Standard of care drugs do not modulate activity of senescent primary human lung fibroblasts.

Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is not evident whether the standard-of-care (SOC) drugs, nintedanib and pirfenidone, have senolytic properties. To address this question, we performed colorimetric and fluorimetric assays, qRT-PCR, and western blotting to evaluate the effect of SOC drugs and D + Q on senescent normal and IPF lung fibroblasts. In this study, we found that SOC drugs did not provoke apoptosis in the absence of death ligand in normal or IPF senescent lung fibroblasts. Nintedanib increased caspase-3 activity in the presence of Fas Ligand in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma 2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone induced mixed lineage kinase domain-like pseudokinase phosphorylation, provoking necroptosis. Furthermore, pirfenidone increased transcript levels of FN1 and COL1A1 in senescent IPF fibroblasts. Lastly, D + Q augmented growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Taken together, these results establish that SOC drugs failed to trigger apoptosis in senescent primary human lung fibroblasts, possibly due to enhanced Bcl-2 levels by nintedanib and the activation of the necroptosis pathway by pirfenidone. Together, these data revealed the inefficacy of SOC drugs to target senescent cells in IPF.

Cellular Senescence: Pathogenic Mechanisms in Lung Fibrosis.

Pulmonary fibrosis is a chronic and fatal lung disease that significantly impacts the aging population globally. To date, anti-fibrotic, immunosuppressive, and other adjunct therapy demonstrate limited efficacies. Advancing our understanding of the pathogenic mechanisms of lung fibrosis will provide a future path for the cure. Cellular senescence has gained substantial interest in recent decades due to the increased incidence of fibroproliferative lung diseases in the older age group. Furthermore, the pathologic state of cellular senescence that includes maladaptive tissue repair, decreased regeneration, and chronic inflammation resembles key features of progressive lung fibrosis. This review describes regulatory pathways of cellular senescence and discusses the current knowledge on the senescence of critical cellular players of lung fibrosis, including epithelial cells (alveolar type 2 cells, basal cells, etc.), fibroblasts, and immune cells, their phenotypic changes, and the cellular and molecular mechanisms by which these cells contribute to the pathogenesis of pulmonary fibrosis. A few challenges in the field include establishing appropriate in vivo experimental models and identifying senescence-targeted signaling molecules and specific therapies to target senescent cells, known collectively as "senolytic" or "senotherapeutic" agents.

Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.

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

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

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis.

BACKGROUND: Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. METHODS: Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. RESULTS: DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. CONCLUSIONS: Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Phytic Acid Decreases Oxidative Stress and Intestinal Lesions Induced by Fumonisin B1 and Deoxynivalenol in Intestinal Explants of Pigs.

The purpose of the present study was to investigate the effects of phytic acid (IP6) on morphological and immunohistochemical parameters and oxidative stress response in intestinal explants of pigs exposed to fumonisin B1 (FB1) and/or deoxynivalenol (DON). The jejunal explants were exposed to the following treatments: vehicle, IP6 5 mM, DON 10 µM, FB1 70 µM, DON 10 µM + FB1 70 µM, DON 10 µM + IP6 5 mM, FB1 70 µM + IP6 5 mM, and DON 10 µM + FB1 70 µM + IP6 5 mM. The decrease in villus height and goblet cell density was more evident in DON and DON + FB1 treatments. In addition, a significant increase in cell apoptosis and cell proliferation and a decrease in E-cadherin expression were observed in the same groups. DON and FB1 exposure increased cyclooxygenase-2 expression and decreased the cellular antioxidant capacity. An increase in lipid peroxidation was observed in DON- and FB1-treated groups. IP6 showed beneficial effects, such as a reduction in intestinal morphological changes, cell apoptosis, cell proliferation, and cyclooxygenase-2 expression, and an increase in E-cadherin expression when compared with DON, FB1 alone, or DON and FB1 in association. IP6 inhibited oxidative stress and increased the antioxidant capacity in the explants exposed to mycotoxins.

Contribution of Spinal Cord Oligodendrocytes to Neuroinflammatory Diseases and Pain.

BACKGROUND: Neuroinflammatory diseases that affect spinal cord or associated spinal nerves represent challenging conditions for management in current medicine because of their complex pathology, poor prognosis, and high morbidity, which strikingly reduces the quality of life of patients. In this sense, a better understanding of the cellular and molecular mechanisms of spinal cord neuroinflammation might contribute to the development of novel therapies. Oligodendrocytes have unique and vital biological properties in central nervous system (CNS) homeostasis and physiology. A growing body of experimental evidence demonstrates that these glial cells are involved in the pathophysiological mechanisms underlying many chronic, neurodegenerative, and incapacitating CNS disorders. These cells also have important implications for the development and maintenance of neural plasticity and chronic pain states. On the other hand, evidence indicates that oligodendrocytes and their products may act in favor of CNS promoting beneficial effects orchestrating CNS tissue repair after injury. OBJECTIVE: The present review aims to explore the multi-faceted actions of spinal cord oligodendrocyte progenitors cells (OPCs) and mature oligodendrocytes in CNS inflammation and pathology, addressing their roles in experimental and clinical settings. A major focus was given to spinal cord amyotrophic lateral sclerosis, multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), traumatic injury and pain processing. METHODS: This review analyses and discusses published original research articles regarding the role of OPCs/oligodendrocytes in spinal cord inflammation and pain processing. RESULTS AND CONCLUSION: Findings from a number of clinical and experimental paradigms suggest spinal cord OPCs/oligodendrocytes are a potential therapeutic target for the control of neuroinflammation.

Quercetin Enhances Ligand-induced Apoptosis in Senescent Idiopathic Pulmonary Fibrosis Fibroblasts and Reduces Lung Fibrosis In Vivo.

Although cellular senescence may be a protective mechanism in modulating proliferative capacity, fibroblast senescence is now recognized as a key pathogenic mechanism in idiopathic pulmonary fibrosis (IPF). In aged mice, abundance and persistence of apoptosis-resistant senescent fibroblasts play a central role in nonresolving lung fibrosis after bleomycin challenge. Therefore, we investigated whether quercetin can restore the susceptibility of senescent IPF fibroblasts to proapoptotic stimuli and mitigate bleomycin-induced pulmonary fibrosis in aged mice. Unlike senescent normal lung fibroblasts, IPF lung fibroblasts from patients with stable and rapidly progressing disease were highly resistant to Fas ligand (FasL)-induced and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Senescent IPF fibroblasts exhibited decreased expression of FasL and TRAIL receptors and caveolin-1, as well as increased AKT activation, compared with senescent normal lung fibroblasts. Although quercetin alone was not proapoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts. Mechanistically, quercetin upregulated FasL receptor and caveolin-1 expression and modulated AKT activation. In vivo quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype in aged mice. Collectively, these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis by promoting FasL receptor and caveolin-1 expression and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice. Therefore, quercetin may be a viable therapeutic option for IPF and other age-related diseases that progress with the accumulation of senescent fibroblasts.

Contribution of Nrf2 Modulation to the Mechanism of Action of Analgesic and Anti-inflammatory Drugs in Pre-clinical and Clinical Stages.

Despite the progress that has occurred in recent years in the development of therapies to treat painful and inflammatory diseases, there is still a need for effective and potent analgesics and anti-inflammatory drugs. It has long been known that several types of antioxidants also possess analgesic and anti-inflammatory properties, indicating a strong relationship between inflammation and oxidative stress. Understanding the underlying mechanisms of action of anti-inflammatory and analgesic drugs, as well as essential targets in disease physiopathology, is essential to the development of novel therapeutic strategies. The Nuclear factor-2 erythroid related factor-2 (Nrf2) is a transcription factor that regulates cellular redox status through endogenous antioxidant systems with simultaneous anti-inflammatory activity. This review summarizes the molecular mechanisms and pharmacological actions screened that link analgesic, anti-inflammatory, natural products, and other therapies to Nrf2 as a regulatory system based on emerging evidences from experimental disease models and new clinical trial data.

Naringenin Eye Drops Inhibit Corneal Neovascularization by Anti-Inflammatory and Antioxidant Mechanisms.

Purpose: To investigate the effect of naringenin eye drops in corneal neovascularization induced by alkali (1 N NaOH) burn in mice. Methods: Corneal neovascularization in the right eye of male Swiss mice was induced by alkali. Treatment with naringenin eye drops (0.08-80 µg; 8 µL of 0.01-10 g/L solution) or vehicle (saline) started 2 days before corneal neovascularization was induced and was performed twice a day. Mice were treated up until the time animals were euthanized and cornea tissue was collected for testing, which was 2, 4, and 6 hours after alkali stimulus for cytokine and antioxidant capacity measurements, and 3 and/or 7 days after alkali stimulus for the assessment of corneal epithelial thickness and neovascularization, neutrophil, and macrophage recruitment, and vascular endothelial growth factor (Vegf), platelet-derived growth factor (Pdgf), matrix metalloproteinase-14 (Mmp14), and pigment epithelium-derived factor (Pedf) mRNA expression. Results: Naringenin eye drops inhibited alkali burn-induced neutrophil (myeloperoxidase activity and recruitment of Lysm-GFP+ cells) and macrophage (N-acetyl-ß-D glucosaminidase activity) recruitment into the eye, decrease in epithelial thickness, and neovascularization in the cornea. Further, naringenin inhibited alkali-induced cytokine (IL-1ß and IL-6) production, Vegf, Pdgf, and Mmp14 mRNA expression, and the reduction of ferric reducing antioxidant power and Azinobis-(3-Ethylbenzothiazoline 6-Sulfonic acid) radical scavenging capacity as well as increased the reduced glutathione and protein-bound sulfhydryl groups levels. Conclusions: Collectively, these results indicate that naringenin eye drops are protective in alkali-induced corneal burn by inhibiting leukocyte recruitment, the proangiogenic factor expression, inflammatory cytokine production, and loss of antioxidant defenses.

Targeting IL-33/ST2 signaling: regulation of immune function and analgesia.

INTRODUCTION: IL-33 signals through ST2 receptor and promotes inflammation by activating downstream pathways culminating in the production of pro-inflammatory mediators such as IL-1ß, TNF-α, and IL-6 in an NF-κB-dependent manner. In fact, compelling evidence has demonstrated the importance of IL-33/ST2 in both innate and adaptive immune responses in diseases presenting pain as an important clinical symptom. Areas covered: IL-33 is a pleiotropic cytokine with varied immune functions. Dysregulation of this pathway has been described as a key step in varied immune responses. Further, IL-33 contributes to peripheral and spinal cord nociceptor neuron sensitization in innate and adaptive inflammatory immune responses as well as in neuropathic and cancer pain. In this sense, targeting IL-33/ST2 signaling is a promising therapeutic approach. Expert opinion: The modulation of IL-33/ST2 signaling represents a possible approach in regulating immune functions. In addition to immune function, strategies targeting IL-33/ST2 signaling pathway display a favorable preclinical analgesic profile in both acute and chronic models of pain. Therefore, IL-33-targeting therapies represent a potential target for the development of novel analgesic drugs given that IL-33 activates, for instance, neutrophils, mast cells, macrophages, astrocytes, and microglia that are important cells in the induction and maintenance of chronic pain states.

Capsaicin: Current Understanding of Its Mechanisms and Therapy of Pain and Other Pre-Clinical and Clinical Uses.

In this review, we discuss the importance of capsaicin to the current understanding of neuronal modulation of pain and explore the mechanisms of capsaicin-induced pain. We will focus on the analgesic effects of capsaicin and its clinical applicability in treating pain. Furthermore, we will draw attention to the rationale for other clinical therapeutic uses and implications of capsaicin in diseases such as obesity, diabetes, cardiovascular conditions, cancer, airway diseases, itch, gastric, and urological disorders.

Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production.

Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1ß. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation.

Quercetin reduces Ehrlich tumor-induced cancer pain in mice.

Cancer pain directly affects the patient's quality of life. We have previously demonstrated that the subcutaneous administration of the mammary adenocarcinoma known as Ehrlich tumor induces pain in mice. Several studies have shown that the flavonoid quercetin presents important biological effects, including anti-inflammatory, antioxidant, analgesic, and antitumor activity. Therefore, the analgesic effect and mechanisms of quercetin were evaluated in Ehrlich tumor-induced cancer pain in mice. Intraperitoneal (i.p.) treatments with quercetin reduced Ehrlich tumor-induced mechanical and thermal hyperalgesia, but not paw thickness or histological alterations, indicating an analgesic effect without affecting tumor growth. Regarding the analgesic mechanisms of quercetin, it inhibited the production of hyperalgesic cytokines IL-1ß and TNFα and decreased neutrophil recruitment (myeloperoxidase activity) and oxidative stress. Naloxone (opioid receptor antagonist) inhibited quercetin analgesia without interfering with neutrophil recruitment, cytokine production, and oxidative stress. Importantly, cotreatment with morphine and quercetin at doses that were ineffective as single treatment reduced the nociceptive responses. Concluding, quercetin reduces the Ehrlich tumor-induced cancer pain by reducing the production of hyperalgesic cytokines, neutrophil recruitment, and oxidative stress as well as by activating an opioid-dependent analgesic pathway and potentiation of morphine analgesia. Thus, quercetin treatment seems a suitable therapeutic approach for cancer pain that merits further investigation.

Vanillic Acid Inhibits Inflammatory Pain by Inhibiting Neutrophil Recruitment, Oxidative Stress, Cytokine Production, and NFκB Activation in Mice.

Vanillic acid (1) is a flavoring agent found in edible plants and fruits. It is an oxidized form of vanillin. Phenolic compounds form a substantial part of plant foods used as antioxidants with beneficial biological activities. These compounds have received considerable attention because of their role in preventing human diseases. Especially, 1 presents antibacterial, antimicrobial, and chemopreventive effects. However, the mechanisms by which 1 exerts its anti-inflammatory effects in vivo are incompletely understood. Thus, the effect of 1 was evaluated in murine models of inflammatory pain. Treatment with 1 inhibited the overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone, the second phase of the formalin test, and complete Freund's adjuvant (CFA). Treatment with 1 also inhibited carrageenan- and CFA-induced mechanical hyperalgesia, paw edema, myeloperoxidase activity, and N-acetyl-ß-D-glucosaminidase activity. The anti-inflammatory mechanisms of 1 involved the inhibition of oxidative stress, pro-inflammatory cytokine production, and NFκB activation in the carrageenan model. The present study demonstrated 1 presents analgesic and anti-inflammatory effects in a wide range of murine inflammation models, and its mechanisms of action involves antioxidant effects and NFκB-related inhibition of pro-inflammatory cytokine production.

Hypericum perforatum Reduces Paracetamol-Induced Hepatotoxicity and Lethality in Mice by Modulating Inflammation and Oxidative Stress.

Hypericum perforatum is a medicinal plant with anti-inflammatory and antioxidant properties, which is commercially available for therapeutic use in Brazil. Herein the effect of H. perforatum extract on paracetamol (acetaminophen)-induced hepatotoxicity, lethality, inflammation, and oxidative stress in male swiss mice were investigated. HPLC analysis demonstrated the presence of rutin, quercetin, hypericin, pseudohypericin, and hyperforin in H. perforatum extract. Paracetamol (0.15-3.0 g/kg, p.o.) induced dose-dependent mortality. The sub-maximal lethal dose of paracetamol (1.5 g/kg, p.o.) was chosen for the experiments in the study. H. perforatum (30-300 mg/kg, i.p.) dose-dependently reduced paracetamol-induced lethality. Paracetamol-induced increase in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations, and hepatic myeloperoxidase activity, IL-1ß, TNF-α, and IFN-γ concentrations as well as decreased reduced glutathione (GSH) concentrations and capacity to reduce 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate radical cation; ABTS˙(+) ) were inhibited by H. perforatum (300 mg/kg, i.p.) treatment. Therefore, H. perforatum protects mice against paracetamol-induced lethality and liver damage. This effect seems to be related to the reduction of paracetamol-induced cytokine production, neutrophil recruitment, and oxidative stress.

Protective effects of the flavonoid hesperidin methyl chalcone in inflammation and pain in mice: role of TRPV1, oxidative stress, cytokines and NF-κB.

Cytokines and reactive oxygen species are inflammatory mediators that lead to increased sensitivity to painful stimuli, and their inhibition represents a therapeutic approach in controlling acute and chronic pain. The water-soluble flavonone hesperidin methyl chalcone (HMC) is used in the treatment of venous diseases, but its bioactivity as anti-inflammatory and analgesic is poorly understood. The present study evaluated the protective effects of HMC in widely used mouse models of acute and prolonged inflammation and pain. Male Swiss mice were treated with HMC (3-100 or 30 mg/kg, intraperitoneally) or vehicle (saline) 1h before inflammatory stimuli. In overt pain-like behavior tests, HMC inhibited acetic acid- and phenyl-p-benzoquinone-induced writhing, and capsaicin-, Complete Freund's Adjuvant (CFA)- and formalin-induced paw flinching and licking. HMC also inhibited carrageenan-, capsaicin- and CFA-induced mechanical and thermal hyperalgesia. Mechanistically, HMC inhibited carrageenan-induced cytokine (TNF-α, IL-1ß, IL-6, and IL-10) production, oxidative stress and NF-κB activation. Furthermore, HMC did not cause gastric or hepatic injury in a 7 days treatment protocol. Thus, this is the first report that HMC reduces inflammation and inflammatory pain by targeting TRPV1 (transient receptor potential vanilloid type 1) receptor activity, oxidative stress, cytokine production, and NF-κB activity, which suggests its potential applicability in inflammatory diseases.

Pimaradienoic acid inhibits inflammatory pain: inhibition of NF-κB activation and cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Pimaradienoic acid (1) is a pimarane diterpene (ent-pimara-8(14),15-dien-19-oic acid) extracted at high amounts from various plants including Vigueira arenaria Baker. Compound 1 inhibited carrageenan-induced paw edema and acetic acid-induced abdominal writhing, which are its only known anti-inflammatory activities. Therefore, it is important to further investigate the analgesic effects of 1. Oral administration of 1 (1, 3, and 10 mg/kg) inhibited the acetic acid-induced writhing. This was also observed at 10 mg/kg via sc and ip routes. Both phases of the formalin- and complete Freund's adjuvant (CFA)-induced paw flinch and time spent licking the paw were inhibited by 1. Compound 1 inhibited carrageenan-, CFA-, and PGE2-induced mechanical hyperalgesia. Treatment with 1 inhibited carrageenan-induced production of TNF-α, IL-1ß, IL-33, and IL-10 and nuclear factor κB activation. Pharmacological inhibitors also demonstrated that the analgesic effects of 1 depend on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway. Compound 1 did not alter plasma levels of AST, ALT, or myeloperoxidase activity in the stomach. These results demonstrate that 1 causes analgesic effects associated with the inhibition of NF-κB activation, reduction of cytokine production, and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.