Inflammatory Mediator Prostaglandin E2 Research Articles

Endogenous Cholinergic System Involved in Peripheral Analgesic Control in Mice Is Activated by TNF-α, CXCL-1, and IL-1β

Introduction: Tissue injury results in the release of inflammatory mediators, including a cascade of algogenic substances, which contribute to the development of hyperalgesia. During this process, endogenous analgesic substances are peripherally released to counterbalance hyperalgesia. The present study aimed to investigate whether inflammatory mediators TNF-α, IL-1β, CXCL1, norepinephrine (NE), and prostaglandin E2 (PGE2) may be involved in the deflagration of peripheral endogenous modulation of inflammatory pain by activation of the cholinergic system. Methods: Male Swiss mice were subjected to paw withdrawal test. All the substances were injected via the intraplantar route. Results: The main findings of this study were as follows: (1) carrageenan (Cg), TNF-α, CXCL-1, IL1-β, NE, and PGE2 induced hyperalgesia; (2) the acetylcholinesterase enzyme inhibitor, neostigmine, reversed the hyperalgesia observed after Cg, TNF-α, CXCL-1, and IL1-β injection; (3) the non-selective muscarinic receptor antagonist, atropine, and the selective muscarinic type 1 receptor (m1AChr) antagonist, telenzepine, potentiated the hyperalgesia induced by Cg and CXCL-1; (4) mecamylamine, a non-selective nicotinic receptor antagonist, potentiated the hyperalgesia induced by Cg, TNF-α, CXCL-1, and IL1-β; (5) Cg, CXCL-1, and PGE2 increased the expression of the m1AChr and nicotinic receptor subunit α4protein. Conclusion: These results suggest that the cholinergic system may modulate the inflammatory pain induced by Cg, PGE2, TNF-α, CXCL-1, and IL1-β.

Abstract 178: Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling

Abstract Obesity increases the risk of developing cancer and worsens treatment outcome. Our lab has shown that adipose tissue contributes to B-cell acute lymphoblastic leukemia (B-ALL) resistance to chemotherapy both in vitro and in vivo. However, the mechanisms by which adipose tissue causes ALL cell resistance are complex and not completely understood. We therefore implanted obese mice (n=3) with syngeneic BCR/ABL B-ALL cells and performed single-cell RNA sequencing (scRNAseq) of ALL collected from epididymal adipose tissue vs. bone marrow. Our data uncovered a strong adipose microenvironment effect on the ALL transcriptome, identifying 577 differentially expressed genes using a log2 fold threshold of 0.25 and Bonferroni corrected p-values <0.05. Notably, we found that ALL cells in adipose tissue upregulate expression of Tnfsf11, the gene that encodes receptor activator of nuclear factor kappa beta ligand (RANKL). 93% of ALL cells in adipose tissue express RANKL compared to 33% in marrow, along with a log2 fold expression increase of 2.12 (p<1 × 10−100, Wilcoxon test). Because ALL expression of RANKL has been linked to bone resorption and CNS invasion, both of which have increased incidence in obese patients, we elected to explore this further. Ex vivo culture with human visceral adipose tissue validated ALL cell RANKL upregulation, demonstrating a 1.63 and 17.5-fold increase in gene expression after 48 hours in BV173 and RS4;11 B-ALL cell lines, respectively (n=4-6, both p<0.01, paired t test). Soluble RANKL was not detected in media from ALL cells cultured alone, detectable in media conditioned by human adipose tissue (0.018 pM), but increased in media from adipose-ALL cocultures (RS4;11: 0.065 pM, BV173: 0.317 pM, n=1). One potential candidate signal to stimulate ALL cell RANKL expression is the inflammatory mediator prostaglandin E2 (PGE2), which is known to be released by adipose tissue. Physiological concentrations of PGE2 (200 ng/mL) caused a similar increase in ALL cell RANKL gene expression as adipose tissue in both cell lines (n=6). PGE2 treatment also caused a mild resistance of ALL cells to daunorubicin chemotherapy in vitro, increasing the EC50 from 27±4 to 53±12 nM in RS4;11 (n=6, p<0.05), and from 39±11 to 62±19 nM in BV173 (n=6, p=0.06). Adipose tissue induction of RANKL expression was partially blocked by the addition of PGE2 receptor EP2 and EP4 blockers. Taken together, our results suggest that PGE2 produced by the adipose microenvironment activates ALL cell RANKL expression, which could potentially contribute to CNS invasion, bone breakdown, and chemoresistance. Citation Format: Michael Cohen, Jia Tan, Tyler Kuk, Blesscamal Nadeak, In Sook Ahn, Xia Yang, Etan Orgel, Steven D. Mittelman. Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 178.

Reproducible Synthesis of Biocompatible Albumin Nanoparticles Designed for Intra-articular Administration of Celecoxib to Treat Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, with intra-articular (IA) delivery of therapeutics being the current best option to treat pain and inflammation. However, IA delivery is challenging due to the rapid clearance of therapeutics from the joint and the need for repeated injections. Thus, there is a need for long-acting delivery systems that increase the drug retention time in joints with the capacity to penetrate OA cartilage. As pharmaceutical utility also demands that this is achieved using biocompatible materials that provide colloidal stability, our aim was to develop a nanoparticle (NP) delivery system loaded with the COX-2 inhibitor celecoxib that can meet these criteria. We devised a reproducible and economical method to synthesize the colloidally stable albumin NPs loaded with celecoxib without the use of any of the following conditions: high temperatures at which albumin denaturation occurs, polymer coatings, oils, Class 1/2 solvents, and chemical protein cross-linkers. The spherical NP suspensions were biocompatible, monodisperse with average diameters of 72 nm (ideal for OA cartilage penetration), and they were stable over 6 months at 4 °C. Moreover, the NPs loaded celecoxib at higher levels than those required for the therapeutic response in arthritic joints. For these reasons, they are the first of their kind. Labeled NPs were internalized by primary human articular chondrocytes cultured from the knee joints of OA patients. The NPs reduced the concentration of inflammatory mediator prostaglandin E2 released by the primaries, an indication of retained bioactivity following NP synthesis. Similar results were observed in lipopolysaccharide-stimulated human THP-1 monocytes. The IA administration of these NPs is expected to avoid side-effects associated with oral administration of celecoxib and to maintain a high local concentration in the knee joint over a sustained period. They are now ready for evaluation by IA administration in animal models of OA.

Use of Germination to Enhance Resveratrol Content and Its Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is triggered by a variety of danger signals and is now a worldwide concern. Resveratrol, a natural nonflavonoid polyphenol found in naturally consumed plants and foods, has a wide spectrum of bioactive potency. We successfully generated resveratrol-enriched rice by introducing the resveratrol biosynthesis gene into Dongjin rice. In this study, resveratrol- and piceid-enriched rice (DJ526) was investigated for its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells compared to normal rice (DJ). In addition, the 5-day-old germinated DJ526 (DJ526_5) was tested for its anti-inflammatory effects. The piceid and resveratrol amounts increased in DJ526_5 by germination. Treatment of LPS-stimulated RAW264.7 cells with resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) significantly decreased the production of nitric oxide (NO) and the inflammatory mediator prostaglandin E2 (PGE2), downregulated proinflammatory gene expression, and inhibited nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. These findings demonstrated the anti-inflammatory mechanisms of resveratrol-enriched rice in LPS-stimulated RAW264.7 cells. Furthermore, resveratrol-enriched rice could be a potential source of anti-inflammatory agents.

Antiproliferative effects of resveratrol and indole-3-carbinol in HPV-associated diseases prevention

The burden of persistent papillomavirus infection on public healthcare is not limited to cervical cancer (CC). At present, cervical cancer continues to be one of the most common types of cancer worldwide, although HPV also causes anogenital warts. As there is no HPV treatment available, nowadays researchers focus on the search for new therapeutic platforms, new agents for HPV-associated diseases relapse prevention after surgical treatment. Significant anticancer effects of resveratrol have been demonstrated in a variety of cancers, including breast cancer, skin cancer, lung cancer, pancreatic cancer, and stomach cancer. In CC cells, resveratrol stimulates apoptosis of cancer cells, increases expression of HPV E6 and E7, and the expression of p53 tumor suppressor protein. Protective effects on CC cells migration and invasion is achieved by inhibiting NF-κB transcription and AP-1 mediated MMP9 expression. Resveratrol demonstrates a whole range of anti-inflammatory effects, ranging from the suppression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-17 expression and inflammatory mediator prostaglandin E2, to the suppression of the formation of ROS and NO. I3K has a proven antitumor efficacy based on the molecular mechanisms of pathological cell proliferation and tumor transformation in hormone- dependent tissues, including cervical epithelial cells. Resveratrol and I3K have a wide range of studied positive therapeutic effects that can significantly reduce the likelihood of development and progression of HPV-associated lesions.

NAIP—NLRC4 inflammasome activation in tuft cells contributes to host defense against bacteria

Abstract The small intestinal epithelium is exposed to various microbes, and intestinal epithelial cells (IECs) have developed an array of host defense mechanisms to distinguish commensals from pathogenic microbes and initiate an immune response against the later. NAIP—NLRC4 inflammasome induced-pyroptosis, which is activated when bacterial flagellin or type III secretion systems are detected in the cytosol of the host cell, is one such mechanism. Activation of NAIP—NLRC4 induces extrusion of IECs and release of the inflammatory mediators prostaglandin E2 and IL-18. Work on epithelial inflammasome activation thus far has not delineated if different subtypes of IECs have specific roles in the ensuing immune response. Tuft cells are a rare subset of IEC that is primarily known for its role in sensing parasites, and it is unknown whether they are involved in promoting antibacterial host responses. Here we show that upon activation of the NAIP—NLRC4 inflammasome specifically in tuft cells, they uniquely among IECs release prostaglandin D2, and within the small intestine, tissue IL-22 increases. NLRC4 expression only in tuft cells leads to better control of Salmonella Typhimurium compared to NLRC4 null controls. Taken together, these data suggest that NAIP— NLRC4 inflammasome activation in tuft cells leads to lipid mediator induced downstream inflammatory responses that are complementary to signaling pathways that are activated by other IEC subsets. Understanding how NAIP—NLRC4 activation in different epithelial subsets affects downstream inflammatory responses will inform on the different signaling pathways that synergize together to mount pathogen specific host defense mechanisms.

DUSP-1 Induced by PGE2 and PGE1 Attenuates IL-1β-Activated MAPK Signaling, Leading to Suppression of NGF Expression in Human Intervertebral Disc Cells.

The molecular mechanism of discogenic low back pain (LBP) involves nonphysiological nerve invasion into a degenerated intervertebral disc (IVD), induced by nerve growth factor (NGF). Selective cyclooxygenase (COX)-2 inhibitors are mainly used in the treatment of LBP, and act by suppressing the inflammatory mediator prostaglandin E2 (PGE2), which is induced by inflammatory stimuli, such as interleukin-1β (IL-1β). However, in our previous in vitro study using cultured human IVD cells, we demonstrated that the induction of NGF by IL-1β is augmented by a selective COX-2 inhibitor, and that PGE2 and PGE1 suppress NGF expression. Therefore, in this study, to elucidate the mechanism of NGF suppression by PGE2 and PGE1, we focused on mitogen-activated protein kinases (MAPKs) and its phosphatase, dual-specificity phosphatase (DUSP)-1. IL-1β-induced NGF expression was altered in human IVD cells by MAPK pathway inhibitors. PGE2 and PGE1 enhanced IL-1β-induced DUSP-1 expression, and suppressed the phosphorylation of MAPKs in human IVD cells. In DUSP-1 knockdown cells established using small interfering RNA, IL-1β-induced phosphorylation of MAPKs was enhanced and prolonged, and NGF expression was significantly enhanced. These results suggest that PGE2 and PGE1 suppress IL-1β-induced NGF expression by suppression of the MAPK signaling pathway, accompanied by increased DUSP-1 expression.

Cannabinoid Receptor 1 Expression in Human Dorsal Root Ganglia and CB13-Induced Bidirectional Modulation of Sensory Neuron Activity.

Cannabinoid receptors have been identified as potential targets for analgesia from studies on animal physiology and behavior, and from human clinical trials. Here, we sought to improve translational understanding of the mechanisms of cannabinoid-mediated peripheral analgesia. Human lumbar dorsal root ganglia were rapidly recovered from organ donors to perform physiological and anatomical investigations into the potential for cannabinoids to mediate analgesia at the level of the peripheral nervous system. Anatomical characterization of in situ gene expression and immunoreactivity showed that 61 and 53% of human sensory neurons express the CB1 gene and receptor, respectively. Calcium influx evoked by the algogen capsaicin was measured by Fura-2AM in dissociated human sensory neurons pre-exposed to the inflammatory mediator prostaglandin E2 (PGE2) alone or together with CB13 (1 μM), a cannabinoid agonist with limited blood–brain barrier permeability. Both a higher proportion of neurons and a greater magnitude of response to capsaicin were observed after exposure to CB13, indicating cannabinoid-mediated sensitization. In contrast, membrane properties measured by patch-clamp electrophysiology demonstrated that CB13 suppressed excitability and reduced action potential discharge in PGE2-pre-incubated sensory neurons, suggesting the suppression of sensitization. This bidirectional modulation of sensory neuron activity suggests that cannabinoids may suppress overall membrane excitability while simultaneously enhancing responsivity to TRPV1-mediated stimuli. We conclude that peripherally restricted cannabinoids may have both pro- and anti-nociceptive effects in human sensory neurons.

Anti-inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells.

The present study assessed the effect of freshwater hybrid catfish oil (FFO) on the inflammatory status of lipopolysaccharide (LPS)-stimulated RAW264.7 cells and investigated the underlying mechanisms. RAW264.7 cells were supplemented with various concentrations [0.125-2% in 0.5% propylene glycol (v/v)] of FFO with or without LPS (1 µg/ml) for 24 h. Inflammatory cytokines and mediators were quantified using ELISA and reverse transcription-quantitative PCR. The results revealed that FFO treatment inhibited the secretion and mRNA expression of the pro-inflammatory cytokines IL-6, IL-1β, TNF-α. In line with this, FFO suppressed the expression and secretion of the inflammatory mediators cyclooxygenase-2 and prostaglandin E2. FFO also reduced apoptotic body formation and DNA damage. Correspondingly, FFO enhanced the immune response by modulating the cell cycle regulators p53, cyclin D2 and cyclin E2. Accordingly, FFO may be developed as a nutraceutical product to prevent inflammation.

TNF-α, CXCL-1 and IL-1 β as activators of the opioid system involved in peripheral analgesic control in mice

Tissue injury results in the release of inflammatory mediators, including a cascade of nociceptive substances, which contribute to development of hyperalgesia. In addition, during this process endogenous analgesic substances are also peripherally released with the aim of controlling the hyperalgesia. Thus, the present study aimed to investigate whether inflammatory mediators TNF-α, IL-1β, CXCL1, norepinephrine (NE) and prostaglandin E2 (PGE2) may be involved in the deflagration of peripheral endogenous modulation of inflammatory pain by activation of the opioid system. Thus, male Swiss mice and the paw withdrawal test were used. All substances were injected by the intraplantar route. Carrageenan, TNF-α, CXCL-1, IL1-β, NE and PGE2 induced hyperalgesia. Selectives μ (clocinamox), δ (naltrindole) and κ (norbinaltorphimine, nor-BNI) and non-selective (naloxone) opioid receptor antagonists potentiated the hyperalgesia induced by carrageenan, TNF-α, CXCL-1 and IL1-β. In contrast, when the enzyme N-aminopeptidase involved in the degradation of endogenous opioid peptides was inhibited by bestatin, the hyperalgesia was significantly reduced. In addition, the western blotting assay indicated that the expression of the opioid δ receptor was increased after intraplantar injection of carrageenan. The data obtained in this work corroborate the hypothesis that TNF-α, CXCL-1 and IL-β cause, in addition to hyperalgesia, the release of endogenous substances such as opioid peptides, which in turn exert endogenous control over peripheral inflammatory pain.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BackgroundWe aimed to evaluate the effect of low doses (LD) bone morphogenetic protein‐2 (BMP2) and BMP4 micro‐immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration.MethodsWe first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization‐related enzymes (matrix metalloproteinase‐1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long‐term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release.ResultsLD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re‐establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days.ConclusionsLD BMP4 MI treatment shows anti‐inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

Anti-inflammatory action of physalin A by blocking the activation of NF-κB signaling pathway

Ethnopharmacological relevancePhysalis Calyx seu Fructus is typically used to treat inflammatory diseases such as upper respiratory tract infection and acute tonsillitis in clinical practice of China. Physalin A, a main active ingredient of this traditional Chinese medicine (TCM), has been reported for its significant anti-tumor activity. However, most reports focused on the studies of its anti-tumor activity, the anti-inflammatory activity of physalin A and its molecular mechanism are still not elucidated clearly. Aim of the studyThe aim of the study was to investigate the anti-inflammatory activities both in vitro and in vivo and molecular mechanism of physalin A. Materials and methodsThe potential anti-inflammatory properties of physalin A were evaluated in vitro by lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells, and in vivo via two typical acute inflammation murine models. Some important inflammation-related molecules were analyzed by enzyme-linked immuno sorbent assay (ELISA) and Western blotting. ResultsThe results showed that physalin A inhibited carrageenan-induced paw edema of rats and capillary permeability of mice induced by acetic acid in vivo. Furthermore, physalin A also significantly reduced the release of inflammatory mediators nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in RAW 264.7 in vitro. Further investigations indicated that physalin A can down-regulate the high expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Physalin A remarkably blocked the degradation of inhibitor of nuclear factor kappa B alpha (IκB-α) and the nuclear translocation of nuclear factor-κB (NF-κB) p65 induced by LPS in RAW 264.7 cells. However, physalin A did not significantly inhibit the phosphorylation of mitogen-activated protein kinases (MAPKs) family proteins c-Jun N-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK) or p38. ConclusionsAll the results clearly illustrated that the anti-inflammatory action of physalin A is due to the inactivation of NF-κB signal pathway, but is irrelevant to the MAPKs pathway.

Inflammatory pain in peripheral tissue depends on the activation of the TNF-α type 1 receptor in the primary afferent neuron.

The mechanism underlying the role of tumor necrosis factor alpha (TNF-α) in the development of inflammatory hyperalgesia has been extensively studied, mainly the role of TNF-α in the release of pro-inflammatory cytokines. The current concept relies in the fact that TNF-α stimulates the cascade release of other pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8 (CINC-1 in rats), triggering the release of the final inflammatory mediator prostaglandin E2 (PGE2 ) and sympathetic amines that directly sensitize the nociceptors. However, this may not be the sole mechanism involved as the blockade of TNF-α synthesis by thalidomide prevents hyperalgesia without interrupting the synthesis of IL-1β, IL-6, and CINC-1. Therefore, we hypothesized that activation of TNF-α receptor type 1 (TNFR1) by TNF-α increases nociceptors' susceptibility to the action of PGE2 and dopamine. We have found out that intrathecal administration of oligodeoxynucleotide-antisense (ODN-AS) against TNFR1 or thalidomide prevented carrageenan-induced hyperalgesia. The co-administration of TNF-α with a subthreshold dose of PGE2 or dopamine that does not induce hyperalgesia by itself in the hind paw of Wistar rats pretreated with dexamethasone (to prevent the endogenous release of cytokines) induced a robust hyperalgesia that was prevented by intrathecal treatment with ODN-AS against TNFR1. We consider that the activation of neuronal TNFR1 by TNF-α decisively increases the susceptibility of the peripheral afferent neuron to the action of final inflammatory mediators - PGE2 and dopamine - that ultimately induce hyperalgesia. This mechanism may also underlie the analgesic action of thalidomide.

Mechanisms Mediating High-Molecular-Weight Hyaluronan-Induced Antihyperalgesia.

We evaluated the mechanism by which high-molecular-weight hyaluronan (HMWH) attenuates nociceptor sensitization, in the setting of inflammation. HMWH attenuated mechanical hyperalgesia induced by the inflammatory mediator prostaglandin E2 (PGE2) in male and female rats. Intrathecal administration of an oligodeoxynucleotide antisense (AS-ODN) to mRNA for cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, and intradermal administration of A5G27, a CD44 receptor antagonist, both attenuated antihyperalgesia induced by HMWH. In male rats, HMWH also signals via Toll-like receptor 4 (TLR4), and AS-ODN for TLR4 mRNA administered intrathecally, attenuated HMWH-induced antihyperalgesia. Since HMWH signaling is dependent on CD44 clustering in lipid rafts, we pretreated animals with methyl-β-cyclodextrin (MβCD), which disrupts lipid rafts. MβCD markedly attenuated HMWH-induced antihyperalgesia. Inhibitors for components of intracellular signaling pathways activated by CD44, including phospholipase C and phosphoinositide 3-kinase (PI3K), also attenuated HMWH-induced antihyperalgesia. Furthermore, in vitro application of HMWH attenuated PGE2-induced sensitization of tetrodotoxin-resistant sodium current, in small-diameter dorsal root ganglion neurons, an effect that was attenuated by a PI3K inhibitor. Our results indicate a central role of CD44 signaling in HMWH-induced antihyperalgesia and suggest novel therapeutic targets, downstream of CD44, for the treatment of pain generated by nociceptor sensitization.SIGNIFICANCE STATEMENT High-molecular-weight-hyaluronan (HMWH) is used to treat osteoarthritis and other pain syndromes. In this study we demonstrate that attenuation of inflammatory hyperalgesia by HMWH is mediated by its action at cluster of differentiation 44 (CD44) and activation of its downstream signaling pathways, including RhoGTPases (RhoA and Rac1), phospholipases (phospholipases Cε and Cγ1), and phosphoinositide 3-kinase, in nociceptors. These findings contribute to our understanding of the antihyperalgesic effect of HMWH and support the hypothesis that CD44 and its downstream signaling pathways represent novel therapeutic targets for the treatment of inflammatory pain.

In-vivo Anti-inflammatory Activity of Hydrocotyle umbellata L. aerial parts and Isolation of the Main Phytochemicals.

Hydrocotyle umbellata L. (Family Araliaceae) populary known as Acaricoba, is indicated in folk medicine for treatment of several inflammatory disorders. The goal of the present study is to evaluate the anti-inflammatory activity of the defatted ethanolic extract (DEE) of the aerial parts using carrageenan-induced rat paw oedema method. The levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and the inflammatory mediator prostaglandin E2 (PGE2) were assessed using ELISA. The DEE at dose level 100 mg/kg showed significant decrease in oedema volume after 2 and 3 h, equivalent to 70.75% and 95.92% of the activity of the standard anti-inflammatory indomethacin, respectively. DEE significantly decreased the concentrations of the excessively produced IL-6 and PGE2 (24 ± 2.1 and 2374 ± 87 pg/mL compared to 16 ± 2 and 2419 ± 95 pg/mL induced by indomethacin). Chemical investigation was carried out to isolate and identify the bioactive compounds that might be responsible for this activity. The total phenolic (79.28 ± 0.1 mg) and total flavonoid (57.99 ± 0.1 mg) contents of the DEE were quantified spectrophotometricaly and expressed as gallic acid and rutin equivalents/g dry weight, respectively. The DEE was subjected to further fractionation using solvents of increasing polarities. Purification of the ethyl acetate fraction using different chromatographic techniques led to the isolation of five compounds, which were identified through 1D and 2D and UV/Vis spectral data. The five compounds were: quercetin, avicularin, quercitrin, hyperoside, and neochlorogenic acid. Several biological studies confirmed the important role of caffeoyl quinic acid and quercetin derivatives as anti-inflammatory bioactive compounds.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

Suppression of Src and Syk in the NF-κB signaling pathway by Olea europaea methanol extract is leading to its anti-inflammatory effects

Ethnopharmacological relevanceOlea europaea L., (Oleaceae) has been used widely in folk medicine in the European Mediterranean islands, India, Asia, and other parts of the world. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms of how it inhibits the inflammatory response are not fully understood. In this study, we sought to identify the anti-inflammatory mechanisms of this plant. Materials and methodsUsing macrophages, we investigated the effects of O. europaea L. methanol extract (Oe-ME) and ethanol extract (Oe-EE) on the production of inflammatory mediator nitric oxide (NO) and prostaglandin E2 (PGE2), the expression levels of pro-inflammatory genes and intracellular inflammatory signaling activities. ResultsOe-ME and Oe-EE suppressed the production of NO in lipopolysaccharide-(LPS-), Pam3CSK4-, and poly (I:C)-stimulated RAW264.7 cells; importantly, no cytotoxicity was observed. Oe-ME and Oe-EE reduced production of PGE2 without exhibiting cytotoxicity. The mRNA expression levels of cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), IL-6, IL-1β, and tumor necrosis factor (TNF)-α were down-regulated by Oe-ME and Oe-EE. Nuclear fraction and whole lysate immunoblotting analyses and overexpression experiments strongly suggested that Oe-ME decreased the translocation of p65 and p50 (nuclear factors of the NF-κB subunit) as well as Src and Syk. ConclusionThese results suggest that Oe-ME exerts its anti-inflammatory effects by targeting Src and Syk in the NF-κB signaling pathway.

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS-stimulated co-cultures of periodontal ligament and RAW 264.7 cells, and RANKL-mediated osteoclastogenesis and bone resorption in PBMCs.

Inflammatory mediator prostaglandin E2 (PGE 2) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE 2 synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE 2 production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE 2, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE 2 production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE 2 in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.

Prostaglandin E2 Stimulates CREB-Mediated Modification of Histone Variant Nucleosomes at Enhancers to Promote Hematopoietic Stem Cell Fate

Stimulation of hematopoietic stem and progenitor cells (HSPCs) with the inflammatory mediator Prostaglandin E2 (PGE2) enhances self-renewal and stem cell engraftment following transplantation. Currently, the long-acting derivative of PGE2, 16,16-dimethyl-PGE2 (dmPGE2) is in its fourth clinical trial to improve HSC engraftment and reduce graft versus host disease. To understand the effect of dmPGE2, we assessed genome-wide chromatin reorganization and gene expression changes in human CD34+ HSPCs after 2 hours of dmPGE2 treatment, the time period of treatment in the clinical trials. Enhancers are known to regulate gene expression changes in specific environmental contexts such as stress or inflammation, however the regulatory principles by which subsets of enhancers become activated are poorly understood. Here, we mapped active enhancers by ChIP-seq for H2K27ac and found that dmPGE2 activates a discrete set of enhancers in HSPCs. To investigate enhancer chromatin remodeling, we performed micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) to map the occupancy and position of nucleosomes. We found that, contrary to the predominant assumption that open chromatin structures are essentially nucleosome-free, MNase-accessible nucleosomes are retained at inducible enhancers following dmPGE2 stimulation. Through ATAC-seq analysis we mapped changes in open chromatin and found that induced enhancers gain chromatin accessibility following stimulation while maintaining their nucleosome configuration. Surprisingly, this indicates that nucleosomes present at the center of dmPGE2-responsive enhancers play an important function in enhancer accessibility and activity. We then correlated enhancers with gene expression changes by performing RNA-seq and found that genes associated with dmPGE2-induced enhancers display higher gene expression changes after stimulation compared to genes associated with non-responsive enhancers. Transcripts upregulated after dmPGE2 treatment include previously identified regulators of self-renewal and migration such as NR4A2, EGR1 and CXCR4. Moreover, inflammatory chemokines including CXCL2 and CXCL8 as well as members of the activating protein 1 (AP-1) transcription factor gene family such as FOS, FOSL2 and JUNB are increasingly expressed upon stimulation. The gene expression profile included a signature implying CREB as the main transcription factor responsible for the acute dmPGE2 response. Western blot revealed dmPGE2-mediated activation of the signaling transcription factor CREB through phosphorylation in HSPCs. Using ChIP-seq, we found increased genomic binding of phospho-CREB (pCREB) after dmPGE2 treatment in the enhancers. Surprisingly, the binding of pCREB coincided directly with variant histone H2A.Z containing labile nucleosomes in enhancers. We validated the interaction between pCREB and H2A.Z on chromatin in dmPGE2-responsive U937 cells through chromatin fractionation followed by complex immunoprecipitation. This suggests that labile nucleosomes provide sufficient DNA access to allow for binding of pCREB at enhancers. Taken together, our study proposes a novel model for stimulus-mediated activation of enhancers by the inflammatory mediator dmPGE2. dmPGE2 induces the phosphorylation of CREB and subsequently leads to a specific interaction of pCREB with previously deposited H2A.Z-rich nucleosomes at inducible enhancers who regulate genes that promote HSPC fate. This new mechanism of variant histone deposition followed by the interaction with a signaling transcription factor at enhancers supports a rapid inducible response from the environment. DisclosuresNo relevant conflicts of interest to declare.

Synthesis and In Vitro Anti-inflammatory Activity of C20 Epimeric Ocotillol-Type Triterpenes and Protopanaxadiol.

Ginseng is a perennial herb that contains various medicinal substances. The major active constituents of ginseng are ginsenosides, which have multifarious biological activities. Some pharmacological activities are closely dependent on the stereoisomers derived from the configuration at C20. In this study, the in vitro anti-inflammatory activity of C20 epimeric ocotillol-type triterpenes (2, 3, 9: , and 10: ) and protopanaxadiol [20(S/R)-protopanaxadiol] were investigated. Epimers 2: and 3: were prepared starting from 20(S)-protopanaxadiol. Epimers 9: and 10: were synthesized from 20(R)-3-acetylprotopanaxadiol (7: ). The anti-inflammatory activity of 2, 3, 9, 10: , 20(S)-protopanaxadiol, and 20(R)-protopanaxadiol was evaluated in cultured mouse macrophage RAW 264.7 cells. The MTT assay was used to measure the cytotoxicity. RAW 264.7 cells were stimulated by lipopolysaccharide to release the inflammatory mediators nitric oxide, prostaglandin E2, TNF-α, and interleukin-6 and anti-inflammatory mediator interleukin-10. The effect of the compounds on the overproduction of nitric oxide, prostaglandin E2, TNF-α, interleukin-6, and interleukin-10 was determined using Griess and ELISA methods. The results demonstrated that the in vitro anti-inflammatory activities of C20 epimeric ocotillol-type triterpenes and protopanaxadiol were different. Both the 20S-epimers (2: and 3: ) and 20R-epimers (9: and 10: ) inhibited the release of inflammatory mediator nitric oxide, while mainly the 20S-epimers inhibited the release of inflammatory mediator prostaglandin E2, and the 20R-epimers inhibited the release of inflammatory cytokine TNF-α. Both the 20S-epimers [2, 3: , and 20(S)-protopanaxadiol] and 20R-epimers [9, 10: , and 20(R)-protopanaxadiol] inhibited the release of inflammatory cytokine interleukin-6, but mainly the 20S-epimers [2, 3: , and 20(S)-protopanaxadiol] increased the release of anti-inflammatory mediator interleukin-10.