Lipoxin A4 inhibits microglial activation and reduces neuroinflammation and neuropathic pain after spinal cord hemisection.

BACKGROUND: Spinal cord injury (SCI) is a severe neurological disorder with many disabling consequences, including persistent neuropathic pain, which develops in about 40 % of SCI patients and is induced and sustained by excessive and uncontrolled spinal neuroinflammation. Here, we have evaluated the effects of lipoxin A4 (LXA4), a member of a unique class of endogenous lipid mediators with both anti-inflammatory and analgesic properties, on spinal neuroinflammation and chronic pain in an experimental model of SCI. METHODS: Spinal hemisection at T10 was carried out in adult male CD1 mice and Wistar rats. To test if LXA4 can reduce neuroinflammation and neuropathic pain, each animal received two intrathecal injections of LXA4 (300 pmol) or vehicle at 4 and 24 h after SCI. Sensitivity to mechanical stimulation of the hind paws was evaluated using von Frey monofilaments, and neuroinflammation was tested by measuring the mRNA and/or protein expression levels of glial markers and cytokines in the spinal cord samples after SCI. Also, microglia cultures prepared from murine cortical tissue were used to assess the direct effects of LXA4 on microglial activation and release of pro-inflammatory TNF-α. RESULTS: LXA4 treatment caused significant reductions in the intensity of mechanical pain hypersensitivity and spinal expression levels of microglial markers and pro-inflammatory cytokines induced by SCI, when compared to rodents receiving control vehicle injections. Notably, the increased expressions of the microglial marker IBA-1 and of the pro-inflammatory cytokine TNF-α were the most affected by the LXA4 treatment. Furthermore, cortical microglial cultures expressed ALX/FPR2 receptors for LXA4 and displayed potentially anti-inflammatory responses upon challenge with LXA4. CONCLUSIONS: Collectively, our results suggest that LXA4 can effectively modulate microglial activation and TNF-α release through ALX/FPR2 receptors, ultimately reducing neuropathic pain in rodents after spinal cord hemisection. The dual anti-inflammatory and analgesic properties of LXA4, allied to its endogenous nature and safety profile, may render this lipid mediator as new therapeutic approach for treating various neuroinflammatory disorders and chronic pain with only limited side effects.

Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor.

Allosteric modulation of G-protein-coupled receptors represents a key goal of current pharmacology. In particular, endogenous allosteric modulators might represent important targets of interventions aimed at maximizing therapeutic efficacy and reducing side effects of drugs. Here we show that the anti-inflammatory lipid lipoxin A(4) is an endogenous allosteric enhancer of the CB(1) cannabinoid receptor. Lipoxin A(4) was detected in brain tissues, did not compete for the orthosteric binding site of the CB(1) receptor (vs. (3)H-SR141716A), and did not alter endocannabinoid metabolism (as opposed to URB597 and MAFP), but it enhanced affinity of anandamide at the CB1 receptor, thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition, lipoxin A(4) displayed a CB(1) receptor-dependent protective effect against ß-amyloid (1-40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB(1) receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice.

Resolvins of the D series are generated from docosahexaenoic acid, which are enriched in fish oils and are believed to exert beneficial roles on diverse inflammatory disorders, including inflammatory bowel disease (IBD). In this study, we investigated the anti-inflammatory effects of the aspirin-triggered resolvin D1 (AT-RvD1), its precursor (17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) and resolvin D2 (RvD2) in dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Our results showed that the systemic treatment with AT-RvD1, RvD2, or 17R-HDHA in a nanogram range greatly improved disease activity index, body weight loss, colonic damage, and polymorphonuclear infiltration in both colitis experimental models. Moreover, these treatments reduced colonic cytokine levels for TNF-α, IL-1ß, MIP-2, and CXCL1/KC, as well as mRNA expression of NF-κB and the adhesion molecules VCAM-1, ICAM-1, and LFA-1. Furthermore, AT-RvD1, but not RvD2 or 17R-HDHA, depended on lipoxin A4 receptor (ALX) activation to inhibit IL-6, MCP-1, IFN-γ, and TNF-α levels in bone marrow-derived macrophages stimulated with LPS. Similarly, ALX blockade reversed the beneficial effects of AT-RvD1 in DSS-induced colitis. To our knowledge, our findings showed for the first time the anti-inflammatory effects of resolvins of the D series and precursor 17R-HDHA in preventing experimental colitis. We also demonstrated the relevant role exerted by ALX activation on proresolving action of AT-RvD1. Moreover, AT-RvD1 showed a higher potency than 17R-HDHA and RvD2 in preventing DSS-induced colitis. The results suggest that these lipid mediators possess a greater efficacy when compared with other currently used IBD therapies, such as monoclonal anti-TNF, and have the potential to be used for treating IBD.

ß-Caryophyllene inhibits dextran sulfate sodium-induced colitis in mice through CB2 receptor activation and PPARγ pathway.

Cannabinoid receptor 2 (CB2) activation is suggested to trigger the peroxisome proliferator-activated receptor-γ (PPARγ) pathway, and agonists of both receptors improve colitis. Recently, the plant metabolite (E)-ß-caryophyllene (BCP) was shown to bind to and activate CB2. In this study, we examined the anti-inflammatory effect of BCP in dextran sulfate sodium (DSS)-induced colitis and analyzed whether this effect was mediated by CB2 and PPARγ. Oral treatment with BCP reduced disease activity, colonic macro- and microscopic damage, myeloperoxidase and N-acetylglucosaminidase activities, and levels and mRNA expression of colonic tumor necrosis factor-α, IL-1ß, interferon-γ, and keratinocyte-derived chemokine. BCP treatment also inhibited the activation of extracellular signal-regulated kinase 1/2, nuclear factor κB, IκB-kinase α/ß, cAMP response element binding and the expression of caspase-3 and Ki-67. Moreover, BCP enhanced IL-4 levels and forkhead box P3 mRNA expression in the mouse colon and reduced cytokine levels (tumor necrosis factor-α, keratinocyte-derived chemokine, and macrophage-inflammatory protein-2) in a culture of macrophages stimulated with lipopolysaccharide. The use of the CB2 antagonist AM630 or the PPARγ antagonist GW9662 significantly reversed the protective effect of BCP. Confirming our results, AM630 reversed the beneficial effect of BCP on pro-inflammatory cytokine expression in IEC-6 cells. These results demonstrate that the anti-inflammatory effect of BCP involves CB2 and the PPARγ pathway and suggest BCP as a possible therapy for the treatment of inflammatory bowel disease.

TRPA1 receptor modulation attenuates bladder overactivity induced by spinal cord injury.

The ankyrin-repeat transient receptor potential 1 (TRPA1) has been implicated in pathological conditions of the bladder, but its role in overactive bladder (OAB) following spinal cord injury (SCI) remains unknown. In this study, using a rat SCI model, we assessed the relevance of TRPA1 in OAB induced by SCI. SCI resulted in tissue damage, inflammation, and changes in bladder contractility and in voiding behavior. Moreover, SCI caused upregulation of TRPA1 protein and mRNA levels, in bladder and in dorsal root ganglion (DRG; L6-S1), but not in corresponding segment of spinal cord. Alteration in bladder contractility following SCI was evidenced by enhancement in cinnamaldehyde-, capsaicin-, or carbachol-induced bladder contraction as well as in its spontaneous phasic activity. Of relevance to voiding behavior, SCI induced increase in the number of nonvoiding contractions (NVCs), an important parameter associated with the OAB etiology, besides alterations in other urodynamic parameters. HC-030031 (TRPA1 antagonist) treatment decreased the number and the amplitude of NVCs while the TRPA1 antisense oligodeoxynucleotide (AS-ODN) treatment normalized the spontaneous phasic activity, decreased the cinnamaldehyde-induced bladder contraction and the number of NVCs in SCI rats. In addition, the cinnamaldehyde-induced bladder contraction was reduced by exposure of the bladder preparations to HC-030031. The efficacy of TRPA1 AS-ODN treatment was confirmed by means of the reduction of TRPA1 expression in the DRG, in the corresponding segment of the spinal cord and in the bladder, specifically in detrusor muscle. The present data show that the TRPA1 activation and upregulation seem to exert an important role in OAB following SCI.

Endothelins implicated in referred mechanical hyperalgesia associated with colitis induced by TNBS in mice.

This study evaluated the contribution of endothelins to changes in sensitivity to mechanical stimulation of the lower abdomen and hind paw associated with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The frequency of withdrawal responses to 10 consecutive applications of von Frey probes to the lower abdomen (0.07 g) or hind paw (0.4 g) was assessed in male BALB/c mice before and after intracolonic TNBS injection (0.5 mg in 100 microL of 35% ethanol). TNBS (0.5 mg) induced referred mechanical hyperalgesia in the abdomen (response frequencies at 24 h: saline 11.0% +/- 3.1%, TNBS 48.0% +/- 6.9%) and hind paw (frequencies at 24 h: saline 12.5% +/- 4.7%, TNBS 47.1% +/- 7.1%) lasting up to 72 and 48 h, respectively. Mice receiving 1.0 or 1.5 mg TNBS assumed hunch-backed postures and became immobile during abdominal mechanical stimulation, suggestive of excessive ongoing pain. Atrasentan (ETA receptor antagonist; 10 and 30 mg/kg, i.v.) given 24 h after TNBS abolished hind paw and abdominal mechanical hyperalgesia for 2-3 h. A-192621 (ETB receptor antagonist; 20 mg/kg, i.v.) attenuated abdominal mechanical hyperalgesia at the 3 h time point only. Thus, endothelins contribute importantly to abdominal and hind paw referred mechanical hyperalgesia during TNBS-induced colitis mainly through ETA receptor-signaled mechanisms.

The selective nonpeptide CXCR2 antagonist SB225002 ameliorates acute experimental colitis in mice.

Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR2 is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR2 antagonist SB225002 ameliorates experimental colitis, which was induced in mice by 2,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (24 h), mice were treated with SB225002. At later time-points, up to 72 h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB225002 significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB225002 reduced neutrophil influx, MPO activity, IL-1beta, MIP-2, and keratinocyte-derived chemokine (KC) levels and the expression of vascular endothelial growth factor, inducible NO synthase, and cyclooxygenase-2 proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB225002. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR2 consistently reduced TNBS-induced colitis, suggesting that the use of SB225002 is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders.

EPA- and DHA-derived resolvins' actions in inflammatory bowel disease.

Inflammatory bowel diseases are chronic diseases divided into two major forms, ulcerative colitis and Crohn's disease, which are both associated with a chronic inflammatory condition of the gastrointestinal tract. Recent studies have shown that the resolution of inflammatory conditions is a biosynthetically active process where new pro-resolution lipid mediators derived from omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), such as E- and D-series resolvins, protectins, and macrophage mediator in resolving inflammation (maresins), have potent anti-inflammatory activity and serve as specialised mediators that play an important role in the resolution of inflammation. Recent studies have also shown the role of resolvins in referred hyperalgesia associated with different inflammatory processes, such as the visceral pain caused by inflammatory bowel disease. There are many reports describing the principal effects of EPA- and DHA-derived mediators in experimental models of inflammatory bowel diseases. This review focuses on the recent studies on the important role played by pro-resolution lipid mediators in controlling the inflammatory process associated with inflammatory bowel diseases.

Neuroprotective effects of lipoxin A4 in central nervous system pathologies.

Many diseases of the central nervous system are characterized and sometimes worsened by an intense inflammatory response in the affected tissue. It is now accepted that resolution of inflammation is an active process mediated by a group of mediators that can act in synchrony to switch the phenotype of cells, from a proinflammatory one to another that favors the return to homeostasis. This new genus of proresolving mediators includes resolvins, protectins, maresins, and lipoxins, the first to be discovered. In this short review we provide an overview of current knowledge into the cellular and molecular interactions of lipoxins in diseases of the central nervous system in which they appear to facilitate the resolution of inflammation, thus exerting a neuroprotective action.

Preventive and therapeutic oral administration of the pentacyclic triterpene α,ß-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: the relevance of cannabinoid system.

The pentacyclic triterpene α,ß-amyrin has been previously reported as an effective compound in the treatment of several inflammatory conditions. Recent evidence indicates that α,ß-amyrin displayed its effects through interaction with the cannabinoid pathway. We assessed the anti-inflammatory effects of the α,ß-amyrin in the dextran sulfate sodium (DSS)-induced colitis in mice and investigated whether its effects were associated with the interaction with the cannabinoid system. Our results showed that the oral preventive or therapeutic treatment with α,ß-amyrin significantly reduced disease activity, body weight loss, colonic damage, as well as colonic myeloperoxidase and N-acetylglucosaminidase activities. Moreover, α,ß-amyrin decreases the colonic pro-inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and keratinocyte-derived chemokine (CXCL1/KC), while up-regulating the IL-4 levels. Additionally, we also observed that the α,ß-amyrin caused a significant reduction of the adhesion molecules mRNA expression for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet cell adhesion molecule 1 (PCAM-1), ß(2)-integrin and protein expression for proliferation marker Ki67, the macrophage molecule CD68 and for adhesion molecule P-selectin. Interestingly, our results also showed that the cannabinoid receptor 1 (CB(1)), but not CB(2), pharmacological blockade significantly reversed the beneficial effects of α,ß-amyrin in DSS-induced colitis. Besides, our data demonstrated that mRNA expression for both the endocannabinoid hydrolase monoglyceride lipase 1 (MGL1) and fatty acid amide hydrolase (FAAH) were significantly reduced in the colon of α,ß-amyrin-treated mice. Altogether, these results suggest that the α,ß-amyrin might possess potential therapeutic interest for the treatment of IBD, and also provide new insights for the underlying mechanisms.

Oral administration of the flavonoid myricitrin prevents dextran sulfate sodium-induced experimental colitis in mice through modulation of PI3K/Akt signaling pathway.

SCOPE: We investigated the protective effect of the flavonoid myricitrin in dextran sulfate sodium (DSS) induced colitis as promising candidate for the treatment of ulcerative colitis which is considered an important worldwide public health problem. METHODS AND RESULTS: Male CD1 mice were provided with a solution of filtered water containing 3% w/v DSS ad libitum over a 5-day period followed by 2 days with normal drinking water. Myricitrin was administered orally, once a day, at the doses 1, 3, and 10 mg/kg of body weight. At the end of day 7th, the animals were euthanized and the colonic tissue was collected to be analyzed by RT-PCR, immunohistochemistry and Western blot. Our results showed that oral treatment with myricitrin exerts consistent anti-inflammatory action in DSS-induced acute colitis in mice by the inhibition of the Akt/phosphatidylinositol-3 kinase-dependent phosphorylation. Consequently, the phosphorylation of mitogen-activated protein kinases (MAPK) p38, extracellular signal-regulated protein kinase (ERK1/2), and c-Jun N-terminal kinase and of the nuclear factor B (NF-κB) was reduced and prevented an increase in the cytokines/chemokines levels. CONCLUSION: Together, these data reveal that the anti-inflammatory effect of myricitrin in DSS-induced colitis in mice is likely associated with its ability to prevent the activation of upstream kinases, such as phosphatidylinositol-3 kinase-dependent Akt, NF-κB, and mitogen-activated protein kinase.

Euphol prevents experimental autoimmune encephalomyelitis in mice: evidence for the underlying mechanisms.

Multiple sclerosis (MS) is a severe chronic T cell-mediated autoimmune inflammatory disease of the central nervous system (CNS), the existing therapy of which is only partially effective and is associated with undesirable side effects. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. However there are no reports about the effects and mechanisms of euphol in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here we report the effects and the underlying mechanisms of action of euphol in EAE. Euphol (1-10mg/kg) was administered orally at different time-points of EAE. Immunological and inflammatory responses were evaluated by real-time PCR, Western blot and flow cytometry assays. We provide evidence that euphol significantly attenuates neurological signs of EAE. These beneficial effects of euphol seem to be associated with the down-regulation of mRNA and protein expression of some pro-inflammatory mediators such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the CNS. Furthermore, in vitro, euphol consistently inhibited the T cell-mediated immune response including the production of T(H)1 and T(H)17 cytokines in spleen cells of untreated EAE animals. Likewise, oral euphol treatment inhibited the infiltration of T(H)17 myelin-specific cells into the CNS through the adhesion molecule, lymphocyte function-associated antigen 1 (LFA-1). Our findings reveal that oral administration of euphol consistently reduces and limits the severity and development of EAE. Therefore, euphol might represent a potential molecule of interest for the treatment of MS and other T(H)17 cell-mediated inflammatory diseases.

Evaluation of chemical mediators and cellular response during acute and chronic gut inflammatory response induced by dextran sodium sulfate in mice.

Inflammatory bowel disease (IBD) affects millions of people worldwide but its pathophysiology remains unclear. Therefore, experimental models of colitis have contributed crucially for the understanding of IBD, and also in the investigations for effective therapies. Herein we investigated the kinetics of inflammatory mediator production and cell infiltration during acute and chronic dextran sodium sulfate (DSS)-induced colitis. The induction phases with DSS were characterized by severe disease activity with massive colonic polymorphonuclear infiltration and increased levels of tumor necrosis factor-α (TNF-α), keratinocyte-derived chemokine (CXCL1/KC), interleukin (IL)-17 and vascular adhesion molecule-1 (VCAM-1). Interestingly, in the recovery periods, we found marked increase of anti-inflammatory mediators IL-10, IL-4, transforming growth factor-ß (TGF-ß) and cyclooxygenase 2 (COX-2) that seems be essential for the resolution of intestinal inflammation. Furthermore, nuclear factor κB (NFκB) and regulatory T cell marker forkhead box P3 (FoxP3) were increased gradually during experimental colitis, demonstrating a discrepant profile response and evident immune disbalance in the chronic phase of intestinal mucosal inflammation. Taken together, these results provide valuable information for studies on DSS-induced colitis and especially for the identification of biomarkers that predict disease course and possible therapeutic interventions.

Euphol, a tetracyclic triterpene produces antinociceptive effects in inflammatory and neuropathic pain: the involvement of cannabinoid system.

Persistent pains associated with inflammatory and neuropathic states are prevalent and debilitating diseases, which still remain without a safe and adequate treatment. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. Here, we assessed the effects and the underlying mechanisms of action of euphol in preventing inflammatory and neuropathic pain. Oral treatment with euphol (30 and 100 mg/kg) reduced carrageenan-induced mechanical hyperalgesia. Likewise, euphol given through the spinal and intracerebroventricular routes prevented mechanical hyperalgesia induced by carrageenan. Euphol consistently blocked the mechanical hyperalgesia induced by complete Freund's adjuvant, keratinocyte-derived chemokine, interleukin-1ß, interleukin-6 and tumor necrosis factor-alpha associated with the suppression of myeloperoxidase activity in the mouse paw. Oral treatment with euphol was also effective in preventing the mechanical nociceptive response induced by ligation of the sciatic nerve and also significantly reduced the levels and mRNA of cytokines/chemokines in both paw and spinal cord tissues following i.pl. injection of complete Freund's adjuvant. In addition, the pre-treatment with either CB1R or CB2R antagonists, as well as the knockdown gene of the CB1R and CB2R, significantly reversed the antinociceptive effect of euphol. Interestingly, even in higher doses, euphol did not cause any relevant action in the central nervous system. Considering that few drugs are currently available for the treatment of chronic pain states, the present results provided evidence that euphol constitutes a promising molecule for the management of inflammatory and neuropathic pain states.

Activation of cannabinoid receptors by the pentacyclic triterpene α,ß-amyrin inhibits inflammatory and neuropathic persistent pain in mice.

In this study, we report that α,ß-amyrin, a plant-derived pentacyclic triterpene, reduced persistent inflammatory and neuropathic hyperalgesia in mice by a direct activation of the CB(1) and CB(2) cannabinoid receptors (CB(1)R and CB(2)R). The oral treatment with α,ß-amyrin (30 mg/kg) significantly reduced mechanical and thermal hyperalgesia and inflammation induced by complete Freund's adjuvant (CFA) and by partial sciatic nerve ligation (PSNL). The pretreatment with either CB(1)R or CB(2)R antagonists and the knockdown gene of the receptors significantly reverted the antinociceptive effect of α,ß-amyrin. Of note, binding studies showed that α,ß-amyrin directly bound with very high affinity to CB(1)R (K(i)=0.133 nM) and with a lower affinity to CB(2)R (K(i)=1989 nM). Interestingly, α,ß-amyrin, ACEA (CB(1)R agonist), or JWH-133 (CB(2)R agonist), at doses that caused antinociception, failed to provoke any behavioral disturbance, as measured in the tetrad assay. In addition, α,ß-amyrin largely decreased interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), keratinocyte-derived chemokine (KC) and interleukin 6 (IL-6) levels, and myeloperoxidase activity. Likewise, α,ß-amyrin prevented the activation of the transcriptional factors: nuclear factor κB (NF-κB) and cyclic adenosine monophosphate response element binding (CREB) and the expression of cyclooxygenase 2 in mice footpads and spinal cords. The present results demonstrated that α,ß-amyrin exhibits long-lasting antinociceptive and anti-inflammatory properties in 2 models of persistent nociception via activation of cannabinoid receptors and by inhibiting the production of cytokines and expression of NF-κB, CREB and cyclooxygenase 2.