Inhibitors of angiotensin I converting enzyme potentiate fibromyalgia-like pain symptoms via kinin receptors in mice.

Fibromyalgia is a potentially disabling chronic disease, characterized by widespread pain and a range of comorbidities such as hypertension. Among the mechanisms involved in fibromyalgia-like pain symptoms are kinins and their B1 and B2 receptors. Moreover, angiotensin I converting enzyme (ACE) inhibitors, commonly used as antihypertensive drugs, can enhance pain by blocking the degradation of peptides such as substance P and bradykinin, besides enhancing kinin receptors signalling. We investigated the effect of ACE inhibitors on reserpine-induced fibromyalgia-like pain symptoms and the involvement of kinins in this effect in mice. Nociceptive parameters (mechanical and cold allodynia and overt nociception) were evaluated after ACE inhibitors administration in mice previously treated with reserpine. The role of kinin B1 and B2 receptors was investigated using pharmacological antagonism. Additionally, bradykinin levels, as well as the activity of ACE and kininase I, were measured in the sciatic nerve, spinal cord and cerebral cortex of the mice. The ACE inhibitors enalapril and captopril enhanced reserpine-induced mechanical allodynia, and this increase was prevented by kinin B1 and B2 receptor antagonists. Substance P and bradykinin caused overt nociception and increased mechanical allodynia in animals treated with reserpine. Reserpine plus ACE inhibitors increased bradykinin-related peptide levels and inhibited ACE activity in pain modulation structures. Since hypertension is a frequent comorbidity affecting fibromyalgia patients, hypertension treatment with ACE inhibitors in these patients should be reviewed once this could enhance fibromyalgia-like pain symptoms. Thus, the treatment of hypertensive patients with fibromyalgia could include other classes of antihypertensive drugs, different from ACE inhibitors.

Involvement of nNOS in the antinociceptive activity of melatonin in inflammatory pain at the level of sensory neurons.

OBJECTIVE: The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain. MATERIALS AND METHODS: In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry. RESULTS: In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05). CONCLUSIONS: Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

FAAH inhibition attenuates TLR3-mediated hyperthermia, nociceptive- and anxiety-like behaviour in female rats.

Aberrant activation of toll-like receptor (TLR)s results in persistent and prolonged neuroinflammation and has been implicated in the pathogenesis and exacerbation of psychiatric and neurodegenerative disorders. TLR3 coordinates the innate immune response to viral infection and recent data have demonstrated that inhibiting fatty acid amide hydrolase (FAAH), the enzyme that primarily metabolizes anandamide, modulates TLR3-mediated neuroinflammation. However, the physiological and behavioural consequences of such modulation are unknown. The present study examined the effect of URB597, a selective FAAH inhibitor, on neuroinflammation, physiological and behavioural alterations following administration of the TLR3 agonist and viral mimetic poly I:C to female rats. URB597 attenuated TLR3-mediated fever, mechanical and cold allodynia, and anxiety-like behaviour in the elevated plus maze and open field arena. There was no effect of URB597 on TLR3-mediated decreases in body weight and no effect in the sucrose preference or forced swim tests. URB597 attenuated the TLR3-mediated increase in the expression of CD11b and CD68, markers of microglia/macrophage activation. In summary, these data demonstrate that enhancing FAAH substrate levels suppresses TLR3-mediated microglia/macrophage activation and associated changes in fever, nociceptive responding and anxiety-related behaviour. These data provide further support for FAAH as a novel therapeutic target for neuroinflammatory disorders.

p,p'-Methoxyl-diphenyl diselenide-loaded polymeric nanocapsules as a novel approach to inflammatory pain treatment: Behavioral, biochemistry and molecular evidence.

The current study investigated the effect of organoselenium compound p,p'-methoxyl-diphenyl diselenide [(OMePhSe)2], free or incorporated into nanocapsules, on behavioral, biochemical and molecular alterations in an inflammatory pain model induced by complete Freund's adjuvant (CFA). Male Swiss mice received an intraplantar injection of CFA in the hindpaw and 24 h later they were treated via the intragastric route with a single (OMePhSe)2 administration, in its free form (dissolved in canola oil) or (OMePhSe)2 NC. The anti-hypernociceptive time- and dose-response curves were carried out using the von Frey hair test. Biochemical and histological parameters were determined in samples of injected paws and those of cerebral contralateral cortex were collected to determine immuno content of inflammatory proteins. Both (OMePhSe)2 forms reduced the hypernociception induced by CFA as well as attenuated the altered parameters of the inflammatory process in the paw (paw edema, myeloperoxidase and histological). However, the (OMePhSe)2 NC had a more prolonged anti-hypernociceptive action (7h) at a lower dose (10mg/kg) and superior effects on the paw alterations than the free compound form (4h and 25mg/kg). Furthermore, independent of the (OMePhSe)2 form, its administration decreased the MAPKs pathway activation (JNK;ERK1,2; p38) as well as iNOS, COX-2, Nf-κB and IL-1ß protein contents in the cerebral contralateral cortex that were increased by paw CFA injection. Therefore, (OMePhSe)2 NC had superior anti-inflammatory action, which possibly occurs by the inflammatory protein content modulation and also attenuates paw biochemical and histological inflammatory alterations induced by CFA injection.

Diacerein reduces joint damage, pain behavior and inhibits transient receptor potential vanilloid 1, matrix metalloproteinase and glial cells in rat spinal cord.

AIM: To investigate the antinociceptive, antiedematogenic and chondroprotective effects of diacerein (DIA) in a model of joint inflammation induced by complete Freund's adjuvant (CFA), as well as to investigate the involvement of metalloproteinase (MMP)-9, transient receptor potential vanilloid 1 (TRPV1) and glial cells in DIA's action mechanism. METHODS: Complete Freund's adjuvant was injected into the knee joint of male rats. We observed mechanical and cold hypersensitivity, vocalization and spontaneous pain-related behaviors, as well as edema of the knee. Tissue samples of the knee were stained with Cason`s technique and the thickness of the condilus cartilage was measured. Immunohistochemical analysis was performed on the spinal cord using anti-GFAP (glial fibrillary acidic protein), anti-MMP and anti-TRPV1 antibodies. Sections of the dorsal horns of the spinal cord were captured and an optical density was obtained. RESULTS: Complete Freund's adjuvant induced mechanical and thermal hypersensitivity, as well as joint edema and changes in the synovial membrane and cartilage. DIA (30 mg/kg, orally, daily) significantly inhibited mechanical (58 ± 10-87 ± 3%) and thermal (66 ± 12-87 ± 8%) hypersensitivity, vocalization (83 ± 5-41 ± 11%), spontaneous pain score, joint swelling (60 ± 6-40 ± 9%), as well as the histological changes induced by CFA. In addition, DIA inhibited astrocyte activation, and prevented the increase of MMP-9 and TRPV1 expression in the spinal cord of the animals subjected to CFA injections. CONCLUSIONS: In short, this study shows that DIA reduces joint damage and hypersensitivity associated with inflammation induced by CFA through the inhibition of astroglial activation and decreases the expression of TRPV1 and MMP-9 in the rat spinal cord.

Ligand-biased activation of extracellular signal-regulated kinase 1/2 leads to differences in opioid induced antinociception and tolerance.

Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates ß-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by administration of PTX or U0126, and was enhanced by administration of dyn-DN. Microinjection of DAMGO, but not fentanyl, into the vlPAG induced phosphorylation of ERK1/2, which was blocked by inhibiting receptor internalization with administration of dyn-DN, but not by inhibition of Gαi/o proteins. ERK1/2 inhibition also prevented the development and expression of tolerance to repeated DAMGO microinjections, but had no effect on fentanyl tolerance. These data reveal that ERK1/2 activation following MOPr internalization contributes to the antinociceptive effect of some (e.g., DAMGO), but not all opioids (e.g., fentanyl) despite the known similarities for these agonists to induce ß-arrestin recruitment and internalization.

Involvement of nuclear factor kappa B in the maintenance of persistent inflammatory hypernociception.

The pathophysiology of chronic inflammatory pain remains poorly understood. In this context, we developed an experimental model in which successive daily injection of prostaglandin E2 (PGE2) for 14days into rat hind paws produces a persistent state of hypernociception (i.e. decrease in mechanical nociceptive threshold). This state persists for more than 30days after discontinuing PGE2 injection. In the present study, we investigated the participation of nuclear factor kappa B (NF-κB), in the maintenance of this process. Mechanical hypernociception was evaluated using the electronic von Frey test. Activation of NF-κB signaling was measured through the determination of NF-κB p65 subunit translocation to the nucleus of dorsal root ganglion neurons (DRG) by immunofluorescence and western blotting. Herein, we detected an increase in NF-κB p65 subunit translocation to the nucleus of DRG neurons along with persistent inflammatory hypernociception compared with controls. Intrathecal treatment with either dexamethasone or PDTC (NF-κB activation inhibitor) after ending of the induction phase of the persistent inflammatory hypernociception, curtailed the hypernociception period as well as reducing NF-κB p65 subunit translocation. Treatment with antisense oligonucleotides against the NF-κB p65 subunit for 5 consecutive days also reduced persistent inflammatory hypernociception. Inhibition of PKA and PKCε reduced persistent inflammatory hypernociception, which was associated with inhibition of NF-κB p65 subunit translocation. Together these results suggest that peripheral activation of NF-κB by PKA and PKC in primary sensory neurons plays an important role in maintaining persistent inflammatory pain.

Spinal p38 activity and analgesic effect after low- and high-intensity electroacupuncture stimulation in a plantar incision rat model.

AIMS: Postoperative pain is a major problem. Electroacupuncture (EA) has been accepted as a useful and low-risk complementary therapy for post-operative pain. Animal studies indicate that surgical incision activates p38 MAPK in the spinal microglia, which critically contributes to post-incisional nociceptive development. How EA affects incision-induced p38 activation is important but yet to be fully elucidated. METHODS: Male adult rats received plantar incision (PI) at the right hind paw followed by 30-min EA of 4-Hz, one of two intensities (3 and 10mA), and at right ST36 (Zusanli) acupoint immediately after PI and for 3 successive days. EA analgesia was evaluated by von Frey fibers and Hargreaves' tests. Spinal p38 activation was examined by immunostaining. In separate groups, SB203580, a p38 inhibitor, was intrathecally injected alone or with EA to test the combining effect on nociception and spinal phospho-p38. KEY FINDINGS: EA of 10-mA significantly ameliorated mechanical allodynia, but 3-mA did not. None of them altered thermal hyperalgesia. Repeated EA could not inhibit phospho-p38 in the PI rats, contrarily, EA per se significantly induced phospho-p38 in the normal rats. Intrathecal SB203580 injection dose-dependently prevented PI-induced allodynia. Combination of low-dose SB203580 and 3-mA EA, which were ineffective individually, profoundly reduce post-PI allodynia. SIGNIFICANCE: We demonstrated that 10-mA EA exerts a significant inhibition against post-PI mechanical hypersensitivity via a p38-independent pathway. Importantly, co-treatment with low-dose p38 inhibitor and 3-mA EA can counteract spinal phospho-p38 to exert strong analgesic effect. Our finding suggests a novel strategy to improve EA analgesic quality.

Potent natural soluble epoxide hydrolase inhibitors from Pentadiplandra brazzeana baillon: synthesis, quantification, and measurement of biological activities in vitro and in vivo.

We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 µg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana.

Dual effects of Rho-kinase inhibitors on a rat model of inflammatory pain.

BACKGROUND: Rho-kinases (ROCKs), a family of small GTP-dependent enzymes, are involved in a range of pain models, and their inhibition typically leads to antinociceptive effects. OBJECTIVES: To study the effects of inhibiting ROCKs using two known inhibitors, Y27632 and HA1077 (fasudil), administered locally, on nociception and paw edema in rats. METHODS: A range of doses of Y27632 or HA1077 (2.5 µg to 1000 µg) were injected locally into rat paws alone or in combination with carrageenan, a known proinflammatory stimulus. Nociceptive responses to mechanical stimuli and increased paw volume, reflecting edema formation, were measured at 2 h and 3 h, using a Randall-Selitto apparatus and a hydroplethysmometer, respectively. RESULTS: Animals treated with either ROCK inhibitor showed biphasic nociceptive effects, with lower doses being associated with pronociceptive, and higher doses with antinociceptive responses. In contrast, a monophasic dose-dependent increase in edema was observed in the same animals. Local injection of 8-bromo-cyclic (c)GMP, an activator of the nitric oxide/cGMP/protein kinase G pathway, also produced biphasic effects on nociceptive responses in rat paws; however, low doses were antinociceptive and high doses were pronociceptive. Local administration of cytochalasin B, an inhibitor of actin polymerization and a downstream mediator of ROCK activity, reversed the antinociceptive effect of Y27632. CONCLUSIONS: The results of the present study suggest that ROCKs participate in the local mechanisms associated with nociception/antinociception and inflammation, with a possible involvement of the nitric oxide/cGMP/protein kinase G pathway. Also, drug effects following local administration may differ markedly from the effects following systemic administration. Finally, separate treatment of pain and edema may be needed to maximize clinical benefit in inflammatory pain.

Arcuate Src activation-induced phosphorylation of NR2B NMDA subunit contributes to inflammatory pain in rats.

The tyrosine kinases of Src family play an important role in the central sensitization following peripheral inflammation. However, whether the Src family in the arcuate nucleus (ARC) of mediobasal hypothalamus is involved in central sensitization remains unknown. The aim of this study was to investigate the role and mechanisms of tyrosine kinases of Src family in N-methyl-d-aspartate (NMDA) receptor activity in the ARC following peripheral inflammation. Peripheral inflammation was induced by unilateral injection of complete Freund's adjuvant (CFA) into rat hindpaw. The neuronal activities of the ARC were recorded using electrophysiological field recording from the in vitro mediobasal hypothalamic slices from control and CFA rats. Expression of total and phosphorylated Src and NR2B subunit protein was analyzed by Western blot and immuoprecipitation. Our results showed that CFA injection resulted in an increase in mechanical and thermal sensitivity, which was partially blocked by neonatal monosodium glutamate treatment. CFA injection also enhanced spontaneous firings of ARC neurons, which were reversed by the NMDA receptor NR2B subunit specific antagonist Ro25-6981 and by PP2, an Src family tyrosine kinase inhibitor. In addition, peripheral inflammation enhanced Src phosphorylation and NMDA receptor NR2B subunit phosphorylation without alteration of total NR2B subunit expression in the ARC. Peripheral inflammation also increased the association of NR2B protein with p-Src protein in the ARC. Administration of PP2 blocked the upregulation of NR2B phosphorylation induced by CFA injection. Taken together, our present results suggest that the arcuate Src activation-induced tyrosine phosphorylation of NR2B NMDA subunit may contribute to inflammatory pain.