RESUMO
Primary cell cultures are essential tools for elucidating the physiopathological mechanisms of the cardiovascular system. Therefore, a primary culture growth protocol of cardiovascular smooth muscle cells (VSMCs) obtained from human abdominal aortas was standardized. Ten abdominal aorta samples were obtained from patients diagnosed with brain death who were organ and tissue donors with family consent. After surgical ablation to capture the aorta, the aortic tissue was removed, immersed in a Custodiol® solution, and kept between 2 and 8 °C. In the laboratory, in a sterile environment, the tissue was fragmented and incubated in culture plates containing an enriched culture medium (DMEM/G/10% fetal bovine serum, L-glutamine, antibiotics and antifungals) and kept in an oven at 37 °C and 5% CO2. The aorta was removed after 24 h of incubation, and the culture medium was changed every six days for twenty days. Cell growth was confirmed through morphological analysis using an inverted optical microscope (Nikon®) and immunofluorescence for smooth muscle alpha-actin and nuclei. The development of the VSMCs was observed, and from the twelfth day, differentiation, long cytoplasmic projections, and adjacent cell connections occurred. On the twentieth day, the morphology of the VSMCs was confirmed by actin fiber immunofluorescence, which is a typical characteristic of VSMCs. The standardization allowed VSMC growth and the replicability of the in vitro test, providing a protocol that mimics natural physiological environments for a better understanding of the cardiovascular system. Its use is intended for investigation, tissue bioengineering, and pharmacological treatments.
Assuntos
Aorta Abdominal , Doenças Vasculares , Humanos , Morte Encefálica/metabolismo , Morte Encefálica/patologia , Músculo Liso Vascular/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Modelos Teóricos , Miócitos de Músculo Liso , Encéfalo , Células CultivadasRESUMO
BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.
Assuntos
Tratamento Farmacológico da COVID-19 , Armadilhas Extracelulares , Animais , Dissulfiram/metabolismo , Armadilhas Extracelulares/metabolismo , Camundongos , Neutrófilos/metabolismo , SARS-CoV-2RESUMO
Pods of Caesalpinia ferrea, popularly used to treat inflammatory processes, were collected to obtain the polysaccharide-rich extract, presenting anti-inflammatory and antinociceptive effects in acute inflammation models. This study aimed to evaluate the anti-inflammatory, antinociceptive and healing activities of the polysaccharide-rich extract from Caesalpinia ferrea pods (PEp-Cf) in the rat model of cutaneous excisional wound. PEp-Cf (0.025-0.1%) or 0.9% NaCl was topically applied in the wounds at dorsal thoracic region (2×/day) during 21 days for measurement of clinical signs (hyperemia, inflammatory exudate, edema, nociception), wound size, histopathological/histomorphometric, oxidative/inflammatory markers and systemic toxicity. PEp-Cf at 0.1% reduced wound area and increased ulcer contraction [days 2 and 10 (21-78%)]. PEp-Cf reduced clinical signs [days 2 and 5 (2.2-2.8×)] and modulated the healing inflammatory phase via stimulation of epithelialization (days 10 and 14), and inhibition of polymorphonuclears [days 2 and 5 (71-74%)], protein leakage [days 2 and 5 (28-41%)], nitrate [days 2 and 5 (2.2-6×)] and malondialdehyde [days 2 and 5 (46-49%)]. PEp-Cf increased the number of blood vessels [days 5 and 7 (3.1-9.6×)], fibroblasts [days 5 and 7 (2.1-6.4×)] and collagen [days 5 to 14 (1.5-1.8×)]. In conclusion, the topical application of PEp-Cf at 0.1% accelerates the healing process of rat cutaneous wounds via modulation of the inflammatory and proliferative phases, being devoid of systemic alterations.
Assuntos
Caesalpinia , Analgésicos , Animais , Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Malondialdeído , Nitratos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Polissacarídeos/farmacologia , Ratos , Ratos Wistar , Solução SalinaRESUMO
In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% - 3 h, 19% - 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-ß (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-ß levels via the lectin domain.
Assuntos
Anti-Inflamatórios/farmacologia , Bauhinia/química , Galectinas/farmacologia , Neutrófilos/fisiologia , Extratos Vegetais/farmacologia , Lectinas de Plantas/farmacologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Adesão Celular , Citocinas/fisiologia , Avaliação Pré-Clínica de Medicamentos , Galectinas/química , Migração e Rolagem de Leucócitos , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Peritonite/imunologia , Extratos Vegetais/química , Lectinas de Plantas/química , Ratos Wistar , Sementes/químicaRESUMO
Psoriasis is a chronic inflammatory skin disorder driven by the IL-23/type 3 immune response. However, molecular mechanisms sustaining the chronicity of inflammation and psoriatic lesions remain elusive. Combining systematic analyses of several transcriptomic datasets, we delineated gene signatures across human psoriatic skin, identifying S100A9 as one of the most up-regulated genes, which was confirmed in lesioned skin from patients with psoriasis and preclinical psoriasiform skin inflammation models. Genetic ablation or pharmacologic inhibition of S100A9 alleviated Aldara-induced skin inflammation. By single-cell mapping of human psoriatic skin and bone marrow chimeric mice experiments, we identified keratinocytes as the major source of S100A9. Mechanistically, S100A9 induced IL-23 production by dendritic cells, driving the IL-23/type 3 immunity in psoriasiform skin inflammation. In addition, the cutaneous IL-23/IL-17 axis induced epidermal S100A9 expression in human and experimental psoriasis. Thus, we showed an autoregulatory circuit between keratinocyte-derived S100A9 and IL-23/type 3 immunity during psoriasiform inflammation, identifying a crucial function of S100A9 in the chronification of psoriasis.
Assuntos
Psoríase , Humanos , Animais , Camundongos , Pele/patologia , Queratinócitos/metabolismo , Inflamação/patologia , Calgranulina B/genética , Interleucina-23/genética , Interleucina-23/metabolismo , Modelos Animais de DoençasRESUMO
Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.
Assuntos
Neutrófilos , Piruvato Quinase , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neutrófilos/metabolismo , Fosforilação , GlicóliseRESUMO
Th17 cell differentiation and pathogenicity depend on metabolic reprogramming inducing shifts toward glycolysis. Here, we show that the pyruvate kinase M2 (PKM2), a glycolytic enzyme required for cancer cell proliferation and tumor progression, is a key factor mediating Th17 cell differentiation and autoimmune inflammation. We found that PKM2 is highly expressed throughout the differentiation of Th17 cells in vitro and during experimental autoimmune encephalomyelitis (EAE) development. Strikingly, PKM2 is not required for the metabolic reprogramming and proliferative capacity of Th17 cells. However, T cell-specific PKM2 deletion impairs Th17 cell differentiation and ameliorates symptoms of EAE by decreasing Th17 cell-mediated inflammation and demyelination. Mechanistically, PKM2 translocates into the nucleus and interacts with STAT3, enhancing its activation and thereby increasing Th17 cell differentiation. Thus, PKM2 acts as a critical nonmetabolic regulator that fine-tunes Th17 cell differentiation and function in autoimmune-mediated inflammation.