Mecanismos de ação do efeito antinociceptivo e avaliação da toxicidade in vitro de McLTP1, uma proteína isolada de sementes de Morinda citrifolia L.

Abstract

Pain is a critical condition, especially in chronic diseases, and new therapeutic approaches have been demanded. Among these approaches, plant proteins have been highlighted as promising molecules with the potential to be employed in human health. Previous reports have shown that a lipid transfer protein isolated from Morinda citrifolia L. seeds, named McLTP1 (9.4 kDa), displays antinociceptive activity in mice. Additionally, McLTP1 was not able to induce toxicity in mice, even after daily administration (28 days) in tests performed according to guidelines of the Organization for Economic Cooperation and Development (OECD). This work aimed to investigate the mechanisms underlying the antinociceptive action of McLTP1 in pain models and to study its in vitro preclinical toxicity. McLTP1 was purified from noni seeds as previously described. Antinociceptive mechanisms of McLTP1 were studied in different mice models after intraperitoneal or oral administration (8 mg/kg). All experiments were approved by CEUAUFC (n° 65-2015). In this study, thermal stimulus-induced nociception was inhibited by McLTP1 and antagonized by naloxone (2 mg/kg, i.p.), demonstrating a supraspinal effect via the opioid system. In addition, pretreatment with L-NAME (20 mg/kg) or glibenclamide (2 mg/kg) in the acetic acid-induced abdominal writhing test partially reversed the McLTP1 effect (20.50%, i.p.; 33.33%, p.o. and 35.90%, i.p., 47.21% p.o., respectively), indicating the NO/cGMP/K+ATP pathway activation. McLTP1 also reversed the nociceptive response in the glutamate- (3.7 ng/paw) and capsaicin-induced (1.6 µg/paw) paw licking test (78.30%, i.p.; 67.19%, p.o. and 45.45%, i.p.; 30.90%, p.o., respectively), suggesting involvement glutamatergic system and vanilloid receptors. Moreover, the antinociceptive effect of protein involves modulating inflammation by suppressing the carrageenan- (300 µg/paw) or PGE2- induced (0.1 nmol/paw) hypernociception. Interestingly, McLTP1 explores similar antinociceptive mechanisms by both routes (oral or intraperitoneal). In preclinical in vitro toxicity tests, McLTP1 (20, 40, 80, 160, 320 µg/mL) did not display any alterations in cell viability or patterns of DNA damage in cultured human hepatocellular carcinoma (HepG2). Overall, knowledge of the antinociceptive mechanisms of McLTP1 opens the prospect of exploring this protein in other disease models, expanding its therapeutic actions, besides providing an unprecedented contribution to the use of LTPs group.