Peptide Isolated from Noni Seeds Confers Gastroprotective Effect by Improving Inflammation and Oxidative Stress in Mice

Plant molecules are continuously investigated to prevent and treat inflammatory and ulcerative disorders associated with the gastrointestinal tract, such as gastritis, colitis, mucositis, and ulcers. However, most of the published work is devoted to investigating the therapeutic properties of secondary plant metabolites. This work investigated the gastroprotective activity of a lipid transfer protein isolated from Morinda citrifolia L., named McLTP1, when orally administered to mice from the perspective of its use as a novel peptide-based drug for the prevention and treatment of ulcerative gastric lesions. Pretreatment with McLTP1 at different doses (4, 8, or 16 mg/kg) reduced ethanol-induced gastric lesions (p < 0.05) by 40%, 84%, and 88%, respectively. In ethanol-induced gastric lesions, alterations in the levels of glutathione (GSH) (↑100%; p < 0.05) and a reduction of 45% (p < 0.05) in the levels of malondialdehyde (MDA) were demonstrated after McLTP1 administration (8 mg/kg). McLTP1 showed an anti-inflammatory effect through modulation of the cytokines IL-10 (↑33%) and TNF-α (↓54%) and was able to reduce myeloperoxidase (MPO) levels (↓95%) in gastric tissue. In addition, the gastroprotective effect of McLTP1 also involves the production of nitric oxide. The present findings reveal that McLTP1 has a gastroprotective effect dependent, at least in part, on its anti-inflammatory and antioxidant effects.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Also, kindly confirm the details in the metadata are correct.I confirm that the author names are presented accurately and in the correct sequence.


Introduction
Reactive oxygen species (ROS) are products generated from the metabolism of a healthy cell. They are produced mainly by the mitochondria and have a fundamental role in homeostasis (Forrester et al. 2018). However, when cellular ROS overproduction surpasses intracellular antioxidant capacity, oxidative stress occurs, damaging cells, tissues, and biomolecules (Hassan et al. 2017). Several human sufferings are caused by unbalanced oxidative stress status, including cardiovascular and pulmonary diseases, severe liver injury, and gastric lesions (Kumar et al. 2017).
Oxidative stress is recognized as one of the main contributors to lesions in gastric disorders such as peptic ulcers, gastroesophageal reflux diseases, and gastritis (Jin-Shui et al. 2008). In acute gastric lesions induced by ethanol, severe oxidative stress is triggered due to gastric mucosa lesions, infiltration of neutrophils, and the release of proinflammatory cytokines (Sung et al. 2019). Increases in ethanol concentrations lead to increases in ROS, thus causing cell damage and death.
Many plant extracts and isolated molecules have been studied to treat inflammatory and ulcerative gastrointestinal disorders. Regardless, most investigations focus on the gastroprotective activity of secondary plant metabolites, such as saponins, anthraquinones, alkaloids, and flavonoids (Singh et al. 2018;Saha et al. 2019).
Plant peptides are considered a promising therapeutic agent class (Sani et al. 2019). According to Baig et al. (2018), peptide-based drugs are used to treat various diseases, such as diabetes and cancer, along with antimicrobials, antifungals, antivirals, and antioxidants. Thus, peptides have emerged as a significant therapeutic class, with many natural and synthetic analogs undergoing clinical trials (Lau and Dunn 2018).
Our research group recently isolated a peptide from noni (Morinda citrifolia L.; Rubiaceae) seeds (McLTP 1 ; Uni-ProtKB accession number: C0HJH5) with a diverse range of pharmacological activities when orally administered to mice (Campos et al. 2016). McLTP 1 is a thermostable molecule resistant to proteolytic degradation by pepsin, trypsin, and chymotrypsin and has shown antipyretic, anti-inflammatory, antinociceptive, and anti-sepsis effects in mice (Campos et al. 2016;Campos et al. 2017;Souza et al. 2018). The anti-inflammatory action mechanism of McLTP 1 is related to cytokine modulation, such as TNF-α, which is involved in the pathophysiology of ethanol-induced gastric lesions (Campos et al. 2016(Campos et al. , 2017. Hence, we hypothesized that McLTP 1 could also be explored to treat gastric lesions.
This work aimed to evaluate the gastroprotective effects of McLTP 1 and investigate the mechanisms underlying the observed outcomes from the perspective of its future use as a novel peptide-based drug for treating and preventing ulcerative gastric lesions.

Purification of McLTP 1
McLTP 1 was purified from defatted noni (M. citrifolia L.; Rubiaceae) seed flour according to the experimental procedure reported by Campos et al. (2016). The proteins were extracted from defatted noni seeds (0.05 M Tris-HCl/0.25 M NaCl, pH 8.5, 4 °C), and the crude extract was fractioned with trichloroacetic acid (TCA) to a final concentration of 2.5% (w/v) at 30 min on ice. Then, the soluble fraction was centrifuged (10,000×g for 30 min at 4 °C), and the supernatant was dialyzed, lyophilized, applied to a Sephadex G-50 column, and monitored at 280 nm.
After isolation, the purity of McLTP 1 was verified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% SDS-PAGE) under nonreducing conditions (Laemmli 1970). McLTP 1 samples were prepared immediately before use for the biological assays based on total soluble protein concentrations (mg/mL) estimated by Bradford (1976), using bovine serum albumin as a standard.

Animals
Swiss male mice (Mus musculus) (20-25 g; n = 8/group) were housed at 25 ± 2 °C on a 12-h light/dark cycle and received food and water ad libitum. Before evaluating the gastroprotective effects of McLTP 1 , the mice were fasted for a 12 h period, with free access to 5% glucose in water to prevent hypoglycemia and dehydration events. The experiments are reported in compliance with ARRIVE Guidelines and were performed after approval of the Committee for the Ethical Use of Animals of the Federal University of Ceará (CEUA-UFC Proc. N° 109/16).
The stomachs were removed and opened along the greater curvature, rinsed with saline, extended, and photographed (Medeiros et al. 2008). The hemorrhagic areas were measured using Image Processing and Analysis in Java (ImageJ). The lesion index was calculated in comparison to the ethanol control group as follows: lesion index = [lesion area (mm 2 )/total area of stomach mucosa (mm 2 )] × 100 (Alvarez-Suarez et al. 2011).

Histopathological Analysis of the Stomach
Additionally, for histopathological analysis, the stomachs of groups (naïve) vehicle-NaCl 0.15 M; McLTP 1 8 mg/kg p.o.; or NAC-300 mg/kg, p.o.) were fixed in 10% formalin solution and dehydrated in 95% ethanol for 24 h. Finally, the tissues were embedded in paraffin and sectioned. Sections of 4 µm were then stained with hematoxylin and eosin (H&E) for observation under light microscopy (Olympus, Tokyo, Japan). An experienced pathologist analyzed the sections without access to the experimental groups according to previously described criteria (Laine and Weinstein 1988) with the following parameters: edema (score of 0-4), hemorrhagic damage (score of 0-4), and epithelial cell loss (score of 0-3).

Measurement of Malondialdehyde (MDA) and Glutathione (GSH) Levels in Stomach Tissue
For MDA analysis, stomach samples from the different groups (naïve mice; vehicle-NaCl 0.15 M; McLTP 1 8 mg/ kg p.o.; or NAC-300 mg/kg, p.o.) were homogenized in 1.15% KCl (10% w/v). Each stomach homogenate (250 μL) was added to 1.5 mL of 1% phosphoric acid and 500 μL of 0.6% thiobarbituric acid. This mixture was then stirred and heated in a boiling water bath for 45 min. The preparation was then cooled, followed by the addition of 2 mL of n-butanol. This mixture was stirred and centrifuged at 1200 rpm for 10 min, and the absorbance was measured at 535 nm. The results were expressed as µmol MDA/g of tissue (Mihara and Uchiyama 1978).
The GSH concentration was measured from stomach samples homogenized in 0.02 M ethylenediaminetetraacetic acid (1 mL/100 mg of tissue), distilled water, and TCA (50%, w/v). After centrifugation at 4 °C and 3000 rpm for 15 min, the resulting supernatant was mixed with 800 μL of Tris buffer (0.4 M, pH 8.9), followed by the addition of 20 μL of 0.01 M 5,5′-dithiobis-2-nitrobenzoic acid. The absorbance was measured at 412 nm using a spectrophotometer. The results were expressed as µg GSH/g of tissue (Sedlak and Lindsay 1968).

Determination of Myeloperoxidase (MPO) Enzyme Activity
Neutrophil migration on ethanol-induced gastric lesions was determined by the activity of the enzyme MPO according to the method described by Bradley et al. (1982). Samples of the gastric mucosa obtained from different experimental groups (naïve mice; vehicle-NaCl 0.15 M; McLTP 1 8 mg/kg p.o.) were homogenized in 50 mM sodium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium (100 mg tissue/1.5 mL) and ground in a tissue homogenizer. The homogenate was centrifuged for 15 min at 2,500 rpm. The supernatant (10 μL) was then added to a 96-well microplate in duplicate. Subsequently, 200 μL of buffer solution containing o-dianisidine dihydrochloride (16.7 mg), deionized water (90 μL), sodium phosphate buffer (10 μL), and 1% hydrogen peroxide (50 μL) was added to the wells. After 5 min, the absorbance of the samples was measured in a microplate reader (450 nm), and the enzymatic activity of MPO was expressed as units of MPO/mg of tissue.

Effect of McLTP 1 on Gastric Secretion
Mice were pretreated orally with vehicle NaCl 0.15 M (10 mL/kg), ranitidine (50 mg/kg), or McLTP 1 (8 mg/kg). After 1 h, animals were anesthetized with ketamine (80 mg/ kg, i.p.) and xylazine (10 mg/kg, i.p.). The abdomen of the treated animals was opened for the exposition of the stomach, and subsequently, pyloric ligation was performed. Four hours later, the animals were euthanized by cervical dislocation under anesthesia by halothane inhalation. The stomach was opened, and the gastric content was collected using ultrapure water (2 mL) and centrifuged at 1,500 rpm for 20 min at 25 °C. Next, the total acidity (mEq [H + ]/mL) was determined by titrating the homogenates with 0.01 M NaOH using a pH meter. Changes in pH were inferred to be changes in gastric secretion (Shay 1945).

Statistical Analysis
The Shapiro-Wilk test was performed to determine normality. The data with p > 0.05 (Shapiro-Wilk normality test) were considered normal and analyzed using one-way analysis of variance (ANOVA), followed by Tukey's test for multiple comparisons. The nonparametric Kruskal-Wallis comparisons test analyzed nonnormal data. The statistical analysis was performed using Graph Pad Prism 6.0 (Graph Pad Software Inc., CA, United States). The letters of the statistics indicate that the values show a significant difference (p < 0.05) among the compared groups.

Ethics Approval of Animal Use
The animals were treated in compliance with the ethical standards established by the National Guidelines for the Use of Experimental Animals of Brazil and Directive 2010/63/ EU of the European Parliament and the Council of the European Union. The experiments were performed after approval of the Committee for the Ethical Use of Animals of the Federal University of Ceará (CEUA-UFC Proc. N° 109/16).

Gastroprotective Effect of McLTP 1 on Ethanol-Induced Gastric Lesions
Ethanol administration induced intense macroscopic gastric damage in mice (14.57 ± 3.35 of lesion index). The oral treatment with McLTP 1 (4, 8, and 16 mg/kg) significantly decreased (p < 0.05) the appearance of ethanol-induced lesions with reductions of 40% (8.73 ± 1.76 of lesion index), 84% (2.28 ± 0.75 of lesion index), and 88% (1.73 ± 0.71 of lesion index) of the lesion areas, respectively, when compared to the vehicle group. The doses of 8 and 16 mg/kg were not significantly different from each other, and they did not differ from the standard drug N-acetylcysteine (300 mg/ kg), which reduced gastric lesions by 89% (1.59 ± 0.77 of lesion index) (Fig. 1).
Pretreatment of mice with McLTP 1 (8 mg/kg; p.o.) significantly reduced (p < 0.05) the MPO activity compared with the vehicle group (Fig. 3). Pretreatment of mice with McLTP 1 (8 mg/kg; p.o.) also caused a significant decrease in TNF-α levels by 54% and promoted an increase of 33% in IL-10 levels (p < 0.05) when compared to the control group. In contrast, McLTP 1 was unable to significantly reduce the levels of IL-1β (Fig. 4). absence of edema (E), epithelial cell loss (F), and hemorrhagic damage (G). The results are presented as medians, and the Kruskal-Wallis nonparametric test, followed by Dunn's test, was used for multiple comparisons of histological analyses (n = 8). Significance is represented as a p < 0.05 vs naïve and as b p < 0.05 vs. vehicle

Discussion
Numerous recent studies have considered plants as alternative sources for developing new drugs for treating chronic diseases of the gastrointestinal tract because of their high therapeutic potential and few adverse effects (Singh et al. 2018). A plant species rich in bioactive molecules and widely used in traditional medicine is M. citrifolia L. (Rubiaceae), popularly known as noni (Assi et al. 2017). Diverse pharmacological activities have been reported for this species, including antidiabetic, antioxidant, antiinflammatory, gastric ulcer healing, and hepatoprotective activities. Although over 200 different compounds have been identified in noni (Assi et al. 2017), research on proteins isolated from this plant is still scarce. In this work, we demonstrate for the first time that a protein isolated from noni seeds presents promising therapeutic effects against gastric injuries in mice. This is also the first report of the gastroprotective activity of plant LTPs.
McLTP 1 effects were studied after oral administration to mice to provide the first report for future clinical use. Ethanol causes high production of ROS, produces necrotic lesions, and exposes the epithelium to pepsin and hydrochloric acid (Zatorski et al. 2018). Treatment of mice with McLTP 1 decreased the gastric lesion index and reduced histopathological alterations caused by ethanol, demonstrating that McLTP 1 displays a gastroprotective effect. As observed for the anti-inflammatory activity already investigated for McLTP 1 (Campos et al. 2016), there were no significant differences in the effects observed between the treatment of mice with 8 or 16 mg/kg. In addition, the gastroprotective action displayed by McLTP 1 treatment was not different from that presented by the positive control NAC. Considering that one of the most common side effects of the continuous use of anti-inflammatory drugs is the appearance of gastrointestinal injuries, the gastroprotective effects described by McLTP 1 in this study reinforce the potential use of this protein as a less harmful alternative to treat inflammation, as proposed by Campos et al. (2017).
Alcoholic extracts, secondary metabolites, and polysaccharides have been described as potentially gastroprotective molecules from plants (Diniz et al. 2015;Neto et al. 2017;Sidahmed et al. 2013;Carlotto et al. 2019). However, few studies are still related to the gastroprotective activity of plant proteins or peptides. The advantages of proteins/ peptides over molecules from secondary metabolism are mainly because the former has high selectivity, fewer side effects, and reduced toxicity and can be designed to act on vast mechanisms, increasing the possibilities of their application against different diseases. However, a disadvantage associated with proteins/peptides has been their low oral bioavailability due to degradation by gastrointestinal tract enzymes (Bruno et al. 2013). Remarkably, McLTP 1 is stable to the main enzymes of the gastrointestinal tract (trypsin, chymotrypsin, and pepsin) (Campos et al. 2016), which enables the application of McLTP 1 orally by overcoming the main problem associated with the use of therapeutic proteins/peptides.
Oxidative stress and inflammation are parameters related to the pathophysiology of many diseases. Thus, drugs that modulate ROS production and inflammation can act to treat a diverse set of conditions, including peptic ulcers, gastrointestinal cancers, and inflammatory bowel disease (Bhattachayya et al. 2014). Gastric mucosal damage induced by ethanol has been suggested to be mediated through enhanced oxidative stress (Suzuki et al. 2012;Tamura et al. 2013). Oxidative stress disrupts a subtle oxidant/antioxidant balance, promotes lipid peroxidation and mucosal blood extravasation, and increases the infiltration of activated neutrophils (Halliwell 1994;Asmari et al. 2016). The observed prevention of GSH depletion, combined with L-NAME partial reversion of the gastroprotective effect of McLTP 1 , suggests a potential McLTP 1 action mechanism to reduce oxidative stress.
Corroborating the data presented by Campos et al. (2017), McLTP 1 showed a significant reduction in MPO activity, an indirect parameter of neutrophil accumulation in gastric mucosal tissues. Neutrophil release in the inflammatory response leads to increased gastric expression of NF-κB, which stimulates the synthesis of proinflammatory cytokines, including tumor necrosis factor-α (Yoo et al. 2018). In contrast, the anti-inflammatory cytokine IL-10 is considered a potent molecule for eliminating inflammatory processes involved in maintaining the homeostasis of Fig. 6 Effect of McLTP 1 on gastric acid secretion in mice subjected to pyloric ligation. Mice were pretreated orally with vehicle NaCl 0.15 M (10 mL/kg), ranitidine (50 mg/kg), or McLTP 1 (8 mg/kg) 1 h before the pylorus ligation procedure. The results are expressed as the mean ± SD. a p < 0.05 compared with the vehicle group (one-way ANOVA followed by Tukey's test; n = 8) the gastric mucosa, including the synthesis of the regulator TNF-α (Lee et al. 2017). The ratio of pro-(TNF-α) and antiinflammatory (IL-10) cytokines may influence the degree of inflammation, an essential factor for gastric lesions (Kumar et al. 2015). The data presented here reinforce the idea that McLTP 1 may exert anti-inflammatory and antioxidant therapeutic effects via the involvement of cytokines, such as TNF-α and IL-10. In contrast, McLTP 1 could not act over IL-1β, suggesting McLTP 1 selectivity across the tested cytokines.
It is known that inflammatory cells are directly linked to oxidative stress in gastric mucosa injuries, as the ROS generated by neutrophils promote lipid peroxidation (Raish et al. 2018). Given that McLTP 1 did not show any direct antioxidant activity, it can be reasoned that the antioxidant defenses elevated by McLTP 1 described in this study are partially or entirely due to reducing these inflammatory factors.
It is known that NO plays an essential role in maintaining gastric mucosal integrity (Antosova et al. 2012) and reduces acid secretion from gastric parietal cells (Lanas 2008). Previous administration of the NO synthesis inhibitor L-NAME reversed the action of McLTP 1 , suggesting that the gastroprotective activity of this protein may involve the modulation of NO pathways, leading to an increase in NO levels in the gastric mucosa. Our results also showed that McLTP 1 decreased the total acidity of gastric juice in the ligature pylorus model and suggested that the increased pH is indirectly related to NO and the production of GSH in the gastric mucosa.
Pretreatment of mice with ODQ, glibenclamide, indomethacin, or ruthenium red did not modify the gastroprotective activity of McLTP 1 . These results indicate that sGC, K ATP , PGs, and capsaicin-sensitive sensory receptors are not involved in the effects of McLTP 1 . Our findings are similar to those described by Pinheiro et al. (2013). Proteins isolated from the latex of Himatanthus drasticus showed gastroprotective effects against ethanol-induced gastric lesions. They mediated the restoration of GSH and nitrite levels in the mucosa and modulation of the NO/cGMP/KATP pathway.
LTPs are commonly associated with plant defense (Finkina et al. 2016), and they are underestimated from a therapeutic point of view. However, this study showed for the first time an LTP isolated from noni seeds with gastroprotective action and, at the same time, analgesic, antiinflammatory, and antipyretic effects (Campos et al. 2017). Our work demonstrates the therapeutic potential of plant LTPs, evidencing McLTP 1 as a strong candidate for developing a new drug of protein origin to treat gastrointestinal tract disorders.

Conclusion
M. citrifolia lipid transfer protein is the first lipid transfer protein with gastroprotective effects studied. Its pharmacological effects are related to modulating inflammation and ROS production. These modes of action of McLTP 1 are of significant clinical importance. However, standardized pharmacological safety studies will be necessary to ensure safe use.