A reverse translational approach reveals the protective roles of Mangifera indica in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders often characterized by a dysregulation of T cells, specifically T helper (Th) 1, 17 and T regulatory (Treg) repertoire. Increasing evidence demonstrates that dietary polyphenols from Mangifera indica L. extract (MIE, commonly known as mango) mitigate intestinal inflammation and splenic Th17/Treg ratio. In this study, we aimed to dissect the immunomodulatory and anti-inflammatory properties of MIE using a reverse translational approach, by initially using blood from an adult IBD inception cohort and then investigating the mechanism of action in a preclinical model of T cell-driven colitis. Of clinical relevance, MIE modulates TNF-α and IL-17 levels in LPS spiked sera from IBD patients as an ex vivo model of intestinal barrier breakdown. Preclinically, therapeutic administration of MIE significantly reduced colitis severity, pathogenic T-cell intestinal infiltrate and intestinal pro-inflammatory mediators (IL-6, IL-17A, TNF-α, IL-2, IL-22). Moreover, MIE reversed colitis-induced gut permeability and restored tight junction functionality and intestinal metabolites. Mechanistic insights revealed MIE had direct effects on blood vascular endothelial cells, blocking TNF-α/IFN-γ-induced up-regulation of COX-2 and the DP2 receptors. Collectively, we demonstrate the therapeutic potential of MIE to reverse the immunological perturbance during the onset of colitis and dampen the systemic inflammatory response, paving the way for its clinical use as nutraceutical and/or functional food.

New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function.

OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

Interleukin-17 (IL-17) triggers systemic inflammation, peripheral vascular dysfunction, and related prothrombotic state in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most prevalent forms of neurodegenerative disorders. Previously, we have shown that in vivo administration of an IL-17 neutralizing antibody (IL-17Ab) rescues amyloid-ß-induced neuro-inflammation and memory impairment, demonstrating the pivotal role of IL-17 in AD-derived cognitive deficit. Recently, AD has been recognized as a more intriguing pathology affecting vascular networks and platelet function. However, not much is known about peripheral vascular inflammation and how pro-inflammatory circulating cells/mediators could affect peripheral vessels' function. This study aimed to evaluate whether IL-17Ab treatment could also impact peripheral AD features, such as systemic inflammation, peripheral vascular dysfunction, and related pro-thrombotic state in a non-genetic mouse model of AD. Mice were injected intracerebroventricularly with Aß1-42 peptide (3 µg/3 µl). To evaluate the systemic/peripheral protective profile of IL-17Ab, we used an intranasal administration of IL-17Ab (1 µg/10 µl) at 5, 12, and 19 days after Aß1-42 injection. Circulating Th17/Treg cells and related cyto-chemokines, haematological parameters, vascular/endothelial reactivity, platelets and coagulation function in mice were evaluated. IL-17Ab treatment ameliorates the systemic/peripheral inflammation, immunological perturbance, vascular/endothelial impairment and pro-thrombotic state, suggesting a key role for this cytokine in fostering inflammatory processes that characterize the multifaced aspects of AD.

Galectin-9: A novel promoter of atherosclerosis progression.

BACKGROUND AND AIMS: Atherosclerosis is widely accepted to be an inflammatory disease driven by lipid accumulation and leukocyte recruitment. More recently, galectins, a family of ß-galactoside binding proteins, have been shown to play a role in leukocyte recruitment among other immunomodulatory functions. Galectin (Gal) -9, a tandem repeat type galectin expressed by the endothelium in inflammatory environments, has been proposed to promote leukocyte recruitment. However, the role of Gal-9 in the context of monocyte recruitment remains elusive. METHODS AND RESULTS: Here, we characterise the immunomodulatory role of Gal-9 in context of atherosclerosis. We show that ApoE-/-Gal-9-/- mice have a significantly reduced aortic plaque burden compared to their ApoE-/- littermate controls after 12 weeks of high fat diet. RNA sequencing data from two independent studies reveal Lgals9 expression in leukocyte clusters isolated from murine atherosclerotic plaques. Additionally, soluble Gal-9 protein induces monocyte activation and a pro-inflammatory phenotype in macrophages. Furthermore, we show that immobilised recombinant Gal-9 acts as capture and adhesion molecule for CD14+ monocytes in a ß2-integrin and glycan dependent manner, while adhesion of monocytes to stimulated endothelium is reduced when Gal-9 is knocked down. Gal-9 also facilitates enhanced recruitment of leukocytes from peripheral arterial disease (PAD) patients compared to healthy young and aged controls. We further characterise the endothelium as source of circulating Gal-9, which is increased in plasma of PAD patients compared to healthy controls. CONCLUSIONS: These results highlight a pathological role for Gal-9 as promoter of monocyte recruitment and atherosclerotic plaque progression, making it a novel target in the prevention of plaque formation and progression.

Analysis of rheumatoid- vs psoriatic arthritis synovial fluid reveals differential macrophage (CCR2) and T helper subsets (STAT3/4 and FOXP3) activation.

OBJECTIVE: In psoriatic arthritis (PsA) and rheumatoid arthritis (RA), inflammatory responses are characterized by increased production of pro-inflammatory molecules secreted by various immune cells. The main objectives of our study were: i) to measure levels of pro- and anti-inflammatory cyto-chemokines and soluble factors expressed in both PsA and RA SF; ii) to characterize the phenotype of infiltrated leuko-lymphocytes and; iii) to identify specific synovial biomarkers for both diseases. Notably, Synovial Fluid (SF) samples obtained from PsA and RA populations were compared with SF samples collected from clinically active osteoarthritis (OA) joints. METHODS: SF samples were collected from clinically active knee arthritis of PsA, RA and OA patients and assayed for cyto-chemokines profile and macrophage and T helper subsets markers and transcriptional factors by Elisa Spot and western blot. RESULTS: our study revealed that modulation of CCL-2, G-CSF, IL-1ß and TNF-α is peculiar and specific to RA synovial fluid, whereas we detected more significant levels of ICAM-1, IL-2, IL-6, IL-17A, C5a and CXCL-9/12 in PsA compared to RA patients. We also found that CCR2 expression appeared to be significantly upmodulated in PsA and, even more, in RA group, as well as the expression of specific Th and Treg transcriptional factors as STAT3/4 and FOXP3. CONCLUSION: Even though this study has several limitations, we identified a heterogenous scenario of peculiar molecular pathway and soluble mediators' production that characterize PsA and RA SF that may be useful in understanding the complex pattern of macrophages and lymphocytes infiltration in both pathologies and, potentially, pave the way for personalized precision therapies.

Anti-inflammatory and immunomodulatory activity of Mangifera indica L. reveals the modulation of COX-2/mPGES-1 axis and Th17/Treg ratio.

In the context of inflammation and immunity, there are fragmented and observational studies relating to the pharmacological activity of Mangifera indica L. and its main active component, mangiferin. Therefore, we aimed to analyze the potential beneficial effects of this plant extract (MIE, 90 % in mangiferin) in a mouse model of gouty arthritis, to allow the evaluation of cellular immune phenotypes and the biochemical mechanism/s beyond MIE activity. Gouty arthritis was induced by the intra-articular administration of MSU crystals (200 µg 20 µl-1), whereas MIE (0.1-10 mg kg-1) or corresponding vehicle (DMSO/saline 1:3) were orally administrated concomitantly with MSU (time 0), 6 and 12 h after the stimulus. Thereafter, knee joint score and oedema were evaluated in addition to western blot analysis for COX-2/mPGES-1 axis. Moreover, the analysis of pro/anti-inflammatory cyto-chemokines coupled with the phenotyping of the cellular infiltrate was performed. Treatment with MIE revealed a dose-dependent reduction in joint inflammatory scores with maximal inhibition observed at 10 mg kg-1. MIE significantly reduced leukocyte infiltration and activation and the expression of different pro-inflammatory cyto-chemokines in inflamed tissues. Furthermore, biochemical analysis revealed that MIE modulated COX-2/mPGES-1 and mPGDS-1/PPARγ pathways. Flow cytometry analysis also highlighted a prominent modulation of inflammatory monocytes (CD11b+/CD115+/LY6Chi), and Treg cells (CD4+/CD25+/FOXP3+) after MIE treatment. Collectively, the results of this study demonstrate a novel function of MIE to positively affect the local and systemic inflammatory/immunological perturbance in the onset and progression of gouty arthritis.

Galectin-9 supports primary T cell transendothelial migration in a glycan and integrin dependent manner.

Adaptive immunity relies on the efficient recruitment of T cells from the blood into peripheral tissues. However, the current understanding of factor(s) coordinating these events is incomplete. Previous studies on galectin-9 (Gal-9), have proposed a functionally significant role for this lectin in mediating leukocyte adhesion and transmigration. However, very little is known about its function in T cell migration. Here, we have investigated the role of the Gal-9 on the migration behaviour of both human primary CD4+ and CD8+ T cells. Our data indicate that Gal-9 supports both CD4+ and CD8+ T cell adhesion and transmigration in a glycan dependent manner, inducing L-selectin shedding and upregulation of LFA-1 and CXCR4 expression. Additionally, when immobilized, Gal-9 promoted capture and firm adhesion of T cells under flow, in a glycan and integrin-dependent manner. Using an in vivo model, dorsal air pouch, we found that Gal-9 deficient mice display impaired leukocyte trafficking, with a reduction in pro-inflammatory cytokines/chemokines generated locally. Furthermore, we also demonstrate that Gal-9 inhibits the chemotactic function of CXCL12 through direct binding. In conclusion, our study characterises, for the first time, the capture, adhesion, and migration behaviour of CD4+ and CD8+ T cells to immobilised /endothelial presented Gal-9, under static and physiological flow conditions. We also demonstrate the differential binding characteristics of Gal-9 to T cell subtypes, which could be of potential therapeutic significance, particularly in the treatment of inflammatory-based diseases, given Gal-9 ability to promote apoptosis in pathogenic T cell subsets.

Phosphodiesterases S-sulfhydration contributes to human skeletal muscle function.

The increase in intracellular calcium is influenced by cyclic nucleotides (cAMP and cGMP) content, which rating is governed by phosphodiesterases (PDEs) activity.Despite it has been demonstrated a beneficial effect of PDEs inhibitors in different pathological conditions involving SKM, not much is known on the role exerted by cAMP-cGMP/PDEs axis in human SKM contractility. Here, we show that Ssulfhydration of PDEs modulates human SKM contractility in physiological and pathological conditions. Having previously demonstrated that, in the rare human syndrome Malignant Hyperthermia (MH), there is an overproduction of hydrogen sulfide (H2S) within SKM contributing to hyper-contractility, here we have used MH negative diagnosed biopsies (MHN) as healthy SKM, and MH susceptible diagnosed biopsies (MHS) as a pathological model of SKM hypercontractility. The study has been performed on MHS and MHN human biopsies after diagnosis has been made and on primary SKM cells derived from both MHN and MHS biopsies. Our data demonstrate that in normal conditions PDEs are S-sulfhydrated in both quadriceps' biopsies and primary SKM cells. This post translational modification (PTM) negatively regulates PDEs activity with consequent increase of both cAMP and cGMP levels. In hypercontractile biopsies, due to an excessive H2S content, there is an enhanced Ssulfhydration of PDEs that further increases cyclic nucleotides levels contributing to SKM hyper-contractility. Thus, the identification of a new endogenous PTM modulating PDEs activity represents an advancement in SKM physiopathology understanding.

In Silico, In Vitro, and In Vivo Analysis of Tanshinone IIA and Cryptotanshinone from Salvia miltiorrhiza as Modulators of Cyclooxygenase-2/mPGES-1/Endothelial Prostaglandin EP3 Pathway.

Tanshinone IIA (TIIA) and cryptotanshinone (CRY) from Salvia miltiorrhiza Bunge were investigated for their inhibitory activity against the cyclooxygenase-2 (COX-2)/microsomal prostaglandin E synthase-1 (mPGES-1)/endothelial prostaglandin 3 (EP3) pathway using in silico, in vitro, in vivo, and ex vivo assays. From the analysis of the docking poses, both diterpenoids were able to interact significantly with COX-2, 5-lipoxygenase (5-LO), platelet-activating factor receptor (PAFR), and mPGES-1. This evidence was further corroborated by data obtained from a cell-free assay, where CRY displayed a significant inhibitory potency against mPGES-1 (IC50 = 1.9 ± 0.4 µM) and 5-LO (IC50 = 7.1 µM), while TIIA showed no relevant inhibition of these targets. This was consistent with their activity to increase mice bleeding time (CRY: 2.44 ± 0.13 min, p ≤ 0.001; TIIA: 2.07 ± 0.17 min p ≤ 0.01) and with the capability to modulate mouse clot retraction (CRY: 0.048 ± 0.011 g, p ≤ 0.01; TIIA: 0.068 ± 0.009 g, p ≤ 0.05). For the first time, our results show that TIIA and, in particular, CRY are able to interact significantly with the key proteins involved not only in the onset of inflammation but also in platelet activity (and hyper-reactivity). Future preclinical and clinical investigations, together with this evidence, could provide the scientific basis to consider these compounds as an alternative therapeutic approach for thrombotic- and thromboembolic-based diseases.

IL-17-induced inflammation modulates the mPGES-1/PPAR-γ pathway in monocytes/macrophages.

BACKGROUND AND PURPOSE: Recent biochemical and pharmacological studies have reported that in several tissues and cell types, microsomal PGE2 synthase (mPGES) and PPAR-γ expression are modulated by a variety of inflammatory factors and stimuli. Considering that very little is known about the biological effects promoted by IL-17 in the context of mPGES-1/PPAR-γ modulation, we sought to investigate the contribution of this unique cytokine on this integrated pathway during the onset of inflammation. EXPERIMENTAL APPROACH: We evaluated effects of PF 9184 (mPGES-1 inhibitor) and troglitazone (PPAR-γ agonist) in vitro, using the mouse macrophage cell line J774A.1. In vivo, the dorsal air pouch model in CD1 mice was used, and inflammatory infiltrates were analysed by flow cytometry. Locally produced cyto-chemokines and PGs were assessed using elisa assays. Western blots were also employed to determine the activity of various enzymes involved in downstream signalling pathways. KEY RESULTS: PF 9184 and troglitazone, in a time- and dose-dependent manner, modulated leukocyte infiltration, myeloperoxidase activity, and the expression of COX-2/mPGES-1, NF-кB/IкB-α, and mPTGDS-1/PPAR-γ, induced by IL-17. Moreover, both PF 9184 and troglitazone modulated PG (PGE2 , PGD2 , and PGJ2 ) production, the expression of different pro-inflammatory cyto-chemokines, and the recruitment of inflammatory monocytes, in response to IL-17. CONCLUSIONS AND IMPLICATIONS: Our data suggest that IL-17 may constitute a specific modulator of inflammatory monocytes during later phases of the inflammatory response. The results of this study show, for the first time, that the IL-17/mPGES-1/PPAR-γ pathway could represent a potential therapeutic target for inflammatory-based and immune-mediated diseases. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio.

Gout is caused by depositing monosodium urate (MSU) crystals within the articular area. The infiltration of neutrophils and monocytes drives the initial inflammatory response followed by lymphocytes. Interestingly, emerging evidence supports the view that in situ imbalance of T helper 17 cells (Th17)/regulatory T cells (Treg) impacts the subsequent damage to target tissues. Galectin-9 (Gal-9) is a modulator of innate and adaptive immunity with both pro- and anti-inflammatory functions, dependent upon its expression and cellular location. However, the specific cellular and molecular mechanisms by which Gal-9 modulates the inflammatory response in the onset and progression of gouty arthritis has yet to be elucidated. In this study, we sought to comprehensively characterise the functional role of exogenous Gal-9 in an in vivo model of MSU crystal-induced gouty inflammation by monitoring in situ neutrophils, monocytes and Th17/Treg recruited phenotypes and related cyto-chemokines profile. Treatment with Gal-9 revealed a dose-dependent reduction in joint inflammation scores, knee joint oedema and expression of different pro-inflammatory cyto-chemokines. Furthermore, flow cytometry analysis highlighted a significant modulation of infiltrating inflammatory monocytes (CD11b+/CD115+/LY6-Chi) and Th17 (CD4+/IL-17+)/Treg (CD4+/CD25+/FOXP-3+) cells following Gal-9 treatment. Collectively the results presented in this study indicate that the administration of Gal-9 could provide a new therapeutic strategy for preventing tissue damage in gouty arthritic inflammation and, possibly, in other inflammatory-based diseases.

Safety and Efficacy Evaluation In Vivo of a Cationic Nucleolipid Nanosystem for the Nanodelivery of a Ruthenium(III) Complex with Superior Anticancer Bioactivity.

Selectivity and efficacy towards target cancer cells, as well as biocompatibility, are current challenges of advanced chemotherapy powering the discovery of unconventional metal-based drugs and the search for novel therapeutic approaches. Among second-generation metal-based chemotherapeutics, ruthenium complexes have demonstrated promising anticancer activity coupled to minimal toxicity profiles and peculiar biochemical features. In this context, our research group has recently focused on a bioactive Ru(III) complex-named AziRu-incorporated into a suite of ad hoc designed nucleolipid nanosystems to ensure its chemical stability and delivery. Indeed, we proved that the structure and properties of decorated nucleolipids can have a major impact on the anticancer activity of the ruthenium core. Moving in this direction, here we describe a preclinical study performed by a mouse xenograft model of human breast cancer to establish safety and efficacy in vivo of a cationic Ru(III)-based nucleolipid formulation, named HoThyRu/DOTAP, endowed with superior antiproliferative activity. The results show a remarkable reduction in tumour with no evidence of animal suffering. Blood diagnostics, as well as biochemical analysis in both acute and chronic treated animal groups, demonstrate a good tolerability profile at the therapeutic regimen, with 100% of mice survival and no indication of toxicity. In addition, ruthenium plasma concentration analysis and tissue bioaccumulation were determined via appropriate sampling and ICP-MS analysis. Overall, this study supports both the efficacy of our Ru-containing nanosystem versus a human breast cancer model and its safety in vivo through well-tolerated animal biological responses, envisaging a possible forthcoming use in clinical trials.

Interleukin-17A (IL-17A): A silent amplifier of COVID-19.

One of the hallmarks of COVID-19 is the cytokine storm that provokes primarily pneumonia followed by systemic inflammation. Emerging evidence has identified a potential link between elevated interleukin-17A (IL-17A) levels and disease severity and progression. Considering that per se, IL-17A can activate several inflammatory pathways, it is plausible to hypothesize an involvement of this cytokine in COVID-19 clinical outcomes. Thus, IL-17A could represent a marker of disease progression and/or a target to develop therapeutic strategies. This hypothesis paper aims to propose this "unique" cytokine as a silent amplifier of the COVID-19 immune response and (potentially) related therapy.

Supplementation with ribonucleotide-based ingredient (Ribodiet®) lessens oxidative stress, brain inflammation, and amyloid pathology in a murine model of Alzheimer.

Alzheimer's disease (AD) is the most common type of dementia worldwide, characterized by the deposition of neurofibrillary tangles and amyloid-ß (Aß) peptides in the brain. Additionally, increasing evidence demonstrates that a neuroinflammatory state and oxidative stress, iron-dependent, play a crucial role in the onset and disease progression. Besides conventional therapies, the use of natural-based products represents a future medical option for AD treatment and/or prevention. We, therefore, evaluated the effects of a ribonucleotides-based ingredient (Ribodiet®) in a non-genetic mouse model of AD. To this aim, mice were injected intracerebroventricularly (i.c.v.) with Aß1-42 peptide (3 µg/3 µl) and after with Ribodiet® (0.1-10 mg/mouse) orally (p.o.) 3 times weekly for 21 days following the induction of experimental AD. The mnemonic and cognitive decline was then evaluated, and, successively, we have assessed ex vivo the modulation of different cyto-chemokines on mice brain homogenates. Finally, the level of GFAP, S100ß, and iron-related metabolic proteins were monitored as markers of reactive gliosis, neuro-inflammation, and oxidative stress. Results indicate that Ribodiet® lessens oxidative stress, brain inflammation, and amyloid pathology via modulation of iron-related metabolic proteins paving the way for its rationale use for the treatment of AD and other age-related diseases.

Present Status and Future Trends of Natural-Derived Compounds Targeting T Helper (Th) 17 and Microsomal Prostaglandin E Synthase-1 (mPGES-1) as Alternative Therapies for Autoimmune and Inflammatory-Based Diseases.

Several natural-based compounds and products are reported to possess anti-inflammatory and immunomodulatory activity both in vitro and in vivo. The primary target for these activities is the inhibition of eicosanoid-generating enzymes, including phospholipase A2, cyclooxygenases (COXs), and lipoxygenases, leading to reduced prostanoids and leukotrienes. Other mechanisms include modulation of protein kinases and activation of transcriptases. However, only a limited number of studies and reviews highlight the potential modulation of the coupling enzymatic pathway COX-2/mPGES-1 and Th17/Treg circulating cells. Here, we provide a brief overview of natural products/compounds, currently included in the Italian list of botanicals and the BELFRIT, in different fields of interest such as inflammation and immunity. In this context, we focus our opinion on novel therapeutic targets such as COX-2/mPGES-1 coupling enzymes and Th17/Treg circulating repertoire. This paper is dedicated to the scientific career of Professor Nicola Mascolo for his profound dedication to the study of natural compounds.

New insights on the arylpiperazinylalkyl pyridazinone ET1 as potent antinociceptive and anti-inflammatory agent.

Pyridazine derivatives, such as arylpiperazinylalkyl pyridazinones, display antinociceptive effects to thermal and chemical stimuli. Here, we extended our previous knowledge on the pharmacological profile of 4-amino-6-methyl-2-(3-(4-(4-methylcyclohexa-1,3-dien-1-yl)piperazin-1-yl)propyl)-5-vinylpyridazin-3(2H)-one, here referred as ET1, paving the way for the comprehension of its complete mechanism of action. To this aim, we have evaluated the mouse behavioural responses in several animal models of pain, the effect of ET1 in the murine model of zymosan-induced paw oedema and air-pouch, assessing the cytokines and the cellular phenotype and finally, an in vitro radioligand binding study was performed on a panel of 30 different receptors. In the formalin test, ET1 reduced both neurogenic and inflammatory phase of nociception induced by the aldehyde. Similarly, ET1 strongly reduced paw licking response in the capsaicin test, the abdominal stretching in the writhing test and the carrageenan-induced thermal hyperalgesia. ET1 also evoked a long-lasting reduction of thermal hyperalgesia. Furthermore, ET1 produced a long-lasting anti-inflammatory effect in the zymosan-induced mouse paw oedema and air-pouch through the selective inhibition of inflammatory monocytes recruitment and the modulation of IL-1ß, IL-6, TNF-α and MCP-1. Binding experiments confirmed an inhibitory effect on adrenergic α1A, α1B and α2A receptors subtypes and, for the first time, a moderate affinity was observed for the following receptors: histamine H1, imidazoline I2, sigma non-opioid intracellular receptor 1 and σ2. These results prompt ET1 as a potent analgesic and anti-inflammatory agent, and support the possibility that it may be suitable for clinical applications in a wide-range of inflammatory-based diseases.

The functional link between microsomal prostaglandin E synthase-1 (mPGES-1) and peroxisome proliferator-activated receptor γ (PPARγ) in the onset of inflammation.

Many years have elapsed since the discovery of anti-inflammatories as effective therapeutics for the treatment of inflammatory-related diseases, but we are still uncovering their various mechanisms of action. Recent biochemical and pharmacological studies have shown that in different tissues and cell types lipid mediators from thearachidonic acid cascade, play a crucial role in the initiation and resolution of inflammation by shifting from pro-inflammatory prostaglandin (PG)E2 to anti-inflammatory PGD2 and PGJ2. Considering that until now very little is known about the biological effects evoked by microsomal prostaglandin E synthase-1 (mPGES-1) and contextually by peroxisome proliferator-activated receptor γ (PPARγ) modulation (key enzymes involved in PGE2 and PGD2/PGJ2metabolism), in this opinion paper we sought to define the coordinate functional regulation between these two enzymes at the "crossroads of phlogistic pathway" involved in the induction and resolution of inflammation.