RESUMO
Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.
Assuntos
Macrófagos , Periodontite Periapical , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Humanos , Ligante RANK/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células THP-1 , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Periodontite Periapical/metabolismo , Periodontite Periapical/microbiologia , Periodontite Periapical/patologia , Citocinas/metabolismo , Enterococcus faecalis , Lipopolissacarídeos , Cavidade Pulpar/microbiologia , Cavidade Pulpar/metabolismo , Masculino , Osteoprotegerina/metabolismo , Adulto , Ácidos Teicoicos/farmacologiaRESUMO
The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N-terminal segment (58SYRIF63Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.
Assuntos
Calicreínas , Humanos , Masculino , Calicreínas/metabolismo , Calicreínas/química , Arginina/metabolismo , Arginina/química , Próstata/metabolismo , Próstata/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Animais , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Domínios Proteicos , Interleucina-10/metabolismo , Especificidade por SubstratoRESUMO
The Bacillus Calmette-Guérin (BCG) vaccine is the oldest cancer immunotherapeutic agent in use. Despite its effectiveness, its initial mechanisms of action remain largely unknown. Here, we elucidate the earliest cellular mechanisms involved in BCG-induced tumor clearance. We developed a fast preclinical in vivo assay to visualize in real time and at single-cell resolution the initial interactions among bladder cancer cells, BCG and innate immunity using the zebrafish xenograft model. We show that BCG induced the recruitment and polarization of macrophages towards a pro-inflammatory phenotype, accompanied by induction of the inflammatory cytokines tnfa, il1b and il6 in the tumor microenvironment. Macrophages directly induced apoptosis of human cancer cells through zebrafish TNF signaling. Macrophages were crucial for this response as their depletion completely abrogated the BCG-induced phenotype. Contrary to the general concept that macrophage anti-tumoral activities mostly rely on stimulating an effective adaptive response, we demonstrate that macrophages alone can induce tumor apoptosis and clearance. Thus, our results revealed an additional step to the BCG-induced tumor immunity model, while providing proof-of-concept experiments demonstrating the potential of this unique model to test innate immunomodulators.
Assuntos
Apoptose , Vacina BCG , Macrófagos , Transdução de Sinais , Neoplasias da Bexiga Urinária , Peixe-Zebra , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Animais , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Vacina BCG/farmacologia , Vacina BCG/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Humanos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Microambiente TumoralRESUMO
Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 µM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 µM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds.
Assuntos
Antimaláricos , Croton , Látex , Óxido Nítrico , Plasmodium falciparum , Croton/química , Animais , Camundongos , Látex/química , Plasmodium falciparum/efeitos dos fármacos , Estrutura Molecular , Antimaláricos/farmacologia , Antimaláricos/isolamento & purificação , Antimaláricos/química , Óxido Nítrico/metabolismo , Linhagem Celular , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Macrófagos/efeitos dos fármacosRESUMO
In addition to their pigment properties, the potential health benefits of anthocyanins have made them a subject of interest in recent years. This study aimed to obtain purified anthocyanin fractions from native Mexican black bean cultivars using Amberlite XAD-7 resin column and HPCCC and evaluate their anti-inflammatory properties using RAW 264.7 cells. The major anthocyanins in the purified anthocyanin fractions were delphinidin 3-glucoside (61.8%), petunidin 3-glucoside (25.2%), and malvidin 3-glucoside (12.2%). Purified anthocyanin fractions at 12.5 µg/mL effectively prevented LPS-induced ERK1/ERK2 phosphorylation and reduced the protein expression of COX-2 and mRNA expression of iNOS. Results showed that purified anthocyanin fractions have the potential to modulate the inflammatory response by inhibiting the production of pro-inflammatory mediators through the ERK1/ERK2 and NF-κB pathways. This study suggests that anthocyanins from black beans could be used as a natural strategy to help modulate inflammation-associated diseases.
Assuntos
Antocianinas , Anti-Inflamatórios , NF-kappa B , Extratos Vegetais , Antocianinas/farmacologia , Antocianinas/química , Antocianinas/isolamento & purificação , Camundongos , Células RAW 264.7 , Animais , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/imunologia , Distribuição Contracorrente , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/imunologia , Cromatografia Líquida de Alta Pressão , México , Phaseolus/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Although the labile iron pool (LIP) biochemical identity remains a topic of debate, it serves as a universal homeostatically regulated and essential cellular iron source. The LIP plays crucial cellular roles, being the source of iron that is loaded into nascent apo-iron proteins, a process akin to protein post-translational modification, and implicated in the programmed cell death mechanism known as ferroptosis. The LIP is also recognized for its reactivity with chelators, nitric oxide, and peroxides. Our recent investigations in a macrophage cell line revealed a reaction of the LIP with the oxidant peroxynitrite. In contrast to the LIP's pro-oxidant interaction with hydrogen peroxide, this reaction is rapid and attenuates the peroxynitrite oxidative impact. In this study, we demonstrate the existence and antioxidant characteristic of the LIP and peroxynitrite reaction in various cell types. Beyond its potential role as a ubiquitous complementary or substitute protection system against peroxynitrite for cells, the LIP and peroxynitrite reaction may influence cellular iron homeostasis and ferroptosis by changing the LIP redox state and LIP binding properties and reactivity.
Assuntos
Ferro , Oxirredução , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Ferro/metabolismo , Humanos , Ferroptose/efeitos dos fármacos , Animais , Peróxido de Hidrogênio/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Tagetes erecta L. (Asteraceae), popularly known as Aztec Marigold, is used in folk medicine to treat several ailments including inflammatory processes. Despite its historical use, the specific mechanisms through which it may modulate inflammation, particularly its effects on neutrophils and macrophages activation, have not yet been completely investigated. AIM OF THE STUDY: This study aimed to elucidate the anti-inflammatory mechanism of the hydroalcoholic extract obtained from T. erecta flowers, focusing on its role in the regulation of neutrophil and macrophage functions. MATERIAL AND METHODS: The production of TNF, IL-6, CXCL-1, IL-1ß, IL-10 (ELISA) and NO (Griess reaction), adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry), and chemotaxis were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS) and treated with the extract (1, 10 or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay. The in vivo anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally treated with the T. erecta extract (30, 100 or 300 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (TNF, IL-6 and IL-1ß, ELISA) and protein exudation (Bradford reaction) were quantified in the inflamed exudate. RESULTS: In vitro studies demonstrated that the extract inhibited neutrophil chemotaxis and reduced the production and/or release of cytokines (TNF, IL-1ß, CXCL1, and IL-6) as well as nitric oxide (NO) by neutrophils and macrophages when stimulated with LPS. Neutrophils treated with LPS and incubated with the extract showed an increase in CD62L expression, which leads to the impairment of neutrophil adhesion. The extract also enhanced efferocytosis of apoptotic neutrophils by macrophages, which was accompanied by increased IL-10 secretion and decreased TNF levels. In vivo studies yielded similar results, showing reduction in neutrophil migration, protein exudation, and cytokine release (TNF, IL-6, and IL-1ß). CONCLUSIONS: Together, the data herein obtained shows that T. erecta flower extract has anti-inflammatory effects by regulating inflammatory mediators, limiting neutrophil migration, and promoting efferocytosis. The in vivo results suggest that an herbal medicine made with T. erecta could represent an interesting pharmacological tool for the treatment of acute inflammatory condition.
Assuntos
Anti-Inflamatórios , Carragenina , Citocinas , Inflamação , Medicina Tradicional , Neutrófilos , Extratos Vegetais , Tagetes , Animais , Tagetes/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Camundongos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/isolamento & purificação , Masculino , Células RAW 264.7 , Inflamação/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Citocinas/metabolismo , Medicina Tradicional/métodos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Flores , Lipopolissacarídeos , Fagocitose/efeitos dos fármacos , Óxido Nítrico/metabolismoRESUMO
Research suggests that both tuberculosis (TB) and type 2 diabetes mellitus (T2DM) have an immuno-endocrine imbalance characterized by dysregulated proinflammatory molecules and hormone levels (high cortisol/DHEA ratio), impeding an effective immune response against Mycobacterium tuberculosis (Mtb) driven by cytokines, antimicrobial peptides (AMPs), and androgens like DHEA. Insulin, sulfonylurea derivatives, and metformin are commonly used glucose-lowering drugs in patients suffering from TB and T2DM. For this comorbidity, metformin is an attractive target to restore the immunoendocrine mechanisms dysregulated against Mtb. This study aimed to assess whether metformin influences cortisol and DHEA synthesis in adrenal cells and if these hormones influence the expression of proinflammatory cytokines and AMPs in Mtb-infected macrophages. Our results suggest that metformin may enhance DHEA synthesis while maintaining cortisol homeostasis. In addition, supernatants from metformin-treated adrenal cells decreased mycobacterial loads in macrophages, which related to rising proinflammatory cytokines and AMP expression (HBD-2 and 3). Intriguingly, we find that HBD-3 and LL-37 can modulate steroid synthesis in adrenal cells with diminished levels of cortisol and DHEA, highlighting the importance of crosstalk communication between adrenal hormones and these effectors of innate immunity. We suggest that metformin's effects can promote innate immunity against Mtb straight or through modulation of corticosteroid hormones.
Assuntos
Citocinas , Desidroepiandrosterona , Hidrocortisona , Macrófagos , Metformina , Mycobacterium tuberculosis , Metformina/farmacologia , Humanos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Macrófagos/imunologia , Mycobacterium tuberculosis/efeitos dos fármacos , Hidrocortisona/metabolismo , Desidroepiandrosterona/farmacologia , Citocinas/metabolismo , Imunidade Inata/efeitos dos fármacos , Células THP-1 , Interações Hospedeiro-Patógeno , Células Cultivadas , Hipoglicemiantes/farmacologia , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/microbiologia , Mediadores da Inflamação/metabolismoRESUMO
This study investigated the anti-inflammatory effect of hydrophilic and lipophilic extracts from juçara fruits (Euterpe edulis Martius) through measurement of nitric oxide (NOx) and cytokines (IL-12p70, TNF-α, INF-γ, MCP-1, IL-6, and IL-10). J774 macrophages were stimulated with lipopolysaccharides (1 µg/mL) and treated with various concentrations (1-100 µg/mL) of juçara fruits extracts from crude extracts, and hexane, dichloromethane, ethyl acetate, and butanol fractions. Potential relationships between the phenolic composition of the extracts determined by LC-ESI-MS/MS and their anti-inflammatory capacity were also evaluated. Hexane and dichloromethane fractions inhibited NOx and IL-12p70 while increased IL-10. Hexane fractions also decreased IL-6 and IFN-γ production. Hexane and dichloromethane fractions showed a higher number of phenolic compounds (32 and 34, respectively) than the other extracts tested and were also the only ones that presented benzoic acid and pinocembrin. These results suggest juçara fruits compounds as potential anti-inflammatory agents, especially those of a more apolar nature.
Assuntos
Anti-Inflamatórios , Frutas , Lipopolissacarídeos , Macrófagos , Óxido Nítrico , Extratos Vegetais , Macrófagos/efeitos dos fármacos , Frutas/química , Extratos Vegetais/farmacologia , Anti-Inflamatórios/farmacologia , Óxido Nítrico/metabolismo , Camundongos , Animais , Fenóis/análise , Fenóis/farmacologia , Citocinas/metabolismo , Linhagem CelularRESUMO
Leishmaniases, caused by Leishmania parasites, are widespread and pose significant health risks globally. Visceral leishmaniasis (VL) is particularly prevalent in Brazil, with high morbidity and mortality rates. Traditional treatments, such as pentavalent antimonials, have limitations due to toxicity and resistance. Therefore, exploring new compounds like lectins is crucial. Concanavalin A (ConA) has shown promise in inhibiting Leishmania growth. This study aimed to evaluate its leishmanicidal effect on L. infantum promastigotes and understand its mechanism of action. In vitro tests demonstrated inhibition of promastigote growth when treated with ConA, with IC50 values ranging from 3 to 5 µM over 24-72 h. This study suggests that ConA interacts with L. infantum glycans. Additionally, ConA caused damage to the membrane integrity of parasites and induced ROS production, contributing to parasite death. Scanning electron microscopy confirmed morphological alterations in treated promastigotes. ConA combined with the amphotericin B (AmB) showed synergistic effects, reducing the required dose of AmB, and potentially mitigating its toxicity. ConA demonstrated no cytotoxic effects on macrophages, instead stimulating their proliferation. These findings reinforce that lectin exhibits promising leishmanicidal activity against L. infantum promastigotes, making ConA a potential candidate for leishmaniasis treatment.