Lectina da alga Caulerpa cupressoides: Caracterização parcial, eficácia e possíveis mecanismos envolvidos na hipernocicepcão inflamatória da articulacão temporomandibular de ratos

Abstract

Marine algae are sources of lectins, proteins that bind specifically and reversibly to mono- or oligosaccharides, making lectins valuable tools in many pharmacological and biotechnological processes, including pain control and inflammation. Temporomandibular disorders (TMD), in turn, are the second major cause of orofacial pain, commonly associated with chronic pain. Thus, the aim of this study was to partially characterize the lectin structure of the green seaweed Caulerpa cupressoides (CcL), investigating its efficacy and possible mechanisms in the inflammatory hypernociception of the temporomandibular joint of rats. The CcL was, then, isolated by extraction with Tris-HCl buffer 25 mM (pH 7.5), and isolated by ion exchange chromatography on DEAE-cellulose column. Subsequently, was characterized by circular dichroism (CD) spectroscopy. For the evaluation of inflammatory hypernociception, male Wistar rats received CcL or sterile saline, 30min before the intra-articular (i.art.) injection of Formalin (1.5%/50 uL), Serotonin (5-HT) (225 ug/50 uL), Capsaicin (1.5%/ 20 uL) or Carrageenan (100 μg/25 uL + 5-HT 75 μg/25 uL) into the left TMJ. Control groups received saline (50 μL; i.art.), indomethacin (5 mg/kg, s.c.) or morphine (5 mg/kg, s.c.). Nociceptive behavior was quantified for 45min, for the formalin group, and for 30min for 5-HT, Capsaicin and Carregenana + 5-HT groups. The periarticular tissue was excised to determining cytokine levels (TNF-α and IL-1β) by ELISA, protein determination of ICAM-1, CD55 and COX-2 by Western Blotting and the vascular permeability.To investigate the mechanism of action, the animals were pretreated with selective cannabinoid receptor antagonists AM281 or AM630 (3 mg/kg; ip), naloxone (opioid receptor antagonist, 15 μg/10 μL), aminoguanidine, ODQ, KT5823, glibenclamide and ZnPP-IX (NO/ GMPc/PKG/K+ATP and HO-1 pathways antagonists) prior to administration of CcL (10 mg/kg). The rotarod test evaluated the animal motor impairment. As result, the CD analysis revealed that the CcL is composed, predominantly, by β-sheet. Pretreatment with CcL (0. 1; 1 or 10 mg/kg) promoted a reduction (p<0.05) of inflammatory hypernociception induced by formalin in 63.5; 82 and 90%. CcL reduced (p<0.05) the plasma extravasation, levels TNF-α, IL-1β, ICAM-1 and COX-2 in the periarticular tissue when compared to the formalin group (p<0.05). CcL (10 mg / kg) was able to reduce (p<0.05) nociceptive behavior induced Capsaicin, suggesting that CcL has a direct effect on C-nociceptive fibers. CcL also reduced (p<0.05) the inflammatory hypernociception induced by Carrageenan and 5-HT, confirming its anti-inflammatory effect. In addition, the effects of CcL do not depend on the cannabinoid and opioid system, the HO-1 and NO/cGMP/PKG/K+ATP pathway. In addition, the CcL did not alter the motor coordination of the animals in the rotarod test. Therefore, CcL presented antinociceptive and anti-inflammatory effects on inflammatory hypernociception in TMJ, reducing the production of primary cytokines (TNF-α and IL-1β), adhesion molecule ICAM-1 and COX-2.