Wnt signaling is involved in crotalphine-induced analgesia in a rat model of neuropathic pain.

The aberrant activation of Wnt/ß-catenin and atypical Wnt/Ryk signaling pathways in the spinal cord is critical for the development and maintenance of neuropathic pain. Crotalphine is a structural analog to a peptide first identified in Crotalus durissus terrificus snake venom, which induces antinociception by activating kappa-opioid and CB2 cannabinoid receptors. Consistent with previous data, we showed that the protein levels of the canonical Wnt/ß-catenin and the atypical Wnt/Ryk signaling pathways are increased in neuropathic rats. Importantly, the administration of crotalphine downregulates these protein levels, including its downstream cascades, such as TCF4 from the canonical pathway and NR2B glutamatergic receptor and Ca2+-dependent signals, via the Ryk receptor. The CB2 receptor antagonist, AM630, abolished the crotalphine-induced atypical Wnt/Ryk signaling pathway activation. However, the selective CB2 agonist affects both canonical and non-canonical Wnt signaling in the spinal cord. Next, we showed that crotalphine blocked hypersensitivity and significantly decreased the concentration of IL-1ɑ, IL-1ß, IL-6, IL-10, IL-18, TNF-ɑ, MIP-1ɑ and MIP-2 induced by intrathecal injection of exogenous Wnt-3a agonist. Taken together, our findings show that crotalphine induces analgesia in a neuropathic pain model by down-regulating the canonical Wnt/ß-catenin and the atypical Wnt/Ryk signaling pathways and, consequently controlling neuroinflammation. This effect is, at least in part, mediated by CB2 receptor activation. These results open a perspective for new approaches that can be used to target Wnt signaling in the context of chronic pain. PERSPECTIVE: Our work identified that crotalphine-induced activation of CB2 receptors plays a critical role in the impairment of Wnt signaling during neuropathic pain. This work suggests that drugs with opioid/cannabinoid activity may be a useful strategy to target Wnt signaling in the context of chronic pain.

Mitogen-Activated Protein Kinase Signaling Mediates Morphine Induced-Delayed Hyperalgesia.

The use of morphine, the standard opioid drug, is limited by its undesirable effects, such as tolerance, physical dependence, and hyperalgesia (increased pain sensitivity). Clinical and preclinical studies have reported development of hyperalgesia after prolonged opioid administration or after a single dose of intrathecal (i.t.) morphine in uninjured rats. However, whether a single standard systemic morphine dose is sufficient to decrease the nociceptive threshold in rats is unknown. Here, we showed that a single morphine subcutaneous injection induces analgesia followed by a long-lasting delayed hyperalgesia in uninjured and PGE2 sensitized rats. The i.t injection of extracellular signal-regulated kinase (ERK) inhibitor blocked morphine-induced analgesia, without interfering with the morphine-induced hyperalgesia. However, i.t. injection of SB20358, a p38 inhibitor and SP660125, a JNK inhibitor, decreased the morphine-induced hyperalgesia. Consistently with the behavioral data, Western Blot analysis showed that ERK is more phosphorylated 1 h after morphine, i.e., when the analgesia is detected. Moreover, phospho-p38 and phospho-JNK levels are upregulated 96 h after morphine injection, time that coincides with the hyperalgesic effect. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to cAMP-responsive element binding protein (CREB) attenuated morphine-induced hyperalgesia. Real-time polymerase chain reaction (RT-PCR) analysis showed that CREB downstream genes expressions were significantly up-regulated 96 h after morphine injection in spinal cord. Together, our data suggest that central ERK is involved in the analgesic and hyperalgesic effects of morphine while JNK, p38, and CREB are involved in the morphine-induced delayed hyperalgesia.

Frontline Science: Blood-circulating leukocytes fail to infiltrate the spinal cord parenchyma after spared nerve injury.

The development of neuropathic pain after peripheral nerve injury involves neuroimmune-glial interactions in the spinal cord. However, whether the development of neuropathic pain depends on the infiltration of peripheral immune cells, such as monocytes, into the spinal cord parenchyma after peripheral nerve damage remains unclear. Here, we used a combination of different techniques such as transgenic reporter mouse (Cx3cr1GFP/+ and Ccr2RFP/+ mice), bone marrow chimeric mice, and parabiosis to investigate this issue in spared nerve injury (SNI) model. Herein, we provided robust evidence that, although microglial cells are activated/proliferate at the dorsal horn of the spinal cord after SNI, peripheral hematopoietic cells (including monocytes) are not able to infiltrate into the spinal cord parenchyma. Furthermore, there was no evidence of CCR2 expression in intrinsic cells of the spinal cord. However, microglial cells activation/proliferation in the spinal cord and mechanical allodynia after SNI were reduced in Ccr2-deficient mice. These results suggest that blood-circulating leukocytes cells are not able to infiltrate the spinal cord parenchyma after distal peripheral nerve injury. Nevertheless, they indicate that CCR2-expressing cells might be indirectly regulating microglia activation/proliferation in the spinal cord after SNI. In conclusion, our study supports that CCR2 inhibition could be explored as an interventional approach to reduce microglia activation and consequently neuropathic pain development after peripheral nerve injury.

The Neuroprotective Effect of Doxycycline on Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) are a group of chronic, progressive disorders characterized by the gradual loss of neurons that affect specific regions of the brain, which leads to deficits in specific functions (e.g., memory, movement, cognition). The mechanism that drives chronic progression of NDs remains elusive. Among the proposed underlying pathophysiological mechanisms, aggregation and accumulation of misfolded proteins and neuroinflammation have been credited to contribute to extensive neural loss. Therapeutic agents that confer neuroprotection by downregulating these shared characteristics could therefore have beneficial effects on a wide range of NDs. In this regard, a commonly used antibiotic, doxycycline (Doxy), has been shown to reduce the progression and severity of disease in different experimental models of neurodegeneration by counteracting these common features. This review will focus on the effects reported for Doxy regarding its neuroprotective properties, the "off-target" effects, thereby supporting its evaluation as a new therapeutic approach for diseases associated with a neurodegeneration.

IL-27 Counteracts Neuropathic Pain Development Through Induction of IL-10.

Neuroimmune-glia interactions have been implicated in the development of neuropathic pain. Interleukin-27 (IL-27) is a cytokine that presents regulatory activity in inflammatory conditions of the central nervous system. Thus, we hypothesized that IL-27 would participate in the neuropathic pain process. Here, we found that neuropathic pain caused by peripheral nerve injury (spared nerve injury model; SNI), was enhanced in IL-27-deficient(-/-) mice, whereas nociceptive pain is similar to that of wild-type mice. SNI induced an increase in the expression of IL-27 and its receptor subunit (Wsx1) in the sensory ganglia and spinal cord. IL-27 receptor was expressed mainly in resident macrophage, microglia, and astrocytes of the sensory ganglia and spinal cord, respectively. Finally, we identify that the antinociceptive effect of IL-27 was not observed in IL-10-/- mice. These results provided evidence that IL-27 is a cytokine produced after peripheral nerve injury that counteracts neuropathic pain development through induction of the antinociceptive cytokine IL-10. In summary, our study unraveled the role of IL-27 as a regulatory cytokine that counteracts the development of neuropathic pain after peripheral nerve damage. In conclusion, they indicate that immunotherapies based on IL-27 could emerge as possible therapeutic approaches for the prevention of neuropathic pain development after peripheral nerve injury.

The NOD2 signaling in peripheral macrophages contributes to neuropathic pain development.

Neuropathic pain is one of the most important types of chronic pain. It is caused by neuronal damage. Clinical and experimental studies suggest a critical role for neuroimmune interactions in the development of neuropathic pain. In this article, we have shown that the cytoplasmic receptor Nod-like receptor-2, NOD2, and its adaptor-signaling molecule RIPK2 participate in the development of neuropathic pain after peripheral nerve injury (spared nerve injury model). The activation of NOD2 signaling in peripheral macrophage mediates the development of neuropathic pain through the production of pronociceptive cytokines (tumor necrosis factor and IL-1ß). This study found that peripheral nerve injury promoted a systemic increase in the NOD2 ligand. These results highlight a previously undetermined role for NOD2 signaling in the development of neuropathic pain, suggesting a new potential target for preventing neuropathic pain.

The spider toxin Phα1ß recombinant possesses strong analgesic activity.

The native Phα1ß - a Voltage-Gated Calcium Channel (VGCC) blocker - and its Recombinant Version - were both tested in rodent pain models with an intraplantar injections of capsaicin or formalin, a chronic constriction injury, and melanoma cancer related pain. The formalin nociceptive behaviour in the neurogenic phase was not affected by the toxin pre-treatments, while in the inflammatory phase, Phα1ß and the Recombinant form caused a significant reduction. The nociception that was triggered by capsaicin, an agonist of the TRPV1 vanilloid receptor, was totally blocked by 100 pmol/site, i.t. of Phα1ß or the recombinant version. For the neuropathic pain that was induced by a chronic constriction injury of the sciatic nerve, Phα1ß and its Recombinant reduced the allodynia that was induced by the CCI procedure in the rats and the hypersensitivity lasted for 4 h. Fourteen days after the inoculation of the B16-F10 melanoma cells in the mice, a marked hyperalgesia was induced in the melanoma cancer pain model. Phα1ß and the Recombinant form reduced the hyperalgesia with a full reversion at 100 pmol/site i.t. The inhibitory effects of the nociception that was induced by native Phα1ß and the Recombinant in the studied pain models were not statistically different and they developed with no side effects.

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways.

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson's disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1ß). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

Oleanane-type triterpenoid: an anti-inflammatory compound of the roots Arrabidaea brachypoda

AbstractArrabidaea brachypoda Bureau, Bignoniaceae, known as "cipó-una", is widely used in traditional medicine in Southeastern and Northeastern Brazil for kidney stones and painful joints. This study was aimed at evaluating the anti-inflammatory proprieties of the oleanane-type triterpenoid 3β-estearioxy-olean-12-ene isolated from the roots of A. brachypoda. Carrageenan-induced paw oedema, formalin test and hot plate test were used to investigate the antiinflammatory activity of 3β-estearioxy-olean-12-ene in animals. We observed that 3β-estearioxy-olean-12-ene at doses of 5, 10 and 15 mg/kg p.o. demonstrated anti-inflammatory effects, by reduced (p < 0.05) paw oedema induced by carrageenan and by decreased (p < 0.05) licking time caused by a subplantar injection of formalin. In conclusion, 3β-estearioxy-olean-12-ene, a triterpene isolated from the roots of A. brachypoda, demonstrate anti-inflammatory effect in different tests. Thus, it may be useful in the treatment of inflammatory disorders, which supports previous claims of its traditional use.

Anti-inflammatory and antinociceptive effects of the hydroethanolic extract of the flowers of Pyrostegia venusta in mice

Pyrostegia venusta (Ker Gawl.) Miers, Bignoniaceae, is native to the Brazilian Cerrado and popularly known as "cipó-de-são-joão". In Brazilian folk medicine, the flowers of P. venusta are used as a general tonic and a treatment for diarrhea, vitiligo, cough, and common infections and inflammatory diseases of the respiratory system. Nevertheless, there are still no studies on its possible anti-inflammatory and antinociceptive effects. The P. venusta hydroethanolic extract (PvHE) was used to evaluate the anti-inflammatory and analgesic effects in carrageenan-induced paw edema, peritonitis induced by lipopolysaccharide, acetic acid-induced writhing, and formalin-induced paw-licking tests in Swiss male mice. PvHE at doses of 30-300 mg/kg p.o. demonstrated anti-inflammatory effect. PvHE reduced paw edema induced by carrageenan and inhibited leukocyte recruitment into the peritoneal cavity. The extracts showed antinociceptive activity in acetic acid-induced writhing and formalin tests. Our results showed that the PvHE demonstrated anti-inflammatory and antinociceptive action in mice. All the anti-inflammatory actions obtained are also suggested to due the presence of acacetin-7-O-β-glucopyranoside.

7-epiclusianone, the natural prenylated benzophenone, inhibits superoxide anions in the neutrophil respiratory burst.

Despite defenses by polymorphonuclear neutrophils in the host against invading agents, overproduction of oxidant species by phagocytes can lead to damage in the surrounding tissues. Several benzophenones have been shown to possess anti-inflammatory properties. The effect of the natural benzophenone 7-epiclusianone isolated from leaves of Garcinia brasiliensis was investigated by using in vitro antioxidant and ex vivo anti-inflammatory assays, focusing on the neutrophil respiratory burst and on the biochemical pathways involved. The bioactive extract, 7-epiclusianone, showed low in vitro antioxidant activity as evaluated by the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay, the reducing power test, or the chelating power assay. However, the benzophenone displayed potent activity in the ex vivo model of the neutrophil respiratory burst, inhibiting the generation of superoxide anions in a dose-dependent manner. When the respiratory burst was triggered by N-formyl-methionyl-leucyl-phenylalanine, a chemotactic peptide, the 50% effective concentration (EC(50)) was 41.18 µg/10(7) cells. When phagocytes were stimulated directly through protein kinase C via phorbol, the EC(50) was 34.3 µg/10(6) cells. The results indicated that 7-epiclusianone was able to down-regulate inflammatory phagocyte superoxide anion release through a mechanism controlled by tyrosine protein phosphorylation and by a direct stimulation of protein kinase C. These findings could lead to new therapeutic approaches for inflammation management and the development of new drugs.

Antinociceptive and anti-inflammatory properties of 7-epiclusianone, a prenylated benzophenone from Garcinia brasiliensis.

7-Epiclusianone, a natural prenylated benzophenone, was extracted from Garcinia brasiliensis Planch. & Triana (Clusiaceae), a native plant commonly known as bacupari and used in traditional Brazilian medicine for the treatment of inflammatory diseases. As a result of the wide spectrum of biological activities attributed to polyisoprenylated benzophenones, the aim of this study was to evaluate the analgesic and anti-inflammatory effects of 7-epiclusianone using two animal models. Carrageenan-induced paw oedema and peritonitis were used to investigate the anti-inflammatory activity of 7-epiclusianone in rats. The acetic acid-induced writhing, formalin and hot-plate tests were used to investigate its antinociceptive activity in mice. At test doses of 5, 10 and 15 mg/kg p.o., 7-epiclusianone had an anti-inflammatory effect as demonstrated by the reduction of paw oedema induced by carrageenan and the inhibition of leukocyte recruitment into the peritoneal cavity. At the same doses, 7-epiclusianone inhibited nociception induced by an intraperitoneal injection of acetic acid, observed by the decrease in the number of writhing episodes. Additionally, 7-epiclusianone decreased licking time caused by a subplantar injection of formalin. Moreover, the hot plate test produced a significant increase in latency reaction, demonstrating an antinociceptive effect. The experimental data demonstrated that the polyisoprenylated benzophenone 7-epiclusianone has remarkable anti-inflammatory and antinociceptive activities.

Anti-inflammatory and antinociceptive effects of the stem bark of Byrsonima intermedia A. Juss.

ETHNOPHARMACOLOGICAL RELEVANCE: Byrsonima intermedia A. Juss. is popularly known as "murici pequeno" and is native to the Brazilian Cerrado. This species has been used as an antimicrobial, anti-hemorrhagic, anti-diarrheal and anti-inflammatory. Nevertheless, scientific information regarding Byrsonima intermedia is limited; there are no reports related to its possible anti inflammatory and antinociceptive effects. This study employed in vivo inflammatory and nociceptive models to evaluate the scientific basis for the traditional use of Byrsonima intermedia. MATERIALS AND METHODS: Carrageenan-induced paw edema, peritonitis and fibrovascular tissue growth induced by s.c. cotton pellet implantation tests were used to investigate the anti-inflammatory activity of Byrsonima intermedia aqueous extract (BiAE) in rats. Mechanical nociceptive paw, formalin and hot plate tests were used to evaluate the antinociceptive activity in mice. High-performance liquid chromatography (HPLC), phytochemistry screening and determination of total phenolics and flavonoids were used to determine the chemical profile of the BiAE. RESULTS: BiAE at test doses of 30-300 mg/kg p.o. clearly demonstrated anti-inflammatory effects by reduced carrageenan-induced paw edema, by inhibited leukocyte recruitment into the peritoneal cavity and, in the model of chronic inflammation, by using the cotton pellet-induced fibrovascular tissue growth in rats. The extracts at test doses of 30-300 mg/kg p.o. clearly demonstrated antinociceptive activity in all tests. Administration of the opioid receptor antagonist naloxone completely inhibited the antinociceptive effect induced by BiAE (100 mg/kg). CONCLUSION: BiAE markedly exhibits anti-inflammatory action in rats and antinociceptive activity in mice. Thus, it may be useful in the treatment of inflammatory hyperalgesic disorders, which supports previous claims of its traditional use.

Anti-inflammatory and antinociceptive effects of Arrabidaea brachypoda (DC.) Bureau roots.

AIM OF THE STUDY: Arrabidaea brachypoda (DC.) Bureau has been used to relieve general pain, painful joints and kidney stones in Brazilian folk medicine. Nevertheless, scientific information regarding this species is scarce; there are no reports related to its possible analgesic and anti-inflammatory effects. This study was aimed at evaluating the traditional use of Arrabidaea brachypoda root using in vivo inflammatory and nociceptive models. MATERIALS AND METHODS: Carrageenan-induced paw edema, peritonitis and fibrovascular tissue growth induced by s.c. cotton pellet implantation were used to investigate the anti-inflammatory activity of Arrabidaea brachypoda roots ethanolic extract (AbEE) in rats. Formalin and acetic acid-induced writhing tests were used to investigate the antinociceptive activity in mice. High-performance liquid chromatography (HPLC) was used to determine the fingerprint chromatogram of AbEE. RESULTS: The AbEE at test doses of 30-300 mg/kg p.o. demonstrated anti-inflammatory effects. AbEE reduced paw edema induced by carrageenan, inhibited leukocyte recruitment into the peritoneal cavity and, in the model of chronic inflammation using the cotton pellet-induced fibrovascular tissue growth in rats, significantly inhibited the formation of granulomatous tissue. The extracts at test doses of 30-300 mg/kg p.o. clearly demonstrated antinociceptive activity, except during the first phase of the formalin test. The presence of quercetin and phenolic compounds in the extract Arrabidaea brachypoda was confirmed using HPLC. CONCLUSION: Arrabidaea brachypoda ethanol extract markedly demonstrated anti-inflammatory action in rats and antinociceptive activity in mice, which supports the previous claims of traditional use.

Anti-inflammatory and antinociceptive effects of Garcinia brasiliensis.

AIM OF THE STUDY: In Brazilian folk medicine, the leaves of Garcinia brasiliensis are used to treat tumors, inflammation of the urinary tract and arthritis as well as to relieve pain. Nevertheless, scientific information regarding Garcinia brasiliensis is limited; there are no reports related to its possible anti-inflammatory and antinociceptive effects. This study employed in vivo inflammatory and nociceptive models to evaluate the scientific basis for the traditional use of Garcinia brasiliensis. MATERIALS AND METHODS: Carrageenan-induced paw edema, peritonitis and fibrovascular tissue growth induced by s.c. cotton pellet implantation were used to investigate the anti-inflammatory activity of Garcinia brasiliensis ethanolic extract (GbEE) in rats. Formalin and acetic acid-induced writhing tests were used to investigate the antinociceptive activity in mice. RESULTS: GbEE at test doses of 30-300 mg/kg p.o. clearly demonstrated anti-inflammatory effects by reduced paw edema induced by carrageenan, inhibited leukocyte recruitment into the peritoneal cavity, and in the model of chronic inflammation using the cotton pellet-induced fibrovascular tissue growth in rats, the GbEE significantly inhibited the formation of granulomatous tissue. The extracts at test doses of 30-300 mg/kg, p.o., clearly demonstrated antinociceptive activity, except for the first phase of the formalin test. CONCLUSION: GbEE markedly demonstrated anti-inflammatory action in rats and antinociceptive activity in mice, which supports previous claims of the traditional use of species of the Garcinia genus for inflammation and pain.