Numerous recent studies have considered plants as alternative sources for developing new drugs for treating the gastrointestinal tract's chronic diseases because of their high therapeutic potential and fewer 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, anti-inflammatory, gastric ulcer healing, and hepatoprotective. 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. It is also the first report of the gastroprotective activity of plant LTPs.
McLTP1 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 McLTP1 decreased the gastric lesion index and reduced histopathological alterations caused by ethanol, demonstrating that McLTP1 displays a gastroprotective effect. As observed for the anti-inflammatory activity already investigated for McLTP1 (Campos et al. 2016), there were no significant differences in the effects observed between the treatment of mice with 8 or 16 mg/kg. Besides, the gastroprotective action displayed by the McLTP1 treatment is not different from that presented by the positive control, NAC. Considering one of the most common side-effects of continuous use of anti-inflammatory drugs is the appearance of gastrointestinal injuries, the described gastroprotective effects performed by McLTP1 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, 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, McLTP1 is stable to the main enzymes of the gastrointestinal tract (trypsin, chymotrypsin, and pepsin) (Campos et al. 2016), which enables the application of McLTP1 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 infiltration of activated neutrophils (Halliwell, 1994; Asmari et al., 2016). The observed prevention of GSH depletion, combined with L-NAME partial reversion of McLTP1 gastroprotective effect, suggests a potential McLTP1 action mechanism to reduce oxidative stress.
Corroborating with the data presented by Campos et al. (2017), McLTP1 showed a significant reduction of 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 pro-inflammatory 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 the gastric mucosa, including the synthesis of the regulator TNF-α (Lee et al. 2017). The ratio of pro- (TNF-α) and anti-inflammatory (IL-10) cytokines may influence the degree of inflammation, an essential factor for gastric lesions (Kumar et al. 2015). The data presented here reinforces the idea that McLTP1 may exert anti-inflammatory and antioxidant therapeutic effects via the involvement of cytokines, such as TNF-α and IL-10. In contrast, McLTP1 could not act over IL-1β, suggesting McLTP1 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 promotes lipid peroxidation (Raish et al. 2018). Given that McLTP1 did not show any direct antioxidant activity, it can be reasoned that the antioxidant defenses elevated by McLTP1 described in this study are partially or entirely due to reducing these inflammatory factors.
It has been 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, reverted the action of McLTP1, suggesting that the gastroprotective activity of this protein may involve the modulation of NO pathways, leading to an increase in the NO levels on the gastric mucosa. Our results also showed that McLTP1 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 McLTP1. These results indicate that sGC, KATP, PGs, and capsaicin-sensitive sensory receptors are not involved in the effects of McLTP1. Our findings are similar to those described by Pinheiro et al. (2013). Proteins isolated from the latex of Himatanthus drasticus showed a gastroprotective effect 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, anti-inflammatory, and antipyretic effects (Campos et al. 2017). Our work demonstrates the therapeutic potential of plant LTPs, evidencing McLTP1 as a strong candidate for developing a new drug of protein origin to treat gastrointestinal tract disorders.