Rheumatoid arthritis molecular targets and their importance to flavonoid-based therapy.

Rheumatoid arthritis (RA) is a progressive, chronic, autoimmune, inflammatory, and systemic condition that primarily affects the synovial joints and adjacent tissues, including bone, muscle, and tendons. The World Health Organization recognizes RA as one of the most prevalent chronic inflammatory diseases. In the last decade, there was an expansion on the available RA therapeutic options which aimed to improve patient's quality of life. Despite the extensive research and the emergence of new therapeutic approaches and drugs, there are still significant unwanted side effects associated to these drugs and still a vast number of patients that do not respond positively to the existing therapeutic strategies. Over the years, several references to the use of flavonoids in the quest for new treatments for RA have emerged. This review aimed to summarize the existing literature about the flavonoids' effects on the major pathogenic/molecular targets of RA and their potential use as lead compounds for the development of new effective molecules for RA treatment. It is demonstrated that flavonoids can modulate various players in synovial inflammation, regulate immune cell function, decrease synoviocytes proliferation and balance the apoptotic process, decrease angiogenesis, and stop/prevent bone and cartilage degradation, which are all dominant features of RA. Although further investigation is necessary to determine the effectiveness of flavonoids in humans, the available data from in vitro and in vivo models suggest their potential as new disease-modifying anti-rheumatic drugs. This review highlights the use of flavonoids as a promising avenue for future research in the treatment of RA.

Methodologies for Assessing Pancreatic Lipase Catalytic Activity: A Review.

Obesity is a disease of epidemic proportions with a concerning increasing trend. Regarded as one of the main sources of energy, lipids can also represent a big part of an unnecessary intake of calories and be, therefore, directly related to the problem of obesity. Pancreatic lipase is an enzyme that is essential in the absorption and digestion of dietary fats and has been explored as an alternative for the reduction of fat absorption and consequent weigh loss.Literature describes a great variability of methodologies and experimental conditions used in research to evaluate the in vitro inhibitory activity of compounds against pancreatic lipase. However, in an attempt to choose the best approach, it is necessary to know all the reaction conditions and understand how these can affect the enzymatic assay.The objective of this review is to understand and summarize the methodologies and respective experimental conditions that are mainly used to evaluate pancreatic lipase catalytic activity.156 studies were included in this work and a detailed description of the most commonly used UV/Vis spectrophotometric and fluorimetric instrumental techniques are presented, including a discussion regarding the differences found in the parameters used in both techniques, namely enzyme, substrate, buffer solutions, kinetics conditions, temperature and pH.This works shows that both UV/Vis spectrophotometry and fluorimetry are useful instrumental techniques for the evaluation of pancreatic lipase catalytic activity, presenting several advantages and limitations, which make the choice of parameters and experimental conditions a crucial decision to obtain the most reliable results.

The role of flavonoids in the regulation of epithelial-mesenchymal transition in cancer: A review on targeting signaling pathways and metastasis.

One of the hallmarks of cancer is metastasis, a process that entails the spread of cancer cells to distant regions in the body, culminating in tumor formation in secondary organs. Importantly, the proinflammatory environment surrounding cancer cells further contributes to cancer cell transformation and extracellular matrix destruction. During metastasis, front-rear polarity and emergence of migratory and invasive features are manifestations of epithelial-mesenchymal transition (EMT). A variety of transcription factors (TFs) are implicated in the execution of EMT, the most prominent belonging to the Snail Family Transcriptional Repressor (SNAI) and Zinc Finger E-Box Binding Homeobox (ZEB) families of TFs. These TFs are regulated by interaction with specific microRNAs (miRNAs), as miR34 and miR200. Among the several secondary metabolites produced in plants, flavonoids constitute a major group of bioactive molecules, with several described effects including antioxidant, antiinflammatory, antidiabetic, antiobesogenic, and anticancer effects. This review scrutinizes the modulatory role of flavonoids on the activity of SNAI/ZEB TFs and on their regulatory miRNAs, miR-34, and miR-200. The modulatory role of flavonoids can attenuate mesenchymal features and stimulate epithelial features, thereby inhibiting and reversing EMT. Moreover, this modulation is concomitant with the attenuation of signaling pathways involved in diverse processes as cell proliferation, cell growth, cell cycle progression, apoptosis inhibition, morphogenesis, cell fate, cell migration, cell polarity, and wound healing. The antimetastatic potential of these versatile compounds is emerging and represents an opportunity for the synthesis of more specific and potent agents.

Silver nanoparticles exert toxic effects in human monocytes and macrophages associated with the disruption of Δψm and release of pro-inflammatory cytokines.

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50 nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0-25 µg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect.

Inhibitory activity of flavonoids against human sucrase-isomaltase (α-glucosidase) activity in a Caco-2/TC7 cellular model.

Type 2 diabetes (T2D) is the most common form of diabetes, and the number of people with this metabolic disease is steadily increasing worldwide. Among the available antidiabetic agents, α-glucosidase inhibitors are the most effective at reducing postprandial hyperglycaemia (PPHG), one of the main characteristics of T2D. However, most of the studies that have been performed have used the more readily available rat intestinal preparations or yeast α-glucosidase as the enzyme source, which despite being useful and cost effective, have a questionable physiological value. The present study evaluates the inhibitory activity of a selected group of flavonoids against human sucrase-isomaltase (SI), the α-glucosidase found in Caco-2/TC7 cells. A microassay using the physiological substrates sucrose and maltose, and a synthetic substrate, p-nitrophenyl-α-D-glucopyranoside (pNPG) was performed. The most active flavonoid was compound 4 (melanoxetin), presenting an IC50 value similar using the two natural substrates. In contrast, the tested flavonoids were not effective at inhibiting SI, when pNPG was used as a substrate. Hydroxylation of flavonoids at C-3 of the C ring, at C-3' and C-4' of the B ring, and at C-7 and C-8 of the A ring were the features that improved the inhibitory activity of flavonoids against human SI. These phenolic compounds deserve further exploration as alternatives to the currently available α-glucosidase inhibitors. The present study also demonstrates that the non-clinical in vitro studies conducted for the evaluation of α-glucosidase activity should use the human source rather than surrogate sources of α-glucosidase.

Protective Role of Flavonoids against Intestinal Pro-Inflammatory Effects of Silver Nanoparticles.

Silver nanoparticles (AgNP) have been increasingly incorporated into food-related and hygiene products for their unique antimicrobial and preservative properties. The consequent oral exposure may then result in unpredicted harmful effects in the gastrointestinal tract (GIT), which should be considered in the risk assessment and risk management of these materials. In the present study, the toxic effects of polyethyleneimine (PEI)-coated AgNP (4 and 19 nm) were evaluated in GIT-relevant cells (Caco-2 cell line as a model of human intestinal cells, and neutrophils as a model of the intestinal inflammatory response). This study also evaluated the putative protective action of dietary flavonoids against such harmful effects. The obtained results showed that AgNP of 4 and 19 nm effectively induced Caco-2 cell death by apoptosis with concomitant production of nitric oxide, irrespective of the size. It was also observed that AgNP induced human neutrophil oxidative burst. Interestingly, some flavonoids, namely quercetin and quercetagetin, prevented the deleterious effects of AgNP in both cell types. Overall, the data of the present study provide a first insight into the promising protective role of flavonoids against the potentially toxic effects of AgNP at the intestinal level.

Flavonoids as antiobesity agents: A review.

Obesity is a global health problem that affects all age groups in both developing and developed countries. In recent years, the prevalence of overweight and obesity has reached pandemic levels, resulting in a dramatic increase in the incidence of various comorbidities, such as cardiovascular diseases, type 2 diabetes, and cancer, consequently leading to massive health and socioeconomic burdens. Together with lifestyle changes, antiobesity pharmacotherapy is gaining momentum as an adjunctive treatment. However, the available pharmacological approaches have limited use owing to either significant adverse effects or low efficacy. Over the years, natural products have been an important source of lead compounds for drug discovery. Among these, flavonoids are associated with important biological effects and health-promoting activities. In this review, we discuss the modulatory effects of flavonoids on obesity and their potential mechanisms of action. The literature strongly suggests that most common flavonoids demonstrate a pronounced effect on obesity as shown by their ability to lower body weight, fat mass, and plasma triglycerides/cholesterol, both in in vitro and in vivo models. The impact of flavonoids on obesity can be observed through different mechanisms: reducing food intake and fat absorption, increasing energy expenditure, modulating lipid metabolism, or regulating gut microbiota profile. A better understanding of the known antiobesity mechanisms of flavonoids will enable their potential use to treat this medical condition. Therefore, this review focuses on the putative biological mechanisms through which flavonoids may prevent or treat obesity and highlights new perspectives on future pharmacological use.

ß-Carotene and its physiological metabolites: Effects on oxidative status regulation and genotoxicity in in vitro models.

Carotenoids are ubiquitously distributed in nature, ß-carotene being the most frequently found carotenoid in the human diet. In the human body, ß-carotene is absorbed, distributed and metabolized by enzymatic and/or non-enzymatic oxidant cleavage into several metabolites. Despite the broadly accepted biological value of ß-carotene, it has also been considered a double-edged sword, mainly due to its potential antioxidant versus pro-oxidant behaviour. In this sense, the aim of this work was to scrutinize the antioxidant or pro-oxidant potential of ß-carotene and its metabolites, namely trans-ß-apo-8'-carotenal and ß-ionone. Several parameters were evaluated in this study, viz. their effects on reactive species production, both in human whole blood and neutrophils; their effects on lipid peroxidation, in the absence and presence of peroxynitrite anion (ONOO-) or hydrogen peroxide (H2O2), using a synaptosomal model; and finally, their putative genotoxic effects in the human hepatic HepG2 cell line. In general, depending on the cellular model and conditions tested, ß-carotene and its metabolites revealed antioxidant effects to varying degrees without significant pro-oxidant or genotoxic effects.

Expression and function of the nonclassical estrogen receptor, GPR30, in human cartilage and chondrocytes.

Estrogen hormones are important for cartilage homeostasis, but nothing is known regarding the expression and role of the membrane G protein-coupled estrogen receptor (GPER), G protein-coupled receptor 30 (GPR30), in adult articular chondrocytes. Using immunohistochemistry of cartilage sections, quantitative real-time polymerase chain reaction and Western blot of chondrocyte extracts, we found that these cells express GPR30. Nonetheless, the pattern of bands detected by two distinct antibodies does not overlap, suggesting that the proteins detected represent partially degraded forms of the receptor. Treatment with GPR30 agonists did not induce Akt or ERK1/2 phosphorylation, two known GPR30-activated signaling pathways, suggesting that GPR30 is not functional in human chondrocytes. Therefore, the protective anti-osteoarthritic role of estrogen hormones in cartilage homeostasis is likely independent of GPR30. This study was performed using human cartilage collected from the distal femoral condyles of multiorgan donors at the Bone and Tissue Bank of the University and Hospital Center of Coimbra.

Differential effects of the essential oils of Lavandula luisieri and Eryngium duriaei subsp. juresianum in cell models of two chronic inflammatory diseases.

CONTEXT: Effective drugs to treat osteoarthritis (OA) and inflammatory bowel disease (IBD) are needed. OBJECTIVE: To identify essential oils (EOs) with anti-inflammatory activity in cell models of OA and IBD. MATERIALS AND METHODS: EOs from Eryngium duriaei subsp. juresianum (M. Laínz) M. Laínz (Apiaceae), Laserpitium eliasii subsp. thalictrifolium Sennen & Pau (Apiaceae), Lavandula luisieri (Rozeira) Rivas-Martínez (Lamiaceae), Othantus maritimus (L.) Hoff. & Link (Asteraceae), and Thapsia villosa L. (Apiaceae) were analyzed by GC and GC/MS. The anti-inflammatory activity of EOs (5-200 µg/mL) was evaluated by measuring inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) activation (total and phosphorylated IκB-α), in primary human chondrocytes and the intestinal cell line, C2BBe1, stimulated with interleukin-1ß (IL-1ß) or interferon-γ (IFN-γ), IL-1ß and tumor necrosis factor-α (TNF-α), respectively. RESULTS: The EO of L. luisieri significantly reduced iNOS (by 54.9 and 81.0%, respectively) and phosphorylated IκB-α (by 87.4% and 62.3%, respectively) in both cell models. The EO of E. duriaei subsp. juresianum caused similar effects in human chondrocytes, but was inactive in intestinal cells, even at higher concentrations. The EOs of L. eliasii subsp. thalictrifolium and O. maritimus decreased iNOS expression by 45.2 ± 8.7% and 45.2 ± 6.2%, respectively, in C2BBe1 cells and were inactive in chondrocytes. The EO of T. villosa was inactive in both cell types. DISCUSSION AND CONCLUSION: This is the first study showing anti-inflammatory effects of the EOs of L. luisieri and E. duriaei subsp. juresianum. These effects are specific of the cell type and may be valuable to develop new therapies or as sources of active compounds with improved efficacy and selectivity towards OA and IBD.

Resveratrol modulates cytokine-induced Jak/STAT activation more efficiently than 5-aminosalicylic acid: an in vitro approach.

BACKGROUND: Many advances have been recently made focused on the valuable help of dietary polyphenols in chronic inflammatory diseases. On the other hand, current treatment options for intestinal bowel disease patients are unsatisfying and, for this reason, it is estimated that many patients use dietary supplements to achieve extra benefits. AIM: The aim of this work was to analyze under a mechanistic perspective the anti-inflammatory potential of resveratrol, a natural polyphenolic compound, and to compare it with a pharmaceutical agent, 5-aminosalicylic acid, using the intestinal HT-29 cell line, as a cellular model. METHODOLOGY AND PRINCIPAL FINDINGS: In the present study, HT-29 colon epithelial cells were pre-treated with 25 µM resveratrol and/or 500 µM 5-aminosalicylic acid and then exposed to a combination of cytokines (IL-1α, TNF-α, IFN-γ) for a certain period of time. Our data showed that resveratrol, used in a concentration 20 times lower than 5-aminosalicylic acid, was able to significantly reduce NO and PGE2 production, iNOS and COX-2 expression and reactive oxidant species formation induced by the cytokine challenge. However, as already verified with 5-aminosalicylic acid, in spite of not exhibiting any effect on IkB-α degradation, resveratrol down-regulated JAK-STAT pathway, decreasing the levels of activated STAT1 in the nucleus. Additionally, resveratrol decreased the cytokine-stimulated activation of SAPK/JNK pathway but did not counteract the cytokine-triggered negative feedback mechanism of STAT1, through p38 MAPK. CONCLUSION/SIGNIFICANCE: Taken together, our results show that resveratrol may be considered a future nutraceutical approach, promoting remission periods, limiting the inflammatory process and preventing colorectal cancer, which is common in these patients.

Anti-inflammatory and chondroprotective activity of (+)-α-pinene: structural and enantiomeric selectivity.

Previous studies have suggested that α-pinene, a common volatile plant metabolite, may have anti-inflammatory effects in human chondrocytes, thus exhibiting potential antiosteoarthritic activity. The objective of this study was to further characterize the potential antiosteoarthritic activity of selected pinene derivatives by evaluating their ability to modulate inflammation and extracellular matrix remodeling in human chondrocytes and to correlate the biological and chemical properties by determining whether the effects are isomer- and/or enantiomer-selective. To further elucidate chemicopharmacological interactions, the activities of other naturally occurring monoterpenes with the pinane nucleus were also investigated. At noncytotoxic concentrations, (+)-α-pinene (1) elicited the most potent inhibition of the IL-1ß-induced inflammatory and catabolic pathways, namely, NF-κB and JNK activation and the expression of the inflammatory (iNOS) and catabolic (MMP-1 and -13) genes. (-)-α-Pinene (2) was less active than the (+)-enantiomer (1), and ß-pinene (3) was inactive. E-Pinane (4) and oxygenated pinane-derived compounds, pinocarveol (5), myrtenal (6), (E)-myrtanol (7), myrtenol (8), and (Z)-verbenol (9), were less effective or even completely inactive and more cytotoxic than the pinenes tested (1-3). The data obtained show isomer- and enantiomer-selective anti-inflammatory and anticatabolic effects of α-pinene in human chondrocytes, (+)-α-pinene (1) being the most promising for further studies to determine its potential value as an antiosteoarthritic drug.

Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: possible role in glucose sensing.

ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.

Role of glucose as a modulator of anabolic and catabolic gene expression in normal and osteoarthritic human chondrocytes.

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro-anabolic stimuli, namely Transforming Growth Factor-ß (TGF). For this, we analyzed by real time RT-PCR the expression of articular cartilage matrix-specific and non-specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)-1 and -13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP-1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage.