RESUMO
Malaria is a parasitic infection responsible for high morbidity and mortality rates worldwide. During the disease, phagocytosis of infected red blood cells by the macrophages induces the production of reactive oxygen (ROS) and nitrogen species (RNS), culminating in parasite death. Curcumin (CUR) is a bioactive compound that has been demonstrated to reduce the production of pro-inflammatory cytokines and chemokines produced by macrophages but to reduce parasitemia in infected mice. Hence, the main purpose of this study is to investigate whether curcumin may interfere with macrophage function and polarization after Plasmodium berghei infection in vitro. In our findings, non-polarized macrophage (M0), classically activated (M1), and alternatively activated (M2) phenotypes showed significantly increased phagocytosis of infected red blood cells (iRBCs) when compared to phagocytosis of uninfected red blood cells (RBCs) 3 h after infection. After 24 h, M1 macrophages exposed to RBCs + CUR showed greater elimination capacity when compared to macrophages exposed to iRBCs + CUR, suggesting the interference of curcumin with the microbicidal activity. Additionally, curcumin increased the phagocytic activity of macrophages when used in non-inflammatory conditions (M0) and reduced the inducible nitric oxide synthase (iNOS) and arginase activities in all macrophage phenotypes infected (M0, M1, and M2), suggesting interference in arginine availability by curcumin and balance promotion in macrophage polarization in neutral phenotype (M0). These results support the view of curcumin treatment in malaria as an adjuvant, promoting a balance between pro- and anti-inflammatory responses for a better clinical outcome.
RESUMO
The potentiation of pharmacological effects can be achieved through several strategies, such as the association of substances and delivery in nanostructured systems. In practice, potentiation can be measured by the law of mass action and joint evaluation of the combination index (CI) and dose-response curves. In this context, this study aimed to evaluate the anti-inflammatory effect of the association of ß-caryophyllene and indomethacin in the free form and delivered in nanoemulsions using the in vitro model of LPS-stimulated murine macrophage. The results indicated potentiation of the anti-inflammatory effect of nanoemulsified substances compared to free substances, as well as synergistic action between the sesquiterpene and the selected NSAID. In comparison, the association of ß-caryophyllene and indomethacin in the free form inhibited the production of nitric oxide by 50% at 48.60 µg/mL (CI = 0.21), while the nanoemulsified association of these substances resulted in an IC50 of 1.45 µg/mL (CI = 0.14). In parallel, cytotoxicity assays on HaCaT and MRC-5 cell lines demonstrated the safety of IC50-equivalent concentrations of the anti-inflammatory action, and no irritating effects on the chorioallantoic membrane of embryonated eggs were observed (HET-CAM assay). The results suggest that ß-caryophyllene may be an alternative to replace an inert oily core in nanoemulsion systems when anti-inflammatory effects are desirable.
Assuntos
Indometacina , Lipopolissacarídeos , Camundongos , Animais , Indometacina/farmacologia , Indometacina/metabolismo , Lipopolissacarídeos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismo , MacrófagosRESUMO
BACKGROUND: Jackfruit seed flour can be used as a cocoa aroma replacer with similar technological properties. The purpose of this study was to investigate the in vivo toxicity and in vitro antioxidant activity of fermented jackfruit seed flour (Fjs) and non-alkaline cocoa powder (Nac). RESULTS: Fjs and Nac extracts (Fjs-E and Nac-E) were produced and submitted to in vitro gastrointestinal digestion producing digested fractions named Fjs-D and Nac-D, respectively. Nac-E showed over two-fold higher oxygen radical absorbance capacity (ORAC) than Fjs-E. However, after simulated gastrointestinal digestion (in vitro), there were no significant differences between Nac-D and Fjs-D (P < 0.01). Similarly, the cellular antioxidant activity (CAA) of Nac-D and Fjs-D was not significantly different (P < 0.01). The anti-inflammatory assay in transgenic RAW 264.7 murine macrophages showed that Fjs-E did not affect cell viability up to 300 µg mL-1 (P > 0.05) and reduced by 15% the release of TNF-α (P < 0.05). Fjs-D did not affect cell viability up to 300 µg mL-1 (P > 0.05) and showed 58% reduction of NF-κB activation (P < 0.05), with no effects on TNF-α levels. Treatment with Nac-E up to 300 µg mL-1 did not decrease cell viability (P > 0.05) and reduced the release of TNF-α levels by 34% and 66% at 100 and 300 µg mL-1 , respectively (P < 0.05). Nac-D did not reduce the NF-κB activation or TNF-α levels at any tested concentration. CONCLUSION: Collectively, these findings indicate that Fjs is a safe and promising functional ingredient with biological activities even after gastrointestinal digestion. © 2023 Society of Chemical Industry.
Assuntos
Artocarpus , Chocolate , Camundongos , Animais , Antioxidantes/farmacologia , Antioxidantes/análise , Artocarpus/química , Farinha/análise , Fator de Necrose Tumoral alfa/genética , NF-kappa B/genética , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/análise , Sementes/química , DigestãoRESUMO
Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease, shed extracellular vesicles (EVs) that promote the susceptibility of host cells to infection. During T. cruzi infection, the immune response of the host is important for controlling parasitism, which is necessary for survival. Macrophages produce inflammatory mediators, such as eicosanoids and nitric oxide (NO), with trypanocidal effects that control the parasite load in the early stages of the disease. In this study, we evaluated the contribution of host cyclooxygenase (COX) to the actions of EVs shed by T. cruzi strain Y (EVs-Y) in infected macrophages. RAW 264.7 macrophages exposed to EVs-Y and then infected with trypomastigote forms of T. cruzi produced less NO, and an increased number of trypomastigote forms were internalized in the cell compared to the controls, indicating that the effects exerted by EVs-Y favor the parasite. Interestingly, when macrophages were pretreated with acetylsalicylic acid, a dual COX inhibitor, before exposure to EVs-Y and subsequent infection with trypomastigote forms, there was an increase in NO production and a decrease in trypomastigote uptake compared to the controls. These results suggest that EVs-Y modulates the macrophage response in favor of T. cruzi and indicate a role for COX in the effects of EVs.
Assuntos
Doença de Chagas , Vesículas Extracelulares , Trypanosoma cruzi , Humanos , Aspirina/farmacologia , Macrófagos , Ciclo-Oxigenase 2 , Óxido NítricoRESUMO
BACKGROUND AND AIM: Inflammation is the immune response to a harmful stimulus, and its purpose is to destroy foreign agents so that the affected site can be repair. When uncontrolled or unresolved, inflammation can lead to significant tissue damage. Many classes of compounds are used today as anti-inflammatory drugs. However, there is an ongoing demand for new, more effective molecules with higher safety margins. In this regard, the anti-inflammatory effect of six synthetic compounds of N-antipyrine-3,4-dichloromaleimide was evaluated. METHODS: RAW 264.7 cells were used to evaluate the cytotoxicity and the anti-inflammatory activity, by measuring the effect of these molecules on nitric oxide, IL-1ß, IL-6, MCP-1 (CCL2), TNF-α, INF-γ, IL-4, and IL-13 levels, as well as under NF-κB activation. RESULTS: Some of the tested compounds showed significant cytotoxicity (CC50 < 100 µM). Subsequently, the potential of nitric oxide (NO) inhibition as screening for potential anti-inflammatory action was evaluated. Three of the compounds tested showed a promising profile (1, 3, and 5). When the effect of these compounds was evaluated on the production of IL-1ß, IL-6, MCP-1 (CCL2), TNF-α, and INF-γ, only N-antipyrine-3,4-dichloromaleimide (1) and N-antipyrine-3-chloro-4-(3,4-dichloroaniline) maleimide (3) showed significant inhibition profiles. These two compounds were also able to increase the production of cytokines known for having an anti-inflammatory profile (IL-4 and IL-13) and inhibit the phosphorylation of the p-p65 NF-κB subunit significantly. CONCLUSION: In conclusion, these two compounds present a significant and unusual anti-inflammatory mechanism (increasing the production of anti-inflammatory mediators). They are therefore considered promising prototypes for the development of new anti-inflammatory drugs with immunomodulatory characteristics.
Assuntos
Citocinas , NF-kappa B , Humanos , Citocinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-6 , Óxido Nítrico , Interleucina-13/farmacologia , Interleucina-13/uso terapêutico , Interleucina-4 , Macrófagos , Lipopolissacarídeos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Antipirina/farmacologia , Antipirina/uso terapêutico , ImunidadeRESUMO
The inflammatory response is a common feature of many pathological conditions, and there is urgent necessity for new substances that minimize the harmful effects of inflammation. Chromenes represent a class of compounds with multiple pharmacological actions that have already been described and may be potential candidates for studies of therapeutic action. This study aimed to test novel 4-aryl-4H-chromene-derived molecules in an in vitro model of inflammation using lipopolysaccharide (LPS)-induced Raw 264.7 cells. Seven compounds derived from 4-aryl-4H-chromene were tested on Raw 264.7 cells to evaluate their cytotoxic effects. Next, the effect of the selected compounds on the pro-inflammatory mediators (tumor necrosis factor-alpha [TNF-α], monocyte chemoattractant protein-1 [MCP-1], interleukin [IL]-6) and on the anti-inflammatory mediators (IL-10 and IL-13) was analyzed, and finally, the effect of the compounds on macrophage apoptosis and expression of surface receptors (toll-like receptor 4 [TLR-4] and mannose) was evaluated. The results of this study demonstrated that changes in the molecular structure of 4-aryl-4H-chromene altered its cytotoxic profile. Therefore, derivatives that showed safe results were selected for further analyses (named compounds: 4-6). In these experiments, the compounds were able to decrease nitric oxide (NO) levels and production of MCP-1, IL-6, IL-10, and IL-13. Furthermore, these derivatives were effective in reducing macrophage apoptosis and the expression of surface receptors, as TLR-4/CD284. Moreover, compounds 5 and 6 also were effective in increasing mannose receptor (CD206) expression. The results indicate, for the first time to our knowledge, that the anti-inflammatory effect produced by chromenes is linked to macrophage repolarization (M1 to M2).
Assuntos
Anti-Inflamatórios , Benzopiranos , Macrófagos , Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like , Animais , Camundongos , Células RAW 264.7RESUMO
INTRODUCTION: Several experimental models have been designed to promote the development of new anti-inflammatory drugs. The in vitro model using RAW 264.7 cells has been widely used. However, there is still no consensus on which inflammatory mediators should initially be measured to screen for possible anti-inflammatory effects. To determine the rationality of measuring inflammatory mediators together with NO, such as the levels of tumor necrosis factor (TNF)-α, and interleukins (IL) 1ß and 6, we carried out this systematic review (SR) and meta-analysis (MA). METHODOLOGY: We conducted this SR and MA in accordance with the Preferred Reporting of Systematic Reviews and Meta-Analysis and the Cochrane Handbook for Systematic Reviews of Intervention. This review was registered in the Open Science Framework ( https://doi.org/10.17605/OSF.IO/8C3HT ). RESULTS: LPS-induced cells produced high NO levels compared to non-LPS induced, and this production was not related to cell density. TNF-α, IL-1ß, and IL-6, also showed high levels after cells had been stimulated with LPS. Though with some restrictions, all studies were reliable, as the risk of bias was detected in the test compounds and systems. CONCLUSION: Measurement of NO levels may be sufficient to screen for possible anti-inflammatory action in the context of LPS-induced RAW 264.7 cells.
Assuntos
Lipopolissacarídeos , Macrófagos , Animais , Anti-Inflamatórios/farmacologia , Biomarcadores , Mediadores da Inflamação , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B , Óxido Nítrico , Células RAW 264.7 , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 µM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis.
Assuntos
Macrófagos , Nanopartículas Metálicas , Prata , Toxoplasmose , Animais , Proliferação de Células , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Prata/farmacologia , Toxoplasma , Toxoplasmose/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The inflammatory process is a mammalian physiological reaction against infectious agents or injuries. Among the cells involved, the macrophages have a highlighted role during this process. Depending on the inflammatory context, they can polarize into pro- or anti-inflammatory profiles (M1 and M2). In this context, compounds derived from cinnamic acid have demonstrated strong evidence of anti-inflammatory activity; however, the mechanism responsible for this effect remains unclear. In this study, we investigated the anti-inflammatory activity of five cinnamate-derived dienes of synthetic origin. The compounds that did not demonstrate significant cytotoxicity were tested to assess anti-inflammatory activity (NOx ) in RAW 264.7 cells stimulated with LPS. Then, the selected compound (diene 1) was evaluated as to its ability to inhibit the secretion of pro-inflammatory cytokines (IL-1ß, TNF-α, INF-γ, MCP-1, and IL-6) and increase the production of anti-inflammatory cytokines (IL-13, IL-4, and IL-10). Finally, diene 1 was able to reduce the expression of TLR4 and increase the phagocytic activity of the macrophages. Gathering these results together, we conclude that diene 1 showed an important anti-inflammatory effect, and this effect is linked to its immunomodulatory characteristic. Since the M1 markers were reduced at the same time, M2 markers were increased by the treatment of the macrophages with diene 1.
Assuntos
Anti-Inflamatórios , Macrófagos , Animais , Anti-Inflamatórios/farmacologia , Cinamatos/metabolismo , Cinamatos/farmacologia , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Mamíferos/metabolismo , Camundongos , Células RAW 264.7RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, Echinodorus macrophyllus (Alismataceae), popularly known as chapéu-de-couro, is used to treat inflammatory diseases. Previous studies have shown a significant decrease in the acute inflammation for the aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20). AIM OF THE STUDY: This work fractionated Fr20, identified the fraction and substances responsible for the in vivo anti-inflammatory property, and demonstrated important immunomodulatory mechanisms of action. MATERIALS AND METHODS: Fr20 was fractionated using Sephadex LH-20, and the most active fraction was chromatographically analyzed (HPLC-DAD and UPLC-ESI-TOF-MS). Leukotriene B4, Prostaglandin E2, and cytokines were determined by the enzyme-linked immunosorbent assay and in vivo acute inflammation by the air pouch model. RESULTS: The subfractions SF1, SF3, and mainly the SF4 decreased NO levels (p < 0.05). SF3 and SF4 showed high DPPH scavenger activity. SF1 was more effective than SF4 in reducing vasodilation, redness, and leukocyte migration into the 4-h air pouch. SF1 inhibited 90.5% (100 mg/kg) and SF4 54.0% (50 mg/kg), mainly affecting the number of neutrophils. SF1 and SF4 reduced the protein level in the exudate. SF1 was also more effective in inhibiting neutrophil migration in a transwell assay (46.3%) and reduced (86.1%) the Leukotriene B4 level in the exudate. After five days of treatment, some SF1 anti-inflammatory mechanisms were evaluated in the air pouch's 24 h exudate and tissue. Despite the high level of inflammation of the control group in this condition, SF1 confirmed the decrease in the protein level and neutrophils migration into the pouch. It decreased the number of bone marrow cells, indicating a systemic effect of SF1. SF1 also decreased TNF-α (87%), IL-1ß (77%), CKCL1/KC (71.3%), and PGE2 (97.8%) and increased IL-10 (74.1%) levels in the air pouch exudate. Phytochemical analysis of SF1 indicates mainly hydroxycinnamoyl derivatives. CONCLUSION: Hydroxycinnamoyl derivatives present in SF1 are related to the crucial anti-inflammatory mechanisms of E. macrophyllus, decreasing the levels of TNF-α, IL-1ß, CKCL1/KC, LTB4, and PGE2 on the exudate. These results explain the reduction of vasodilatation, erythema, and neutrophil migration into the air pouch model, confirming this plant's anti-inflammatory potential.
Assuntos
Inflamação/tratamento farmacológico , Macrófagos/metabolismo , Extratos Vegetais/farmacologia , Prostaglandinas/metabolismo , Alismataceae/química , Animais , Carragenina/toxicidade , Movimento Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Prostaglandinas/genética , Células RAW 264.7RESUMO
The best conservation method for native Chilean berries has been investigated in combination with an implemented large-scale extract of maqui berry, rich in total polyphenols and anthocyanin to be tested in intestinal epithelial and immune cells. The methanolic extract was obtained from lyophilized and analyzed maqui berries using Folin-Ciocalteu to quantify the total polyphenol content, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) to measure the antioxidant capacity. Determination of maqui's anthocyanins profile was performed by ultra-high-performance liquid chromatography (UHPLC-MS/MS). Viability, cytotoxicity, and percent oxidation in epithelial colon cells (HT-29) and macrophages cells (RAW 264.7) were evaluated. In conclusion, preservation studies confirmed that the maqui properties and composition in fresh or frozen conditions are preserved and a more efficient and convenient extraction methodology was achieved. In vitro studies of epithelial cells have shown that this extract has a powerful antioxidant strength exhibiting a dose-dependent behavior. When lipopolysaccharide (LPS)-macrophages were activated, noncytotoxic effects were observed, and a relationship between oxidative stress and inflammation response was demonstrated. The maqui extract along with 5-aminosalicylic acid (5-ASA) have a synergistic effect. All of the compiled data pointed out to the use of this extract as a potential nutraceutical agent with physiological benefits for the treatment of inflammatory bowel disease (IBD).
RESUMO
BACKGROUND: The macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregulatory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii. METHODS AND RESULTS: We demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-α in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-α even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control. CONCLUSION: Polarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen.
RESUMO
V. meridionale Swartz is an underutilized Andean Berry that has been linked to several health benefits potentially derived from its anti-inflammatory effects. This research aimed to evaluate the impact of Andean Berry Juice (ABJ) combined with Aspirin in the modulation of anti-inflammatory markers from LPS-stimulated RAW 264.7 macrophages. The chemical characterization of ABJ showed a high content of polyphenols, mainly gallic acid (659-75 µg/g) and cyanidin chloride (418.61 µg/mL). Compared to LPS-stimulated macrophages, ABJ, Aspirin, and its combination reduced NO and ROS production (3.26-42.55 and 17.59-65.68%, respectively). In comparison, the half inhibitory concentration of NO reduction (IC50) was found at 7.69% v/v (ABJ) and 24.48 mM (Aspirin). Compared to the pro-inflammatory control (LPS), ABJ reduced IL-1ß, MCP-1, and GCSF; Aspirin decreased IL1R1, MCP-1, GMCSF, GCSF, and TNF-α; and the ABJ + Aspirin treatment reduced IL1R, GMCSF, and CXCL10. The in silico interaction of cytokines and the prediction of potential binding interactions suggested CCR1, CCR5, and NF-kB modulation. These results showed the anti-inflammatory potential of underutilized South American berries and their co-adjuvant effect with known drugs such as Aspirin in the resolution of inflammatory-derived conditions. This is the first report of the anti-inflammatory effects of V. meridionale Swartz juice in combination with Aspirin on LPS-challenged RAW 264.7 macrophages.
Assuntos
Lipopolissacarídeos , Vaccinium , Animais , Anti-Inflamatórios/farmacologia , Aspirina/farmacologia , Frutas , Macrófagos , CamundongosRESUMO
Aim: Plant products have been evaluated to control opportunistic micro-organisms, as well as fortify immune system cells. Thus, Curcuma longa L. (turmeric) extract was evaluated in interactions of murine macrophages (RAW 264.7) with Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, in order to establish cooperation with defense cells. Materials & methods: Effects of minimal inhibitory concentrations (MIC) of the plant extract were analyzed on phagocytosis, cell viability of RAW 264.7 and production of inflammation-related molecules (IL-1ß, TNF-α, IL-10 and NO). Results: The plant extract was cytocompatible and promoted significant reductions of micro-organisms, and synthesis of inflammation-related molecules, during interactions. Conclusion:C. longa L. extract showed significant antimicrobial response and cooperation with macrophages, by fighting bacteria and yeasts during host-microbe interactions.
Assuntos
Anti-Infecciosos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Extratos Vegetais/farmacologia , Animais , Anti-Infecciosos/química , Candida albicans/efeitos dos fármacos , Candida albicans/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Extratos Vegetais/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Células RAW 264.7 , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologiaRESUMO
Leishmaniasis is an anthropozoonotic disease, and dogs are considered the main urban reservoir of the parasite. Macrophages, the target cells of Leishmania sp., play an important role during infection. Although dogs have a major importance in the epidemiology of the disease, the majority of the current knowledge about Leishmania-macrophage interaction comes from murine experimental models. To assess whether the canine macrophage strain DH82 is an accurate model for the study of Leishmania interaction, we compared its infection by two species of Leishmania (Leishmania infantum and L. amazonensis) with the murine macrophage cell line (RAW264.7). Our results demonstrated that L. amazonensis survival was around 40% at 24 h of infection inside both macrophage cell lines; however, a reduction of 4.3 times in L. amazonensis infection at 48 h post-infection in RAW 264.7 macrophages was observed. The survival index of L. infantum in DH82 canine macrophages was around 3 times higher than that in RAW264.7 murine cells at 24 and 48 h post-infection; however, at 48 h a reduction in both macrophages was observed. Surprisingly at 24 h post-infection, NO and ROS production by DH82 canine cells stimulated with LPS or menadione or during Leishmania infection was minor compared to murine RAW264.7. However, basal arginase activity was higher in DH82 cells when compared to murine RAW264.7 cells. Analysis of the cytokines showed that these macrophages present a different response profile. L. infantum induced IL-12, and L. amazonensis induced IL-10 in both cell lines. However, L. infantum and L. amazonensis also induced TGF-ß in RAW 264.7. CD86 and MHC expression showed that L. amazonensis modulated them in both cell lines. Conversely, the parasite load profile did not show significant difference between both macrophage cell lines after 48 h of infection, which suggests that other mechanisms of Leishmania control could be involved in DH82 cells.
Assuntos
Leishmania infantum , Leishmania mexicana , Animais , Linhagem Celular , Cães , Macrófagos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary. PURPOSE: The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages. METHODS: For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-ß by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique. RESULTS: The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-ß and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA. CONCLUSION: Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.
Assuntos
Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Extratos Vegetais/farmacologia , Anacardiaceae/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocinas/análise , Citocinas/imunologia , Interleucina-10/análise , Macrófagos/parasitologia , Camundongos , Células RAW 264.7RESUMO
The objective of this study was to investigate and quantify the composition phenolic, reducing capacity, the free radical scavenging activity, as well as, the anti-inflammatory effect evaluated against lipopolysaccharides-stimulated RAW264.7 macrophages through modulation of inflammatory mediators, in eight stingless bee honey types (Meliponinae) from southern Brazil. Stingless bee honey did not show to be cytotoxic at the tested concentrations (1-100 µM) and also reduced nitric oxide and the secretion of the pro-inflammatory cytokine in the inflamed macrophages. Two honey samples showed the ability to increase the secretion of anti-inflammatory cytokine (interleukin-10), suggesting a significant anti-inflammatory effect. All these findings indicate that stingless bee honey could be an important source of natural compounds presenting anti-inflammatory and antioxidant effect, which could would provide health benefits when included in the diet.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Abelhas/metabolismo , Mel/análise , Fenóis/farmacologia , Animais , Anti-Inflamatórios/análise , Antioxidantes/análise , Brasil , Sobrevivência Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cromatografia Líquida de Alta Pressão , Citocinas/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Fenóis/análise , Células RAW 264.7 , Espectrometria de Massas em TandemRESUMO
The biocompatibility, bionanointeraction, uptake efficiency, and entry pathway of luminescent nanomaterials are the key factors to understand development of an efficient bionanoprobe. The foremost objective of this work is to explore the potential of 3-mercaptopropionic acid (3-MPA) capped ZnSe:xMn2+ (x = 5, 10, and 15 mol %) quantum dots (QDs) for the development of bionanoprobe used in future biological and clinical applications. For this purpose, highly intense orange-emitting activator Mn2+ ion doped ZnSe QDs were synthesized via a high-temperature organometallic method and rendered water-soluble by a ligand exchange approach. The morphological and physicochemical characterizations displayed the ultrasmall zinc-blend cubic crystal structure of QDs with an elliptical shape nanocrystals and average diameter of 4 nm. The luminescent nanomaterials exhibited orange emission centered at 584 nm under excitation at 385 nm. The biocompatibility, time-dependent cellular uptake, and the uptake mechanism of QDs were studied in RAW 264.7 macrophages, accomplished by various cytotoxicity assays, CytoViva hyperspectral enhanced dark-field and dual-mode fluorescence (DMF) microscopy, and transmission electron microscopy (TEM) images. The cytotoxicity study did not confirm any noticeable deleterious effect of QDs within incubation for 6 h. The fluorescence images of cells incubated with QDs showed efficient emission, which is a manifestation that QDs are photochemically stable in the intracellular environment. The cellular uptake findings demonstrated that the QDs were predominantly internalized via clathrin- and caveolae-mediated pathways. After the uptake, QDs aggregates appeared inside the vesicles in the cytoplasm, and their number and size gradually increased as a function of time. Nevertheless, the fluorescent QDs presented remarkable colloidal stability in various media, biocompatibility within the designated time, efficient time-dependent uptake, and distinct entry pathway in RAW macrophages, suggesting promising candidates to explore for the development of future bionanoprobes.
RESUMO
El ministerio de salud tiene como una de las prioridades en investigación a las enfermedades infecciosas, entre las que incluye a las leishmaniasis y la tripanosomiasis americana. En la Facultad Ciencias Farmacéuticas y Bioquímicas se estudian especies vegetales medicinales utilizadas por la cultura Tacana como fuente de agentes antiparasitarios potenciales. Los laboratorios dedicados al descubrimiento de moléculas naturales con actividad antiparasitaria, tienen el desafío de desarrollar protocolos que permitan detectar biomoléculas selectivas, efectivas y menos tóxicas que las disponibles. Por ende, las evaluaciones antiparasitarias in vitro (CI50), deberían ir acompañadas de evaluaciones de citotoxicidad (DL50), con el fin de calcular un Índice de Selectividad (IS=DL50 / CI50) como parámetro de especificidad biológica. La citotoxicidad fue medida sobre líneas de macrófagos (RAW 264.7, murino), células involucradas en las infecciones por parásitos intracelulares. El protocolo fluorométrico parte con una población de 5x104 células/mL, incubada a 37ºC en DMEM (Dulbecco's Modified Eagle's médium), por 96 horas, con adición de resazurina (2mM) 3 horas antes de las lecturas finales. Bajo estas condiciones se evaluó la citotoxicidad de drogas control y 15 extractos vegetales seleccionados por su actividad anti-kinetoplastida. El extracto de Cosmailu fue el más citotóxico, Ejije bid'u resultó selectivo para T. cruzi y Leishmania amazonensis, mientras que Id'ene eidhue, fue selectivo para L. amazonensis. Finalmente, los otros 12 extractos resultaron ser poco selectivos o citotóxicos.
The Ministry of Health has infectious diseases as one of the research priorities, including leishmaniasis and American trypanosomiasis. The Faculty of Pharmaceutical and Biochemical Sciences develops evaluations of medicinal plant species used by the Tacana culture as a source of potential antiparasitic agents. Laboratories dedicated to the discovery of natural molecules with antiparasitic activity, have the challenge of developing protocols that allow the detection of selective, effective and less toxic biomolecules than those available. Therefore, in vitro antiparasitic evaluations (IC50) should be accompanied by cytotoxicity evaluations (LD50), in order to calculate a Selectivity Index (IS = LD50/ IC50) as a parameter of biological specificity. The cytotoxicity was measured on macrophage line (RAW 264.7, murine), cells engaged on intracellular parasites infections. The fluorometric protocol starts with an initial population of 5x104 cells/mL, incubated at 37ºC, in DMEM (Dulbecco's Modified Eagle's medium) for 96 hours with addition of resazurin (2mM) 3 hours, before final readings. Under these conditions the cytotoxicity of control drugs and 15 plant extracts, selected by their anti-kinetoplastid activity, was evaluated. Cosumailu extract was the most cytotoxic, Ejije bid'u was selective for T. cruzi, and Leishmania amazonensis, while Id'ene eidhue, was selective for L. amazonensis. Finally, the other 12 extracts were little selective or cytotoxic.
Assuntos
Plantas Medicinais , Preparações Farmacêuticas , Leishmaniose , Doenças Parasitárias , Extratos Vegetais , DiagnósticoRESUMO
Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-γ and tumor necrosis factor [TNF]-α) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-α and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.