Synthesis of Carvone Derivatives and In Silico and In Vitro Screening of Anti-Inflammatory Activity in Murine Macrophages.

The chemical modification of natural compounds is a promising strategy to improve their frequently poor bioavailability and low potency. This study aimed at synthesizing chemical derivatives of carvone, a natural monoterpene with anti-inflammatory properties, which we recently identified, and evaluating their potential anti-inflammatory activity. Fourteen chemical derivatives of carvone were synthesized, purified and their chemical structures confirmed. Noncytotoxic concentrations of the test compounds were selected based on the resazurin reduction assay. Among the tested compounds, four significantly reduced the lipopolysaccharides-induced protein levels of the inducible isoform of the nitric oxide synthase and nitric oxide production and showed a dual effect on pro-IL-1 protein levels in the Raw 264.7 cell line. The Ligand Express drug discovery platform was used to predict the targets of the test compounds, and an enrichment analysis was performed to group the different biological processes and molecular and cellular functions of the tested compounds. Moreover, Ligand Express also predicted that all chemicals evaluated have intestinal and blood-brain barrier permeability, do not inhibit P-gp and do not interact with major receptors. Although presenting anti-inflammatory and some advantageous ADME properties, the tested compounds still have low potency and specificity but may provide novel structures the further chemical modification of which may yield more promising drugs.

Molecular Mechanisms Underlying the Anti-Inflammatory Properties of (R)-(-)-Carvone: Potential Roles of JNK1, Nrf2 and NF-κB.

To explore the molecular mechanisms underlying the anti-inflammatory activity of (R)-(-)-carvone, we evaluated its ability to inhibit the signaling pathways involving the mitogen-activated protein kinases (MAPKs) and the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). (R)-(-)-carvone significantly decreased c-Jun N-terminal kinase (JNK) 1phosphorylation, but not that of the other MAPKs, induced by bacterial lipopolysaccharides (LPS) in the RAW 264.7 macrophage cell line. Although (R)-(-)-carvone significantly inhibited resynthesis of the inhibitor of NF-κB (IκB)-α induced by LPS, it did not interfere with the canonical NF-κB activation pathway, suggesting that it may interfere with its transcriptional activity. (R)-(-)-carvone also showed a tendency to decrease the levels of acetylated NF-κB/p65 in the nucleus, without affecting the activity and protein levels of Sirtuin-1, the major NF-κB/p65 deacetylating enzyme. Interestingly, the nuclear protein levels of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the expression of its target,, heme oxygenase-1 (HO-1), an antioxidant enzyme, also showed a tendency to increase in the presence of (R)-(-)-carvone. Taken together, these results suggest that the ability of (R)-(-)-carvone to inhibit JNK1 and to activate Nrf2 can underlie its capacity to inhibit the transcriptional activity of NF-κB and the expression of its target genes. This study highlights the diversity of molecular mechanisms that can be involved in the anti-inflammatory activity of monoterpenes.

Monoterpenes as Sirtuin-1 Activators: Therapeutic Potential in Aging and Related Diseases.

Sirtuin 1 (SIRT) is a class III, NAD+-dependent histone deacetylase that also modulates the activity of numerous non-histone proteins through deacylation. SIRT1 plays critical roles in regulating and integrating cellular energy metabolism, response to stress, and circadian rhythm by modulating epigenetic and transcriptional regulation, mitochondrial homeostasis, proteostasis, telomere maintenance, inflammation, and the response to hypoxia. SIRT1 expression and activity decrease with aging, and enhancing its activity extends life span in various organisms, including mammals, and improves many age-related diseases, including cancer, metabolic, cardiovascular, neurodegenerative, respiratory, musculoskeletal, and renal diseases, but the opposite, that is, aggravation of various diseases, such as some cancers and neurodegenerative diseases, has also been reported. Accordingly, many natural and synthetic SIRT1 activators and inhibitors have been developed. Known SIRT1 activators of natural origin are mainly polyphenols. Nonetheless, various classes of non-polyphenolic monoterpenoids have been identified as inducers of SIRT1 expression and/or activity. This narrative review discusses current information on the evidence that supports the role of those compounds as SIRT1 activators and their potential both as tools for research and as pharmaceuticals for therapeutic application in age-related diseases.

Elucidation of the Mechanism Underlying the Anti-Inflammatory Properties of (S)-(+)-Carvone Identifies a Novel Class of Sirtuin-1 Activators in a Murine Macrophage Cell Line.

The signaling pathways involved in age-related inflammation are increasingly recognized as targets for the development of preventive and therapeutic strategies. Our previous study elucidated the structure-activity relationship of monoterpene compounds derived from p-menthane as potential anti-inflammatory drugs and identified (S)-(+)-carvone as the most potent among the compounds tested. This study aims at identifying the molecular mechanism underlying the anti-inflammatory properties of (S)-(+)-carvone. The murine macrophage cell line, Raw 264.7, was stimulated with bacterial lipopolysaccharide (LPS) to simulate inflammation. Western blot was used to assess protein levels and post-translational modifications. The subcellular localization of NF-κB/p65 was visualized by immunocytochemistry. An in vitro fluorometric assay was used to measure Sirtuin-1 (SIRT1) activity. (S)-(+)-carvone inhibited LPS-induced JNK1 phosphorylation, but not that of p38 and ERK1/2 and also did not affect the phosphorylation and degradation of the NF-κB inhibitor, IκB-α. Accordingly, (S)-(+)-carvone did not affect LPS-induced phosphorylation of NF-κB/p65 on Ser536 and its nuclear translocation, but it significantly decreased LPS-induced IκB-α resynthesis, a NF-κB-dependent process, and NF-κB/p65 acetylation on lysine (Lys) 310. Deacetylation of that Lys residue is dependent on the activity of SIRT1, which was found to be increased by (S)-(+)-carvone, while its protein levels were unaffected. Taken together, these results show that (S)-(+)-carvone is a new SIRT1 activator with the potential to counteract the chronic low-grade inflammation characteristic of age-related diseases.

Common risk factors and therapeutic targets in obstructive sleep apnea and osteoarthritis: An unexpectable link?

Osteoarthritis (OA) and Obstructive Sleep Apnea (OSA) are two highly prevalent chronic diseases for which effective therapies are urgently needed. Recent epidemiologic studies, although scarce, suggest that the concomitant occurrence of OA and OSA is associated with more severe manifestations of both diseases. Moreover, OA and OSA share risk factors, such as aging and metabolic disturbances, and co-morbidities, including cardiovascular and metabolic diseases, sleep deprivation and depression. Whether this coincidental occurrence is fortuitous or involves cause-effect relationships is unknown. This review aims at collating and integrating present knowledge on both diseases by providing a brief overview of their epidemiology and pathophysiology, analyzing current evidences relating OA and OSA and discussing potential common mechanisms by which they can aggravate each other. Such mechanisms constitute potential therapeutic targets whose pharmacological modulation may provide more efficient ways of reducing the consequences of OA and OSA and, thus, lessen the huge individual and social burden that they impose.

Standardised comparison of limonene-derived monoterpenes identifies structural determinants of anti-inflammatory activity.

Mint species are widely used in traditional and conventional medicine as topical analgesics for osteoarthritic pain and for disorders of the gastrointestinal and respiratory tracts which are all associated with chronic inflammation. To identify the structural determinants of anti-inflammatory activity and potency which are required for chemical optimization towards development of new anti-inflammatory drugs, a selected group of monoterpenes especially abundant in mint species was screened by measuring bacterial lipopolysacharide (LPS)-induced nitric oxide (NO) production in murine macrophages. Nine compounds significantly decreased LPS-induced NO production by more than 30%. IC50 values were calculated showing that the order of potency is: (S)-(+)-carvone > (R)-(-)-carvone > (+)-dihydrocarveol > (S)-8-hydroxycarvotanacetone > (R)-8-hydroxycarvotanacetone > (+)-dihydrocarvone > (-)-carveol > (-)-dihydrocarveol > (S)-(-)-pulegone. Considering the carbon numbering relative to the common precursor, limonene, the presence of an oxygenated group at C6 conjugated to a double bond at C1 and an isopropenyl group and S configuration at C4 are the major chemical features relevant for activity and potency. The most potent compound, (S)-(+)-carvone, significantly decreased the expression of NOS2 and IL-1ß in macrophages and in a cell model of osteoarthritis using primary human chondrocytes. (S)-(+)-carvone may be efficient in halting inflammation-related diseases, like osteoarthritis.

The importance of determining circadian parameters in pharmacological studies.

In mammals, most molecular and cellular processes show circadian changes, leading to daily variations in physiology and ultimately in behaviour. Such daily variations induce a temporal coordination of processes that is essential to ensure homeostasis and health. Thus, it is of no surprise that pharmacokinetics (PK) and pharmacodynamics (PD) of many drugs are also subject to circadian variations, profoundly affecting their efficacy and tolerability. Understanding how circadian rhythms influence drug PK, PD, and toxicity might significantly improve treatment efficacy and decrease related side effects. Therefore, it is essential to take circadian variations into account and to determine circadian parameters in pharmacological studies, especially when drugs have a short half-life or target rhythmic processes. This review provides an overview of the current knowledge on circadian rhythms and their relevance to the field of pharmacology. Methodologies to evaluate circadian rhythms in vitro, in rodent models and in humans, from experimental to computational approaches, are described and discussed. Lastly, we aim at alerting the scientific, medical, and regulatory communities to the relevance of the physiological time, as a key parameter to be considered when designing pharmacological studies. This will eventually lead to more successful preclinical and clinical trials and pave the way to a more personalized treatment to the benefit of the patients.

The "Journal of Functional Morphology and Kinesiology" Journal Club Series: Highlights on Recent Papers in Exercise-Induced Immune Response.

We are glad to introduce the ninth Journal Club. This edition is focused on several relevant studies published in the last few years in the field of Exercise-Induced Immune Response, chosen by our Editorial Board members and their colleagues. We hope to stimulate your curiosity in this field and to share with you the passion for sport seen also from the scientific point of view. The Editorial Board members wish you an inspiring lecture.

Assessment of cell line competence for studies of pharmacological GPR30 modulation.

CONTEXT/OBJECTIVE: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. MATERIALS AND METHODS: The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17ß-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. RESULTS: None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17ß-estradiol and G-1. DISCUSSION AND CONCLUSION: Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis.

Osteoarthritis is a progressive joint disease and a major cause of disability for which no curative therapies are yet available. To identify compounds with potential anti-osteoarthritic properties, in this study, we screened one sesquiterpene, E-caryophyllene, and two monoterpenes, myrcene and limonene, hydrocarbon compounds for anti-inflammatory, anti-catabolic and pro-anabolic activities in human chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1ß-induced nitric oxide production (IC50=37.3µg/ml and 85.3µg/ml, respectively), but E-caryophyllene was inactive. Myrcene, and limonene to a lesser extent, also decreased IL-1ß-induced NF-κB, JNK and p38 activation and the expression of inflammatory (iNOS) and catabolic (MMP-1 and MMP-13) genes, while increasing the expression of anti-catabolic genes (TIMP-1 and -3 by myrcene and TIMP-1 by limonene). Limonene increased ERK1/2 activation by 30%, while myrcene decreased it by 26%, relative to IL-1ß-treated cells. None of the compounds tested was able to increase the expression of cartilage matrix-specific genes (collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage specific, collagen I, induced by IL-1ß. These data show that myrcene has significant anti-inflammatory and anti-catabolic effects in human chondrocytes and, thus, its ability to halt or, at least, slow down cartilage destruction and osteoarthritis progression warrants further investigation.

Screening of five essential oils for identification of potential inhibitors of IL-1-induced Nf-kappaB activation and NO production in human chondrocytes: characterization of the inhibitory activity of alpha-pinene.

Nuclear factor-kappaB is a key transcription factor activated by pro-inflammatory signals, like interleukin-1beta (IL-1), being required for the expression of many inflammatory and catabolic mediators, such as nitric oxide (NO), that play an important role in arthritic diseases. This work aimed at screening and identifying natural inhibitors of IL-induced NF-kappaB activation and NO production in human articular chondrocytes. Five essential oils obtained from four plants of the Iberian flora, Mentha x piperita L. (Lamiaceae), Origanum virens L. (Lamiaceae), Lavandula luiseri L. (Lamiaceae), and Juniperus oxycedrus L. subsp. oxycedrus (Cupressaceae), were screened for their ability to prevent IL-1-induced NO production. The oil showing higher inhibitory activity was fractionated, concentrated, analyzed for composition elucidation and prepared for further assays. For this purpose, the human chondrocytic cell line C-28/I2 was used to evaluate NF-kappaB activation by determining the cytoplasmic levels of the total and phosphorylated forms of the inhibitory protein, I kappaB-alpha, and the NF-kappaB-DNA binding activity. The essential oil from the leaves of J. oxycedrus in a concentration of 0.02 % (v/v) achieved the greatest inhibition (80 +/- 8%) of IL-1-induced NO production. Chemical analysis showed that this essential oil is predominantly composed of monoterpene hydrocabons, being alpha-pinene [2,6,6-trimethyl-bicyclo(3.1.1)hept-3-ene] the major constituent (76 %). Similarly to the effect of the whole oil, a fraction containing 93% alpha-pinene reduced significantly IL-1-induced I kappaB-alpha degradation. Moreover, alpha-pinene also decreased I kappaB-alpha phosphorylation, NF-kappaB-DNA binding activity, and NO production. Another fraction containing oxygenated mono- and sesquiterpenes was nearly as effective as alpha-pinene. The ability of the alpha-pinene-containing fraction to reduce IL-1-induced NF-kappaB activation and NO production warrants further studies to demonstrate the usefulness of alpha-pinene in the treatment of arthritic diseases and other conditions in which NF-kappaB and NO play pathological roles.

Facilitative glucose transporters in articular chondrocytes. Expression, distribution and functional regulation of GLUT isoforms by hypoxia, hypoxia mimetics, growth factors and pro-inflammatory cytokines.

Articular cartilage is a unique and highly specialized avascular connective tissue in which the availability of oxygen and glucose is significantly lower than synovial fluid and plasma. Glucose is an essential source of energy during embryonic growth and fetal development and is vital for mesenchymal cell differentiation, chondrogenesis, and skeletal morphogenesis. Glucose is an important metabolic fuel for differentiated chondrocytes during postnatal development and in adult articular cartilage and is a common structural precursor for the synthesis of extracellular matrix glycosaminoglycans. Glucose metabolism is critical for growth plate chondrocytes which participate in long bone growth. Glucose concentrations in articular cartilage can fluctuate depending on age, physical activity, and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the concentration of oxygen and glucose in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Consequently chondrocytes must have the capacity to survive in an extracellular matrix with limited nutrients and low oxygen tensions. Published data from our laboratories suggest that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. In other tissues GLUT proteins are expressed in a cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. Several GLUTs expressed in chondrocytes are regulated by hypoxia, hypoxia mimetics, metabolic hormones, and proinflammatory cytokines. In this multidisciplinary text we review the molecular and morphological aspects of GLUT expression and function in chondrocytes and their mesenchymal and embryonic stem cell precursors and propose key roles for these proteins in glucose sensing and metabolic regulation in cartilage.

Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-kappa B activation and iNOS expression in bovine articular chondrocytes.

Our previous studies showed that reactive oxygen species (ROS) are required for the pro-inflammatory cytokine interleukin-1 beta (IL-1) to induce the activity of the Nuclear transcription Factor-kappa B (NF-kappa B) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H(2)O(2)) and the superoxide radical, two major ROS, in mediating those IL-1-induced responses. The results obtained show that chondrocytes produce both H(2)O(2) and superoxide radical in response to IL-1. Treatment of the chondrocyte cultures with H(2)O(2) alone did not induce NF-kappa B activation or iNOS expression. Addition of H(2)O(2) simultaneously with IL-1 did neither enhance nor inhibit NF-kappa B activation and iNOS expression, relatively to treatment with IL-1 alone. Accordingly, treatment with catalase did not inhibit those IL-1-induced responses. Treatment with superoxide dismutase, however, effectively prevented IL-1-induced I kappa B-alpha degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL-1-induced I kappa B-alpha degradation and the consequent NF-kappa B activation and iNOS expression in chondrocytes, whereas H(2)O(2) does not seem to participate in those IL-1-induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL-1-induced signaling pathway that leads to NF-kappa B activation and to the expression of NF-kappa B-dependent genes in bovine articular chondrocytes.

Diacerhein and rhein prevent interleukin-1beta-induced nuclear factor-kappaB activation by inhibiting the degradation of inhibitor kappaB-alpha.

Diacerhein and rhein are anthraquinone compounds that ameliorate the course of osteoarthritis. Recent reports also suggest that these compounds may have antiinflammatory properties, but the cellular mechanisms by which they exert antiosteoarthritic and possibly antiinflammatory effects are still incompletely understood. The purpose of this study was to investigate the ability of diacerhein and rhein to inhibit the activation of the transcription factor nuclear factor kappaB, induced by the proinflammatory cytokine interleukin-1beta, in primary monolayer cultures of bovine articular chondrocytes. We also studied the ability of diacerhein and rhein to prevent the expression of the inducible nitric oxide synthase gene, which is driven by nuclear factor-kappaB. We observed that interleukin-1beta induced the degradation of the inhibitor kappaB-alpha protein and the translocation of the protein p65 (a member of the nuclear factor-kappaB family) to the nucleus, which were inhibited by diacerhein and rhein, in a dose-dependent manner. Interleukin-1beta-induced nuclear factor-kappaB binding to a specific (gamma-(32)P)-labelled oligonucleotide probe was also inhibited by treatment of chondrocytes with diacerhein or rhein, as revealed by electrophoretic mobility shift assay. Inducible nitric oxide synthase mRNA and protein synthesis and nitric oxide production were also inhibited by diacerhein and rhein, in a dose-dependent manner. The half-maximal inhibitory concentrations of diacerhein and rhein, relative to nitric oxide production, were 8.2 microM ;and 7.7 microM, respectively. These results suggest that diacerhein and rhein inhibit nuclear factor-kappaB activation and, consequently, the expression of nuclear factor-kappaB-dependent genes, such as the inducible nitric oxide synthase gene, which can explain their antiosteoarthritic and antiinflammatory effects.