Analgesic activity and mechanism of action of a Beta vulgaris dye enriched in betalains in inflammatory models in mice.

Evidence demonstrates the pronounced anti-inflammatory activity of a beetroot (Beta vulgaris) dye enriched in betalains obtained using precipitation with ethanol. Herein, we expand upon our previous observations and demonstrate the analgesic and antioxidant effect of betalains. Betalains [10-1000 mg/kg; intraperitoneal route (i.p.)] diminished acetic acid- and PBQ-induced abdominal contortions, and the overt pain-like behaviour induced by complete Freund`s adjuvant (CFA) and formalin (intraplantar; i.pl.) injection. Moreover, betalains (100 mg/kg) administered by various routes [i.p. or subcutaneous (s.c.)] or as a post-treatment reduced carrageenin- or CFA-induced hyperalgesia. Mechanistically, betalains mitigated carrageenin-induced tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, superoxide anion levels, and lipid peroxidation. Betalains also stopped the depletion of reduced glutathione (GSH) levels and ferric reducing ability produced by carrageenin, as well as upregulated Nrf2 and Ho1 transcript expression in the plantar tissue of mice. Furthermore, betalains showed hydroxyl radical, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS+), and 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH•) scavenging ability and iron-chelating activity (bathophenantroline assay), and inhibited iron-independent and iron-dependent lipid peroxidation (LPO) in vitro. Finally, betalains-treated bone marrow-derived macrophages exhibited lower levels of cytokines (TNF-α and IL-1ß), and superoxide anion levels and nuclear factor kappa B (NF-κB) activation following lipopolysaccharide (LPS) stimulation. Therefore, this betalain-rich dye extracted using a novel precipitation approach presents prominent analgesic effect in varied models of pain by mechanisms targeting cytokines and oxidative stress.

[Ru(bpy)2(NO)SO3](PF6), a Nitric Oxide Donating Ruthenium Complex, Reduces Gout Arthritis in Mice.

Monosodium urate crystals (MSU) deposition induces articular inflammation known as gout. This disease is characterized by intense articular inflammation and pain by mechanisms involving the activation of the transcription factor NFκB and inflammasome resulting in the production of cytokines and oxidative stress. Despite evidence that MSU induces iNOS expression, there is no evidence on the effect of nitric oxide (NO) donors in gout. Thus, the present study evaluated the effect of the ruthenium complex donor of NO {[Ru(bpy)2(NO)SO3](PF6)} (complex I) in gout arthritis. Complex I inhibited in a dose-dependent manner MSU-induced hypersensitivity to mechanical stimulation, edema and leukocyte recruitment. These effects were corroborated by a decrease of histological inflammation score and recruitment of Lysm-eGFP+ cells. Mechanistically, complex I inhibited MSU-induced mechanical hypersensitivity and joint edema by triggering the cGMP/PKG/ATP-sensitive K (+) channels signaling pathway. Complex I inhibited MSU-induced oxidative stress and pro-inflammatory cytokine production in the knee joint. These data were supported by the observation that complex I inhibited MSU-induced NFκB activation, and IL-1ß expression and production. Complex I also inhibited MSU-induced activation of pro-IL-1ß processing. Concluding, the present data, to our knowledge, is the first evidence that a NO donating ruthenium complex inhibits MSU-induced articular inflammation and pain. Further, complex I targets the main physiopathological mechanisms of gout arthritis. Therefore, it is envisaged that complex I and other NO donors have therapeutic potential that deserves further investigation.

Diosmin reduces chronic constriction injury-induced neuropathic pain in mice.

Injury or dysfunction of somatosensory system induces a complex syndrome called neuropathic pain, which still needs adequate pharmacological control. The current pharmacological treatments were in part developed from natural compounds. Flavonoids are natural polyphenolic molecules presenting varied biological activities and low toxicity. The flavonoid diosmin is a safe compound with good tolerability and low toxicity. This study evaluated the antinociceptive effect of diosmin in the sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain model. Male Swiss mice were submitted to CCI and 7 days after, diosmin at 1 or 10 mg/kg was administrated intraperitoneally. Mechanical (electronic analgesimeter) and thermal (hot plate) hyperalgesia were evaluated 1-24 h after treatment. The role of the NO/cGMP/PKG/KATP channel signaling pathway in the analgesic effect of diosmin was evaluated using the pretreatment with L-NAME (an inhibitor of NOS), ODQ (an inhibitor of soluble guanylate cyclase), KT5823 (an inhibitor of PKG), or glibenclamide (an ATP-sensitive K+ channels blocker). Single treatment with diosmin inhibited in a dose-dependent manner CCI-induced mechanical and thermal hyperalgesia by activating the NO/cGMP/PKG/KATP channel signaling pathway and inhibiting spinal cord cytokine (Il-1ß and Il-33/St2) and glial cells activation (microglia - Iba-1, oligodendrocytes - Olig2) mRNA expression markers. Daily treatment during 7 days with diosmin inhibited CCI-induced mechanical and thermal hyperalgesia by inhibiting spinal cord cytokine (Il-1ß, Tnfα, and Il-33/St2) and glial cells activation (astrocytes - Gfap, Iba-1, and Olig2) markers mRNA expression. In conclusion, diosmin inhibits neuropathic spinal cord nociceptive mechanisms suggesting this flavonoid as a potential therapeutic molecule to reduce nerve lesion-induced neuropathic pain.

The nitroxyl donor, Angeli's salt, reduces chronic constriction injury-induced neuropathic pain.

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1ß (IL-1ß), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1ß, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1ß and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.

Anti-inflammatory activity of betalain-rich dye of Beta vulgaris: effect on edema, leukocyte recruitment, superoxide anion and cytokine production.

We have recently developed betalain-rich beetroot (Beta vulgaris) dye (betalain) to be used in food products. Betalain (30-300 mg/kg) intraperitoneal (i.p.) treatment diminished carrageenan (100 µg/paw)-induced paw edema and neutrophil migration to the paw skin tissue. Betalain (100 mg/kg) treatment by subcutaneous or per oral routes also inhibited the carrageenan-induced paw edema. Importantly, the post-treatment with betalain (100 mg/kg, i.p.) significantly inhibited carrageenan- and complete Freund's adjuvant (10 µl/paw)-induced paw edema. Betalain (100 mg/kg) also reduced carrageenan (500 µg/cavity)-induced recruitment of total leukocytes, including mononuclear cells and neutrophils, as well as increasing vascular permeability in the peritoneal cavity. Furthermore, betalain significantly reduced carrageenan-induced superoxide anion, tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß levels in the peritoneal fluid, as well as augmenting IL-10 levels. Therefore, this compound presents prominent anti-inflammatory effect on carrageenan-induced paw edema and peritonitis by reducing the production of superoxide anion and the cytokines TNF-α and IL-1ß, in addition to increasing IL-10 levels. These results suggest that betalain shows therapeutic potential that could be utilized in the treatment of inflammation-associated diseases.

Nitroxyl inhibits overt pain-like behavior in mice: role of cGMP/PKG/ATP-sensitive potassium channel signaling pathway.

BACKGROUND: Several lines of evidence have indicated that nitric oxide (NO) plays complex and diverse roles in modulation of pain/analgesia. However, the roles of charged and uncharged congeners of NO are less well understood. In the present study, the antinociceptive effect of the nitroxyl (HNO) donor, Angeli's salt (Na2N2O3; AS) was investigated in models of overt pain-like behavior. Moreover, whether the antinociceptive effect of nitroxyl was dependent on the activation of cGMP (cyclic guanosine monophosphate)/PKG (protein kinase G)/ATP-sensitive potassium channels was addressed. METHODS: The antinociceptive effect of AS was evaluated on phenyl-p-benzoquinone (PBQ)- and acetic acid-induced writhings and via the formalin test. In addition, pharmacological treatments targeting guanylate cyclase (ODQ), PKG (KT5923) and ATP-sensitive potassium channel (glybenclamide) were used. RESULTS: PBQ and acetic acid induced significant writhing responses over 20min. The nociceptive response in these models were significantly reduced in a dose-dependent manner by subcutaneous pre-treatment with AS. Furthermore, AS also inhibited both phases of the formalin test. Subsequently, the inhibitory effect of AS in writhing and flinching responses were prevented by ODQ, KT5823 and glybenclamide, although these inhibitors alone did not alter the writhing score. Furthermore, pretreatment with L-cysteine, an HNO scavenger, confirmed that the antinociceptive effect of AS depends on HNO. CONCLUSION: The present study demonstrates the efficacy of a nitroxyl donor and its analgesic mechanisms in overt pain-like behavior by activating the cGMP/PKG/ATP-sensitive potassium channel (K(+)) signaling pathway.

Anti-inflammatory effect of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene obtained from Combretum leprosum Mart & Eich in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from a medicinal plant named Combretum leprosum. In folk medicine, this plant is used to treat several diseases associated with inflammation and pain. We previously demonstrated that TTHL presents a significant antinociceptive effect, suggesting the involvement of the glutamatergic system. AIM OF THE STUDY: This study was designed to investigate the effect of TTHL on nociception and vascular permeability induced by acetic acid. We also evaluated the effect of TTHL on carrageenan-induced peritonitis and the levels of cytokines (interleukin 1-ß [IL-1ß], tumor necrosis factor α [TNF-α] and interleukin 10 [IL-10]) on peritoneal fluid. MATERIALS AND METHODS: TTHL was administered orally by intra-gastric gavage (i.g.) 60 min prior to experimentation. Abdominal contractions and vascular permeability were induced by an intraperitoneal (i.p.) injection of acetic acid (0.6%). We also investigated whether TTHL decreases carrageenan-induced peritonitis (750 µg/cavity) by measuring leukocyte migration and vascular permeability. In addition, we evaluated the effects of TTHL on TNF-α, IL-1ß and IL-10 release induced by carrageenan on peritoneal fluid. The levels of these cytokines were measured by ELISA. RESULTS: TTHL (0.01-10 mg/kg) administered by intra-gastric (i.g.) gavage inhibited (69±3%) acetic acid-induced abdominal constrictions, with an ID50 of 0.15 (0.03-0.8) mg/kg. TTHL (10mg/kg) also reduced the leukocyte infiltration induced by acetic acid, with an inhibition of 59±9 but had no effect on abdominal vascular permeability. In addition, indomethacin (10 mg/kg, i.p.) reduced the nociceptive behavior (92±1%), total leukocyte migration (29±3%) and capillary permeability (71±3%) induced by acetic acid. While the glucocorticoid dexamethasone (2 mg/kg, s.c.) reduced partially but significantly the nociception (31±1%), besides to promote a marked reduction on total leukocyte migration (60±2%) to the peritoneal cavity caused by acetic acid. In a model of peritonitis induced by carrageenan, TTHL also reduced total leukocyte migration, mainly neutrophils (inhibition of 84±3% and 85±2% at 30 mg/kg and 100 mg/kg, respectively). Likewise, dexamethasone (0.5 mg/kg, i.p.) resulted in an inhibition of 93±3%. Nevertheless, carrageenan-induced abdominal vascular permeability was reduced by dexamethasone but was not altered by TTHL. Furthermore, dexamethasone and TTHL significantly reduced the TNF-α and IL-1ß levels in peritoneal fluid, whereas the IL-10 levels were unchanged. CONCLUSIONS: Altogether, our data confirm the antinociceptive effect of TTHL and demonstrate its effect in inflammatory animal models, providing novel data about this compound, which could be useful as an anti-inflammatory drug.

Involvement of PGE2 and RANTES in Staphylococcus aureus-induced fever in rats.

This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 × 10(10) colony-forming units (CFU)/cavity, and body temperature (T(b)) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T(b), whereas the two higher doses promoted similar long-lasting and sustained T(b) increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T(b) increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 µg/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

Further analyses of mechanisms underlying the antinociceptive effect of the triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene in mice.

The present study investigated the mechanisms involved in the antinociception produced by the triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (TTHL) in mice. TTHL administered by intra-gastric (i.g.) gavage inhibited glutamate-induced nociception with an ID(50) of 19.0 (13.2-27.5) mg/kg. This action started 60 min (inhibition of: 59±6%) after i.g. administration and remained significant up to 6h (inhibition of 37±6%). Moreover, TTHL inhibited both phases of formalin induced pain. The antinociception of TTHL was reversed by the pre-administration of naloxone (1mg/kg; non-selective opioid receptor antagonist), CTOP (1mg/kg; selective µ-opioid receptor antagonist), nor-binaltorphimine (1mg/kg; selective κ-opioid receptor antagonist), naltrindol (3mg/kg; selective δ-opioid receptor antagonist), p-chlorophenylalanine methyl ester (100mg/kg for 4 consecutive days; inhibitor of serotonin synthesis), WAY100635 (0.5mg/kg; selective 5-HT(1A) receptor antagonist) and ketanserin (0.3mg/kg; selective 5-HT(2A) receptor antagonist) but not by L-arginine (600 mg/kg; nitric oxide precursor) or ondansetron (0.5mg/kg; 5-HT(3) receptor antagonist). Furthermore, the TTHL antinociception was prevented by intrathecal (i.t.) pre-treatment with pertussis toxin (0.5 µg/site; inactivator of G(i/o) protein), charybdotoxin (250 pg/site; blocker of large-conductance calcium-gated K(+) channels), tetraethylammonium (1 µg/site; blocker of voltage-gated K(+) channels) and glibenclamide (80 µg/site; blocker of ATP-gated K(+) channels) but not by apamin (50 ng/site; blocker of small-conductance calcium-gated K(+) channels). The antinociception of TTHL was not it associated with locomotor impairment or sedation. These results showed that TTHL presented a pronounced antinociceptive effect, which is dependent on opioid and serotonergic systems, G(i/o) protein activation and the opening of specific K(+) channels.