solution (TBS Tween20 plus 5% skim milk) for 1 hours. After blocking, the membranes were washed five times with TBS and incubated with COX- 2 (1: 100; Santa Cruz Biotechnology, Santa Cruz, CA). 2.6.7. Statistical Analysis Results were expressed as mean ± SEM of six animals per group. The statistical study was performed using analysis of variance (ANOVA) followed by Newman-Keuls post-hoc test. The histopathological parameters were analyzed by Kruskal-Wallis nonparametric test, followed by multiple comparisons with the Dunn test. p < 0.05 was considered statistically significant. 3. Results 3.1. PLS characterization The results of PLS characterization are described by Sousa et al. (2018) (supplementary data). 3.2. Effect of PLS on macroscopic lesion scores in acetic acid-induced colitis Animals with colitis showed a significant increase in the macroscopic scores of lesions (19.57 ± 1.21) compared to the saline group (0.42 ± 0.20). The group treated with the reference anti-inflammatory drug, Dexa (2.0 mg/kg, subcutaneously (sc)), showed a significant decrease in the scores (4.80 ± 0.73) compared to the colitis group. The animals treated with PLS (0.1, 0.3, and 3.0 mg/kg, intraperitoneally (ip)) showed a significant reduction (p < 0.05) in the scores (0.1 mg/kg: 12.00 ± 1.22;

0.3 mg/kg: 9.00 ± 1.00; 3.0 mg/kg: 5.0 ± 0.63) compared to the colitis group, with the maximum effect observed at the dose of 3.0 mg/kg (Fig. 1A and 1B). 3.3. Action of PLS on wet weight in acetic acid-induced colitis A significant increase in colonic wet weight was observed after induction of colitis (0.38 ± 0.01 g/5/cm) compared to the saline group (0.17 ± 0.005 g/5/cm). However, there was a significant (p < 0.05) reduction in the colonic wet weight in PLStreated animals (0.3 mg/kg: 0.27 ± 0.01 g/5/cm and 3.0 mg/kg: 0.22 ± 0.01 g/5/cm), of which the 3.0 mg/kg dose had a better effect on reducing colonic weight compared to the colitis group. There was no significant reduction in the wet weight of the colon in animals treated with 0.1 mg/kg PLS (0.33 ± 0.02 g/5/cm). The group treated with the reference anti-inflammatory drug, Dexa (2.0 mg/kg, sc), had a significant decrease in wet weight of the colon (0.28 ± 0.01 g/5/cm) compared to the colitis group (Fig. 2). The best dose was defined as 3 mg/kg and used in the other experimental trials. 3.4. Effect of PLS on the microscopic lesion scores in the colon Histological analysis showed that the saline group presented minimum scores for all parameters evaluated (Table 1, Fig. 3A). On the other hand, the colitis group presented significant loss of mucosal architecture, intense cell infiltration, thickening of the muscular layer, formation of abscesses in the crypt, and depletion of goblet cells (Table 1, Fig. 3B). In the group treated with PLS (3.0 mg/kg, ip) when purchased from the colitis group, they achieved a significant reduction in loss of mucosal architecture, cell infiltration, thickening of muscular layer, abscess formation in the crypt, and depletion of goblet cells (Table 1, Fig. 3C and 3D). Similar results were observed upon treatment with the reference drug, Dexa (2.0 mg/kg, sc). 3.5. Effect of PLS on myeloperoxidase (MPO) levels in colonic tissue

Fig. 4 shows the MPO levels in animals with colitis. The colitis group showed a significant increase in MPO level (33.20 ± 1.51 UMPO/mg of colon tissue) compared to the saline group (2.31 ± 0.71 UMPO/mg tissue). The administration of 3 mg/kg of Noni PLS reduced (p < 0.05) the level of this enzyme (1.70 ± 0.19 UMPO/mg of colon tissue) compared to the colitis group.

3.6. Effect of PLS on glutathione (GSH) and malondialdehyde (MDA) levels and nitrate/nitrite concentration (NO3/NO2) When we analyzed the effect of PLS on GSH levels (Fig. 5A), MDA (Fig. 5B) levels, and NO3/NO2 concentration (Fig. 5C) in the colonic mucosa, we observed that PLS was able to restore significantly (p < 0.05) the GSH concentration (130.3 ± 10.74 µg/mL) and decrease the level of MDA (196.4 ± 24.13 nmol/mL) and NO3/NO2 level (0.71 ± 0.73 µM) compared to the colitis group (GSH: 170.0 ± 11.95 µg/mL; MDA: 4.00 ± 23.61 nmol/mL; NO3/NO2: 1.20 ± 0.60 µM). 3.7. Effect of Noni PLS on IL-1β and TNF-α in colonic tissue The effect of PLS on IL-1β and TNF-α levels in colon samples is presented in Fig. 6A and 6B, respectively. A significant increase (p < 0.05) in the levels of IL-1β and TNF-α in the colitis group (3.58 ± 0.07 pg/mL; 4.71 ± 0.02 pg/mL, respectively) was observed compared to the group saline (1.79 ± 0.20 pg/mL; 1.94 ± 0.27 pg/mL, respectively). Administration of PLS at the dose of 3 mg/kg significantly reduced the concentration of IL-1β and TNF-α (1.61 ± 0.16 pg/mL; 2.90 ± 0.25 pg/mL, respectively) compared to the colitis group. 3.8. Effect of Noni PLS on the protein expression of COX-2 Western blotting analysis showed that, after the administration of acetic acid, the colon expression of COX-2 increased (0.66 ± 0.05 COX-2/p38) compared to the saline group (0.14 ± 0.01 COX-2/p38). After PLS administration, there was a significant reduction in COX-2 expression (0.32 ± 0.05 COX-2/p38) compared to the colitis group (Fig. 7). 4. Discussion The present study confirms that PLS of Morinda citrifolia L. possesses antiinflammatory properties and acts effectively by reversing acetic acid-induced colitis in mice as shown by macroscopic lesion analysis, colon wet weight investigation, histological analysis, and biochemical assays (levels of GSH, MDA, MPO, IL-1β, TNFα, and NO3/NO2 and COX-2 expression). The acetic acid-induced colitis model has been widely used as it is easy to reproduce in the laboratory. The acetic acid-treated colon shows acute inflammation,

increased vasopermeability, prolonged infiltration of neutrophils, and increased production of inflammatory mediators in a manner similar to UC in humans (Daneshmand et al., 2009; Goyal et al., 2014). In this study, three different doses of PLS (0.1, 0.3, and 3.0 mg/kg) were used ip in the different groups of mice to treat colitis in order to establish the concentration that with the best anti-inflammatory effect. The PLS fraction isolated from Noni fruits (Morinda citrifolia L.) inhibited colitis in the colonic tissue of mice (indicated by the decrease in the macroscopic inflammatory scores) when administered at the dose of 3 mg/kg (ip). Since this dose had the best effect, it was chosen for the other experimental trials. Destruction of the tissue structure was caused by inflammation resulting from the administration of acetic acid in the colon of the animals, as shown in the macroscopic analysis. On the other hand, the animals treated with PLS showed partial preservation of the tissue structure, exhibiting lower inflammation scores attributed according to the technique described by Morris et al. (1989). These findings are consistent with other studies using natural PLS (Brito et al., 2013). The colons of animals submitted to acetic acid colitis present several features related to local inflammation such as increased neutrophil infiltration (Sanei et al., 2014), crypt abscesses, granulomatous inflammation with fibrosis, and massive submucosal thickening (Wang et al., 2013). Since the increase in colonic tissue weight is considered an indirect index of the degree of local inflammation, PLS caused a significant reduction in the colonic wet weight of the mice submitted to acetic acidinduced colitis. In a previous study by Aleisa (2014), these phenomena contributed to the increase in the weight/length relationship in the colon of the animals, because the rectal application of acetic acid was associated with severe tissue ulceration, necrosis, goblet cell hyperplasia, and inflammatory infiltrate. Thus, PLS exerts an antiinflammatory activity, reducing neutrophilic infiltration, contributing to minimize lesions in the colonic tissue. To reinforce our hypothesis, we performed histopathological analysis that measured the insensitivity of neutrophil infiltration and mucosal damage characterized by disruption of intestinal crypt and damage of blood vessels Colitis produced by acetic acid causes destruction of the epithelial, creates mucosal crypts, and causes significant edema in the submucosa, with a notable inflammatory infiltrate at the microscopic level (Hartmann, 2012; Ferreira et al., 2018). Our results showed that the treatment with

acetic acid caused an increase in the microscope criteria. The PLS treatment reduced the histological alterations caused by acetic acid. This result confirmed our hypothesis that PLS acted at the inflammatory site by reducing the infiltration of inflammatory cells in the colonic tissue and the damage on tissue structure. During intestinal damage, the main cells involved in this process is the neutrophil. This cell during its diapedesis releases MPO (Tahan et al., 2011). MPO is an enzyme has been used extensively as a biochemical marker of granulocyte infiltration in various tissues, being found mainly in neutrophil azurophil granules (Araújo, 2011). In the context, we performed an assay for MPO level and observed that, in the acetic acid group, there was an increase in the MPO levels, but this effect was reversed by PLS treatment. The measurement of the activity of this enzyme is of great value in experimental studies, as it is an indirect marker of neutrophilic infiltration during inflammation. Based on our results, we can infer that PLS inhibits intense neutrophil infiltration and is able to reduce the intestinal damage induced by acetic acid. Neutrophil infiltration and accumulation in inflammatory site is one of the factors that lead to increased oxygen production due the ability to produce superoxide radicals, resulting in overproduction of reactive oxygen species (ROS) (Closa and Folch-Puy, 2004; Hartmann et al., 2012; Adil et al., 2014; Aswar et al., 2015). ROS have been reported in colorectal samples of UC patients (Tüzün et al., 2002; Bitiren et al., 2010). Experimentally, acetic acid-induced colitis is characterized by oxidative damage caused by the imbalance between oxidizing substances and antioxidants (Dröge, 2002), and studies have indicated a correlation between the pathogenesis of UC and oxidative stress in preclinical and clinical trials (D’Odorico et al., 2001). An important indicator that reflects the accumulation of ROS in response to oxidative damage is MDA, which causes mucosal injury and generates lipid peroxidation products (Wang et al., 2013; Daneshmand, 2008). In addition, the cells have an antioxidant device (GSH) that protects them against ROS generated during pathological lesions. GSH exerts a cellular protective effect mainly by maintenance of reduced sulfhydryl groups, preventing them from reacting with free radicals (Filippin et al., 2008; Santiago et al., 2015). In human UC as well as in acetic acid-induced colitis, the production of oxidative stress is a key factor in the pathogenesis and perpetuation of mucosal damage, impairing epithelial integrity and causing tissue injury (Tahan, 2011; Takhshid et al., 2012).

Our results clearly demonstrated that Noni PLS was able to restore GSH colonic level and decrease MDA concentration in colitis induced by acetic acid. Thus, PLS decreases acetic acid-induced colonic injury to preserve, partially, GSH levels and decrease lipid peroxidation in the body induced by chemical action of acetic acid. Several other naturally occurring PLS have been the subject of scientific research for their positive actions on the oxidative balance of cells in inflammatory processes (Brito et al., 2013; Dore et al., 2014). The polymer under study was effective in reducing intestinal inflammation, possibly because of the decreased migration of neutrophils, responsible for unbalance of the antioxidant cascade. Another important inflammatory mediator involved in the activation of neutrophils and the lesion of intestinal epithelial cells is NO. In general, excessively produced NO contributes to lesions in the intestine during colitis by direct cytotoxicity (Carvalho et al., 2018). In addition, NO can interact with superoxide and form the highly toxic peroxynitrite radical (ONOO−), which reversibly increases iNOS expression (Blázovics, 2004; Brito, 2016; Vasconcelos et al., 2017). The overproduction of NO derivatives (NO3/NO2) establishes a positive regulation of the induced synthesis of NO (iNOS). Thus, in experimental colitis in mice as well as in IBD in humans, synthesis of NO in the colon and iNOS activity are increased (Brito et al., 2016). In our experimental model of UC, PLS also reduced NO3/NO2 levels. Cytokine production is another factor responsible for UC pathogenesis. This inflammatory marker acts by activating the recruitment of neutrophils. These cells are also activated by other pro-inflammatory mediators such as interleukins and chemokines (Pedersen et al., 2014; Rahimi et al., 2007; Brito, 2016). Thus, in order to investigate the role of Noni PLS in the release of cytokines, we assayed cytokine levels. We observed, in mice treated with acetic acid, a significantly increased expression of IL-1β and TNF-α. On the other hand, PLS was able to decrease the levels of these cytokines in the colon tissue of animals with acetic acid-induced colitis. These results suggest that the immunomodulatory action of natural PLS (Araujo, 2011) can reflect the potential of Noni PLS in decreasing the release and activation of these cytokines. Therefore, the study of enzymes such as COX-2 becomes very important, as it is responsible for the production of pro-inflammatory mediators such as prostaglandins and is considered an important inflammatory marker (Vasconcelos et al., 2017). Our data showed that Noni PLS was able to decrease COX-2 expression, demonstrating that its anti-inflammatory action could occur in several ways, including interference in gene

expression of important precursors of inflammatory mediators responsible for induction of neutrophil activation, disruption of intestinal mucosa, and overproduction of ROS in the acute phase of the UC.

5. Conclusions In this study, PLS exhibited an anti-inflammatory effect in an experimental model of UC induced by acetic acid by reducing neutrophil infiltration, oxidative stress, pro-inflammatory cytokine action, and expression of COX-2 in inflamed colonic cells. Thus, PLS shows great treatment potential against inflammatory disorders during UC.

Acknowledgments

The authors are grateful to the Brazilian Agency for Scientific and Technological Development-CNPq (Brazil), Council for Advanced Professional Training (CAPES) and the Research Foundation for the State of Piauí (FAPEPI).

List of the authors and their respective contributions

Lorena Almeida Oliveira, Judith Pessoa de Andrade Feitosa and Regina Célia Monteiro de Paula carried out the tests related to the extraction and characterization of polysaccharides; Jalles Arruda Batista, Stefany Guimarães Sous and Diva de Aguiar Magalhães evaluated the action of the PLS of Morinda citrifolia L. during acetic acidinduced colitis in the evaluation of the macroscopic lesion and wet weight of the colon score; David Di Lenardo and Daniel Fernando Pereira Vasconcelos performed the histological analysis of the lesions of the colon; Nayonara Lanara Sousa Dutra Bezerra, Jayro dos Santos Ferreira, Cynthia Maria Carvalho Pereira, José Victor do Nascimento Lima and Ieda Figueira de Albuquerque performed the experiments to measure the concentration of myeloperoxidase, glutathione and malondialdehyde in the intestinal segments; Carlos Eduardo da Silva Monteiro, Alvaro Xavier Franco and Pedro Marcos Gomes Soares carried out the tests of measurement of proinflammatory cytokines, nitrate and nitrite and western blotting in the mucosa of the colon; André Luiz dos Reis Barbosa and Jefferson Soares de Oliveira wrote this article. Conflicts of interest The authors declare no conflict of interest.

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Fig. 2. The PLS fraction reduces the wet weight of the colonic tissue of animals treated with acetic acid. The mice were treated with 0.9% saline solution (negative control) and acetic acid (positive control). They were treated with PLS (0.1, 0.3 and 3.0 mg/kg, i.p.) and dexamethasone (2 mg/kg, s.c). Results are expressed as mean ± SEM of 6 animals per group. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (ANOVA followed by Newman-Keuls post-hoc test). Fig. 3. PLS improves histopathological changes in acetic acid-induced colitis. Micrograph (200x, 50 µm scale) representing the Intestine of an animal receiving only 0.9% saline solution (A) and acetic acid (control group) (B); intestine of an animal with colitis and treated with PLS (3.0 mg/kg) (C) and Dexa (2.0 mg/kg) (D). Red arrow: architecture of the mucosa (villi and crypts), yellow arrow: cell infiltration, black arrow: muscle thickening.. Fig. 4. Decreased MPO activity in the colon tissue from treatment with the fraction of PLS in mice. The animals were treated with saline (negative control), acetic acid (positive control) and PLS (3.0 mg/kg, i.p.). Results are expressed as mean ± SEM of 6 animals per group. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (ANOVA followed by Newman-Keuls post-hoc test). Fig. 5. Effect of treatment with PLS fraction on acetic acid-induced colitis on increase of GSH (A), reduction of MDA levels (B) and concentration on colonic tissue of NO3/NO2 concentration (C). The animals were treated with 0.9% saline solution (negative control), acetic acid (positive control) and PLS (3.0 mg/kg, i.p.). Results are expressed as mean ± SEM of 6 animals per group. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (ANOVA followed by Newman-Keuls post-hoc test). Fig. 6. The PLS fraction reduced levels of IL-1β (A) and TNF-α (B) in the gut of mice with acetic acid-induced colitis. The animals were treated with saline (negative control), acetic acid (positive control) and PLS (3.0 mg/kg, i.p.). Values were presented as mean ± SEM of 6 animals per group. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (ANOVA followed by Newman-Keuls post-hoc test). Fig. 7. Intestinal tissue was collected for measurement of COX-2 protein expression by Western blotting. Acetic acid increases the protein expression of COX-2 when compared to the saline group (negative control). Treatment with PLS (3.0 mg / kg, i.p.)

reduced the expression of this protein. The values were presented as mean + DPM of the COX-2 protein expression that resulted from the ratio of the density of these proteins and α-tubulin. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (ANOVA followed by Newman-Keuls post-hoc test). Table 1. Histological evaluation of intestinal damage in mice. Histological damage scores were expressed as mean ± SEM of 6 animals per group. *P<0.05 versus acetic acid group; #P<0.05 versus saline group (Kruskal-Wallis non-parametric test and Dunn test were used for multiple comparisons of histological analyzes).

Table 1

Median score Criteria

Saline

Acetic acid

Acetic acid + PLS

Acetic acid + Dexa

Loss mucosal architecture

0 (0-0)

3 (3-3)#

1 (1-1)*

2 (1-2)

Cellular infiltration

0 (0-0)

3 (2-3) #

1 (0-1)*

1 (1-2)

Muscle thickening

0 (0-0)

3 (3-3) #

1 (1-1)*

1 (1-2)

Crypt abscess

0 (0-0)

1 (1-1) #

0 (0-0)*

0 (0-1)*

Goblet cell depletion

0 (0-0)

1 (1-1) #

0 (0-0)*

0 (0-1)*

Total damage score

0

11#

3*

4*

Fig. 1. A

B

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5. A

B

C

Fig. 6.

A

B

Fig. 7.

A

B