ABSTRACT
FOXP1 syndrome caused by haploinsufficiency of the forkhead box protein P1 (FOXP1) gene is a neurodevelopmental disorder that manifests motor dysfunction, intellectual disability, autism, and language impairment. In this study, we used a Foxp1+/- mouse model to address whether cognitive and motor deficits in FOXP1 syndrome are associated with mitochondrial dysfunction and oxidative stress. Here, we show that genes with a role in mitochondrial biogenesis and dynamics (e.g., Foxo1, Pgc-1α, Tfam, Opa1, and Drp1) were dysregulated in the striatum of Foxp1+/- mice at different postnatal stages. Furthermore, these animals exhibit a reduced mitochondrial membrane potential and complex I activity, as well as decreased expression of the antioxidants superoxide dismutase 2 (Sod2) and glutathione (GSH), resulting in increased oxidative stress and lipid peroxidation. These features can explain the reduced neurite branching, learning and memory, endurance, and motor coordination that we observed in these animals. Taken together, we provide strong evidence of mitochondrial dysfunction in Foxp1+/- mice, suggesting that insufficient energy supply and excessive oxidative stress underlie the cognitive and motor impairment in FOXP1 deficiency.
Subject(s)
Forkhead Transcription Factors/genetics , Intellectual Disability/genetics , Motor Disorders/genetics , Repressor Proteins/genetics , Animals , Autism Spectrum Disorder/genetics , Autistic Disorder/metabolism , Cognition/physiology , Disease Models, Animal , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/metabolism , Haploinsufficiency/genetics , Mice , Mice, Inbred C57BL , Mitochondria/genetics , Mitochondria/metabolism , Motor Activity/genetics , Motor Disorders/metabolism , Neurodevelopmental Disorders/metabolism , Neurogenesis , Oxidative Stress/physiology , Repressor Proteins/deficiency , Repressor Proteins/metabolismABSTRACT
BACKGROUND: Neutrophil extracellular traps (NETs) are a recently discovered neutrophil defense mechanism which modulates several inflammatory conditions contributing to metabolic profile alterations. Therefore, the present study aimed to evaluate the production of NETs in obese patients and mice, verifying the possible mechanisms associated with the release of NETs by the adipose tissue. METHODS AND RESULTS: The present study investigated NETs production in human adipose tissue and also showing the neutrophils using intravital microscopy in mouse epididymal adipose tissue. Blood and white adipose tissues were obtained from eutrophic and obese individuals and from mice. Lipid, glycemic and leukocyte profiles were evaluated, as well as the levels of NETs and its markers. Bioinformatics and proteomics analyses were performed and the identified key proteins were measured. The main findings showed that the inflammatory markers interleukin-8 (IL-8), heat shock protein 90 (HSP90) and the E1 heat shock protein family (HSPE1) can be modulated by the NETs levels in obesity. Obesity has also been associated with increased cholesterol, glucose intolerance, ionic calcium and NETs. We also observed an increase in catalase and a decreased superoxide dismutase activity. Bioinformatics and proteomics analyses revealed that IL-8, HSP90 and HSPE1 were associated with obesity, inflammation and NETs release. CONCLUSIONS: In conclusion, the present study shows an increase in NETs production during obesity associated with important inflammatory markers in adipose.
Subject(s)
Extracellular Traps , Adipose Tissue/metabolism , Animals , Extracellular Traps/metabolism , Humans , Inflammation/metabolism , Mice , Neutrophils/metabolism , Obesity/metabolismABSTRACT
Chemical compounds belonging to the class of coumarins have promising anti-inflammatory potential. Cinnamoyloxy-mammeisin (CNM) is a 4-phenylcoumarin that can be isolated from Brazilian geopropolis. To our knowledge, its anti-inflammatory activity has never been studied. Therefore, the present study investigated the anti-inflammatory activity of CNM and elucidated its mechanism of action on isolated macrophages. Pretreatment with CNM reduced neutrophil migration into the peritoneal and joint cavity of mice. Likewise, CNM reduced the in vitro and in vivo release of TNF-α and CXCL2/MIP-2. Regarding the possible molecular mechanism of action, CNM reduced the phosphorylation of proteins ERK 1/2, JNK, p38 MAPK, and AP-1 (subunit c-jun) in PG-stimulated macrophages. Pretreatment with CNM also reduced NF-κB activation in RAW 264.7 macrophages stably expressing the NF-κB-luciferase reporter gene. On the other hand, it did not alter IκBα degradation or nuclear translocation of p65. Thus, the results of this study demonstrate promising anti-inflammatory activity of CNM and provide an explanation of its mechanism of action in macrophages via inhibition of MAPK signaling, AP-1, and NF-κB.
Subject(s)
Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Coumarins/isolation & purification , Coumarins/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Brazil , Coumarins/chemistry , Cyclooxygenase 2/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Molecular Structure , NF-kappa B/antagonists & inhibitors , Nitric Oxide Synthase Type II/antagonists & inhibitors , Signal Transduction/drug effects , Transcription Factor AP-1 , Tumor Necrosis Factor-alpha/pharmacology , eIF-2 Kinase/drug effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.
Subject(s)
Extracellular Traps/metabolism , Multiple Organ Failure/complications , Shock, Septic/pathology , Animals , Bacterial Load/drug effects , DNA/genetics , DNA/metabolism , Humans , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Shock, Septic/chemically induced , Shock, Septic/genetics , Shock, Septic/microbiologyABSTRACT
The reactive-oxygen-species-(ROS)-generating-enzyme Nox2 is essential for leukocyte anti-microbial activity. However its role in cellular redox homeostasis and, consequently, in modulating intracellular signaling pathways remains unclear. Herein, we show Nox2 activation favors thioredoxin-1 (TRX-1)/p40phox interaction, which leads to exclusion of TRX-1 from the nucleus. In contrast, the genetic deficiency of Nox2 or its pharmacological inhibition with apocynin (APO) results in reductive stress after lipopolysaccharide-(LPS)-cell stimulation, which causes nuclear accumulation of TRX-1 and enhanced transcription of inflammatory mediators through nuclear-factor-(NF)-κB. The NF-κB overactivation is prevented by TRX-1 oxidation using inhibitors of thioredoxin reductase-1 (TrxR-1). The Nox2/TRX-1/NF-κB intracellular signaling pathway is involved in the pathophysiology of chronic granulomatous disease (CGD) and sepsis. In fact, TrxR-1 inhibition prevents nuclear accumulation of TRX-1 and LPS-stimulated hyperproduction of tumor-necrosis-factor-(TNF)-α by monocytes and neutrophils purified from blood of CGD patients, who have deficient Nox2 activity. TrxR-1 inhibitors, either lanthanum chloride (LaCl3) or auranofin (AUR), also increase survival rates of mice undergoing cecal-ligation-and-puncture-(CLP). Therefore, our results identify a hitherto unrecognized Nox2-mediated intracellular signaling pathway that contributes to hyperinflammation in CGD and in septic patients. Additionally, we suggest that TrxR-1 inhibitors could be potential drugs to treat patients with sepsis, particularly in those with CGD.
Subject(s)
Acetophenones/pharmacology , NADPH Oxidase 2/metabolism , NF-kappa B/metabolism , Signal Transduction/drug effects , Thioredoxins/metabolism , Animals , Granulomatous Disease, Chronic/chemically induced , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/pathology , Lipopolysaccharides/toxicity , Male , Mice , Mice, Knockout , NADPH Oxidase 2/genetics , NF-kappa B/genetics , Oxidation-Reduction/drug effects , Sepsis/chemically induced , Sepsis/genetics , Sepsis/metabolism , Sepsis/pathology , Thioredoxins/geneticsABSTRACT
Infusions of Picrolemma sprucei roots, stems and leaves are used in traditional medicine throughout the Amazon region from the Guianas to Brazil and Peru in the treatment of gastritis, intestinal helminths and malaria. As there are no studies describing its mode of action in providing a gastroprotective effect, we determined herein that one of the main constituents found in P. sprucei infusions, the quassinoid isobrucein B (IsoB), reduces some of the pathophysiological effects in a mouse model of non-steroidal anti-inflammatory drug (NSAID)-induced gastritis and provides mechanisms of action. Then, IsoB (1.17 g) was isolated from the roots and stems (6.5 kg) of P. sprucei. Its structure was confirmed by 1D and 2D (1)H and (13)C NMR, ESI-tof-MS, IR and UV. C57BL/6 strain mice were subcutaneously injected with IsoB (0.5-5 mg kg(-1)) or vehicle before oral administration of indomethacin and sacrificed later at different time points. Gastric damage was assessed by measuring lesion length. Leukocyte migration was evaluated based on leukocyte rolling and adhesion using intravital microscopy in the mesenteric microcirculation and tissue MPO activity. Stomach extract cytokine (TNFα, IL-1ß and KC/CXCL1) and prostaglandin E2 (PGE2) levels were measured by ELISA and RIA, respectively. IsoB pre-treatment (0.5-5.0 mg kg(-1)) significantly reduced the formation of indomethacin-induced stomach lesions in a dose-dependent manner. The decrease in stomach lesions was associated with less observed leukocyte rolling, decreased leukocyte adhesion and less neutrophil infiltration (MPO activity). IsoB (1 mg kg(-1)) pre-treatment did not prevent indomethacin-induced decreases in stomach PGE2 levels. However, IL-1ß and KC/CXCL1 levels were inhibited by this same IsoB dosage, whereas TNF-α was unchanged. IsoB may be a prototypic compound to provide protective effects against NSAID-induced gastritis and possibly other gastropathies. Moreover, IsoB gastroprotective action may be due to a reduction in IL-1ß and KC/CXCL1 production/release and leukocyte rolling, adhesion and migration.
Subject(s)
Gastritis/drug therapy , Phytotherapy , Plant Extracts/therapeutic use , Quassins/therapeutic use , Simaroubaceae/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Cell Adhesion/drug effects , Cytokines/metabolism , Dinoprostone/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Gastric Mucosa/drug effects , Gastritis/chemically induced , Indomethacin/metabolism , Leukocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Neutrophils/drug effects , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Roots/chemistry , Plant Stems/chemistry , Plants, Medicinal , Quassins/chemistry , Quassins/isolation & purificationABSTRACT
Melanocortin is a potent anti-inflammatory molecule. However, little is known about the effect of melanocortin on acute inflammatory processes such as neutrophil migration. In the present study, we investigated the ability of [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP-MSH), a semisynthetic melanocortin compound, in the inhibition of neutrophil migration in carrageenin-induced peritonitis model. Herein, subcutaneous pretreatment with NDP-MSH decreased neutrophil trafficking in the peritoneal cavity in a dose-dependent manner. NDP-MSH inhibited vascular leakage, leukocyte rolling, and adhesion and reduced peritoneal macrophage inflammatory protein 2, but not TNF-alpha, IL-1beta, IL-10, and keratinocyte-derived chemokine production. In addition, the effect on neutrophil migration was reverted by the pretreatment with both propranolol (a nonselective beta-adrenergic antagonist) and mecamylamine (a nonselective nicotinic antagonist) but not by splenectomy surgery. Moreover, NDP-MSH intracerebroventricular administration inhibited neutrophil migration, indicating participation of the central nervous system. Our results propose that the NDP-MSH effect may be due to a spleen-independent neuro-immune pathway that efficiently regulates excessive neutrophil recruitment to tissues.
Subject(s)
Neutrophils/drug effects , Peritonitis/immunology , alpha-MSH/analogs & derivatives , Animals , Carrageenan , Cell Adhesion/drug effects , Cell Movement/drug effects , Chemokine CXCL2/immunology , Cytokines/immunology , Leukocyte Rolling/drug effects , Male , Mice , Neutrophils/physiology , Peritonitis/chemically induced , Receptors, Adrenergic/physiology , Receptors, Nicotinic/physiology , alpha-MSH/pharmacologyABSTRACT
Ranitidine is an antisecretory drug with H2 antagonist action useful in treating gastric and duodenal disorders. The dissolution test is used to obtain and compare dissolution profiles and establish similarities of pharmaceutical forms. The aim of this study was to compare the dissolution profiles of 150-mg coated ranitidine tablets of a reference drug (product A) and a generic (product B) and a similar (product C) drug marketed in Bahia, Brazil using a simple, fast and inexpensive ultraviolet method. Dissolution was determined using a USP type 2 apparatus at 50 rpm with 900 mL of distilled water at 37.0 ± 0.5 oC for 1h. The dissolution test was performed in compliance with the American Pharmacopoeia (USP-32). Dissolution efficiency and difference (f1) and similarity (f2) factors were calculated and evaluated. The proposed quantification methodology for drug dissolution test was validated, presenting accuracy, linearity and precision within the acceptance criteria. Products A, B and C showed dissolution efficiency values of 59.29, 73.59 and 66.67%, respectively. Factors f1 and f2 were calculated and showed that the profiles of products A, B and C were dissimilar. However, all the products released ranitidine satisfactorily, with at least 80% of the drug dissolved within 30 min.
A ranitidina é um fármaco antissecretor, antagonista H2, usado no tratamento de desordens gástricas e duodenais. O teste de dissolução é utilizado para obter e comparar perfis de dissolução, estabelecendo semelhança de formas farmacêuticas. Este estudo tem por objetivo comparar perfis de dissolução de comprimidos revestidos contendo 150 mg de ranitidina, em medicamentos de referência (produto A), genérico (produto B) e similar (produto C) comercializados na Bahia-Brasil, usando um método ultravioleta simples, rápido e de baixo custo. As condições que permitiram a determinação da dissolução foram: aparelho USP tipo 2 a 50 rpm, contendo 900 mL de água destilada mantida a 37,0 ± 0,5 °C, durante 1 h. O teste de dissolução foi realizado em conformidade com a Farmacopeia Americana (USP-32). Cálculo da eficiência de dissolução e fatores de diferença (f1) e semelhança (f2) foram avaliados. A metodologia proposta para a quantificação do fármaco no ensaio de dissolução foi validada apresentando precisão, linearidade e exatidão dentro dos critérios de aceitação. Os produtos A, B e C mostraram eficiência de dissolução de 59,29, 73,59 e 66,67%, respectivamente. Calcularam-se os fatores f1 e f2 e mostrou-se que os perfis não foram semelhantes para os comprimidos de produtos A, B e C. No entanto, todos os produtos liberaram o fármaco satisfatoriamente, pois, pelo menos, 80% de ranitidina foram dissolvidos em 30 min.