Atividade antinociceptiva dos ácidos epoxieicosatrienoicos (EETs) no modelo de nocicepção periférica em camundongos

Abstract

INTRODUCTION: Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. The major roles of EETs include modulation of blood pressure and inflammatory cascades, although there are several other physiological functions associated with these substances, including their controversial effects on pain. OBJECTIVES: To verify the peripheral antinociceptive effect of EETs and the mechanisms involved in this event, as well as their participation in peripheral endogenous pain control. METHODS: The mechanical paw pressure test was used, and hyperalgesia was induced by: (i) intraplantar injection of prostaglandin E2 (PGE2) to evaluate the effect of the administration of exogenous EETs; and (ii) intraplantar injection of carrageenan (CG) or ciatic constriction injury (CCI) to assess endogenous EETs. The EETs (5,6-, 8,9-, 11,12- and 14,15-EET) and the drugs of the analgesic systems evaluated were given intraplantarly in male mice (n = 5-8). Statistical analysis was performed using ANOVA followed by Bonferroni post-test. RESULTS: EETs showed a dose-dependent peripheral antinociceptive effect. This effect, except for 8,9-EET, was reversed by Naloxone, a non-selective opioid receptor antagonist, and by Nor-binaltorphimine, a selective κ-opioid receptor antagonist, but not by CTOP and Naltrindole, selective antagonists of µ and δ opioid receptors, respectively. Furthermore, an inhibitor of the enzyme that degrades endogenous opioids, Bestatin, potentiated the antinociceptive effect of low doses of EETs and, through the immunofluorescence technique, the involvement of dynorphin was suggested. The peripheral antinociception induced by EETs was antagonized by AM251, a selective CB1 receptor antagonist, but not by AM630, a selective CB2 receptor antagonist. Besides, an inhibitor of the anandamide (AEA) cellular reuptake (VDM11), as well as an inhibitor of the enzyme responsible for AEA hydrolysis (MAFP), potentiated the peripheral antinociceptive effect of a low dose of EETs. However, an inhibitor of the enzyme responsible for 2-arachidonoylglycerol hydrolysis (JZL184) did not potentiate this effect. Additionally, LNOArg, a non-selective inhibitor of the enzymes nitric oxide synthase (NOS), as well as selective inhibitors of inducible (LNIL) and endothelial (LNIO), but not neuronal (LNPA) NOS, reversed EETs antinociception. Furthermore, an inhibitor of the soluble guanylate cyclase enzyme (ODQ) reduced this effect and a selective inhibitor of GMPc-specific phosphodiesterase (Zaprinast) potentiated the antinociceptive effects of low doses of EETs, suggesting that GMPc is involved in this process. Antinociception triggered by EETS does not seem to be related to opening potassium or chloride channels, as assessed by selective blockers for the different channels: glibenclamide, tetraethylammonium, dequalinium, paxillin and niflumic acid. Regarding the involvement of endogenous EETs in peripheral pain control, it was observed that a non-selective EETs receptor antagonist (14,15-EEZE) and an inhibitor of cytochrome P450 epoxygenase enzymes (MS-PPOH) were responsible for the potentiation of nociception induced by CG or CCI. Furthermore, an inhibitor of the soluble epoxide hydrolase enzyme (TPPU) as well as knockout animals for this enzyme (EHs KO) reduced nociception in inflammatory (CG) and neuropathic (CCI) pain models. In addition to the nociceptive behavior by the mechanical paw pressure test, gait analysis was performed using the CatWalk in C57BL/6 mice submitted to CCI. It was observed that operated animals (WT and EHs KO) had smaller paw print area, reduced mean intensity of paw contact and single stance, and increase in swing time compared to their sham counterparts. The operated animals EHs KO and WT showed significant differences between them in the single stance and swing time. Using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique, we observed an increase in EETs levels in the mice foot pads only in inflammatory pain, in the group treated with TPPU compared to the group treated with vehicle. CONCLUSIONS: Our results provide evidence that EETs have a peripheral antinociceptive effect, whose mechanism of action involves the opioid and cannabinoid systems, and the activation of the NO/GMPc pathway for 5,6-,11,12- and 14,15-EET; while for 8,9-EET, only the last two mechanisms seem to be involved. Furthermore, our findings suggest a modulation of inflammatory and neuropathic pain by endogenous EETs as a control mechanism for the transmission of nociceptive impulses in the periphery, and in neuropathic pain, more AGEPs may participate in this peripheral pain modulation.