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ObjectiveTo explore the mechanisms of Astragali Radix-Curcumae Rhizoma (HQ-EZ) in alleviating hypercoagulability and inhibiting tumor growth and metastasis by modulating the formation of neutrophil extracellular traps (NETs) via the complement component 5a (C5a)/C5a receptor (C5aR) pathway. MethodForty male C57BL/6 mice were randomized into four groups: Blank, model, HQ-EZ (8.2 g·kg-1), and PMX53 (1 mg·kg-1). The mouse model of Lewis lung cancer was established in other three groups except the blank group. Mice were administrated with corresponding drugs from day 3 after modeling. Specifically, the HQ-EZ decoction was administrated for 14 consecutive days, while intraperitoneal injection of PMX53 was implemented on days 3, 6, 9, 12, and 15. Mouse body weight and tumor diameter were measured every two days. On the next day of the last administration, lung microCT was performed to observe the tumor metastasis in vivo. Blood samples were collected from the eyeball after anesthetization, and tumor and lungs were collected after the mice were sacrificed. Tumor weight was measured to calculate the tumor growth inhibitory rate. Enzyme-linked immunosorbent assay was employed to measure the levels of C5a, neutrophil elastase (NE), citrullinated histone-H3 (Cit-H3), myeloperoxidase (MPO), matrix metallopeptidase-9 (MMP-9), NETs, von Willebrand Factor (vWF), tissue factor (TF), and P-selectin in the serum and tumor tissue. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was conducted to assess apoptosis in the tumor tissue. Hematoxylin-eosin staining was conducted to observe lung metastasis, and immunofluorescence (IF) was employed to observe the expression of NETs in the tumor tissue. Western blot was employed to determine the protein levels of C5aR, MPO, and Cit-H3 in the tumor tissue. ResultCompared with the blank group, the model group had nodules in the lung, increased areas with low X-ray transmittance, appearance of nodular foci and multiple hemorrhagic foci in the lungs, and darkening lung color. Furthermore, the modeling elevated the serum levels of C5a, NETs and related proteins, vWF, TF, and P-selectin (P<0.01). Compared with the model group, HQ-EZ and PMX53 reduced the lung metastases, areas with low X-ray transmittance, and nodules in the lungs and lightened the lung color. Compared with the model group, the two drug intervention groups showed flat tumor growth curves, decreased tumor weight (P<0.01), increased apoptosis of tumor cells (P<0.01), lowered levels of C5a, NETs and related proteins, vWF, TF, and P-selectin both in the serum and tumor tissue (P<0.05), and down-regulated protein levels of C5aR, MPO, and Cit-H3 (P<0.05). ConclusionHQ-EZ inhibited the expression of NETs by suppressing the C5a/C5aR pathway, thereby alleviating hypercoagulability and inhibiting tumor growth and metastasis.
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Objective To explore the correlation between acute myocardial infarction(AMI)and neutrophil extracellular traps-os-is(NETosis)in the regulation of cardiomyocyte autophagy in mice.Methods The mouse model of AMI was established in C57BL/6mice by ligation of the left anterior descending coronary artery.Mouse primary cardiomyocytes were treated with oxygen glucose deprivation(OGD)to build an in vitro model of cardiomyocyte injury.Neutrophils were treated with PMA(NETosis inducer)/DNas Ⅰ(NETosis in-hibitor),the supernatant was taken to treat OGD-induced cardiomyocytes,and rapamycin(Rap)was used to treat OGD-induced car-diomyocytes.The myocardial infarction area was detected by TTC staining;serum cTnI level was detected by enzyme-linked immunoad-sordent assay(ELISA);cardiomyocytes apoptosis was detected by TUNEL staining;NETosis marker levels in neutrophil supernatant were detected by ELISA,and related protein expression levels were detected by Western blot.Results TTC staining showed that compared with the sham-operated group,the myocardial infarction area of the mice in the model group was significantly increased,the level of cTnI in serum was significantly increased(P<0.05),the apoptosis of cardiomyocytes was increased,and the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ were significantly increased,the p62 protein expression level was significantly decreased.Compared with the control group,the expression levels of apoptosis and cleaved caspase-3 were significantly increased under OGD conditions(P<0.05).Compared with the control group,the levels of NETosis markers MPO-DNA,MPO,and NE in the neutrophil supernatant in the PMA group were signifi-cantly increased;compared with the PMA-treated group the apoptosis level of cardiomyocytes in the PMA + DNas Ⅰ group was signifi-cantly decreased;the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰwere significantly decreased,and the protein expression level of p62 was significantly increased;compared with the PMA + DNas Ⅰ group,Rap treatment could enhance the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and inhibit the level of p62(P<0.05).It also significantly reversed the decrease in apoptosis induced by PMA + DNas Ⅰ.Conclusion Neutrophil NETosis can promote AMI in mice by regulating cardiomyocyte autophagy,which can provide a new direction and theoretical basis for the research and development of therapeutic drugs for AMI.
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Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.
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Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.
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In recent years, with the progress of research on the molecular mechanism of cell death, it has been discovered that there are many new types of programmed cell death, including non-apoptotic (10 types) and apoptotic (2 types), which are widely involved in the pathogenesis of infectious diseases and tumors. It is also a frontier research topic and provides new ideas for disease prevention and treatment. This article reviews the published literature on programmed cell death, focusing on the characteristics of cell necrosis, apoptosis, pyroptosis, ferroptosis, neutrophil inflammatory cell death (NETosis), cuproptosis, and widespread apoptosis (PANoptosis), as well as their relationship with infectious diseases.
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ABSTRACT Neutrophils play an important role in immune defence against several pathogens. These cells actively participate in the innate immune response through different functions, such as chemotaxis, phagocytosis, oxidative burst and degranulation, which have been widely studied. However, in the last few years, a new function has been described; activated neutrophils are able to release web-like chromatin structures known as neutrophil extracellular traps (NETs). These structures formed by DNA, histones, and proteins, immobilize and kill microorganisms. Disruption in NET formation is associated with the pathophysiology of several disorders, including the autoimmune diseases. NETs are an important source of the autoantigens involved in the production of autoantibodies and maintenance of the inflammatory milieu. This review provides a summary of the contribution of NETs to the pathogenesis of anti-neutrophil cytoplasmic antibodies-associated vasculitis, systemic lupus erythematosus, and rheumatoid arthritis. The preliminary findings on NETs components in Sjögren.'s syndrome will also be described.
RESUMEN Los neutrófilos juegan un papel muy importante en la defensa inmune contra diferentes patógenos. Estas células participan activamente en la respuesta inmune innata a través de diferentes funciones como quimiotaxis, fagocitosis, estallido oxidativo y degranulación, las cuales han sido estudiadas ampliamente. Sin embargo, en los últimos años se ha descrito una nueva función; los neutrófilos activados son capaces de liberar redes de cromatina llamadas trampas extracelulares de neutrófilos (NETs). Estas estructuras están formadas por ADN, histonas y proteínas capaces de inmovilizar y matar microorganismos. Alteraciones en la formación de estas NETs están asociadas con la fisiopatología de varios trastornos, incluyendo las enfermedades autoinmunes (EAI). Las NETs son consideradas una fuente de autoantígenos que ayudan a la producción de autoanticuerpos y al mantenimiento de un ambiente inflamatorio. Esta revisión resume la contribución de las NETs a la patogénesis de vasculitis asociada a anticuerpos contra el citoplasma de los neutrófilos, lupus eritematoso sistémico y artritis reumatoide. Adicionalmente, se describirán los resultados preliminares de la detección de componentes de las NETs en pacientes con síndrome de Sjögren.
Subject(s)
Humans , Autoimmune Diseases , Extracellular Traps , Neutrophils , Homeopathic Pathogenesy , Immunity , NoxaeABSTRACT
Neutrophils are the major antimicrobial cells of the innate immune system, which are recruited rapidly to the sites of infection and provide the primary defense against pathogens. Recent evidence suggests that neutrophils undergo a distinct cell death mechanism called NETosis, which not only contributes to the host defense, but also leads to severe pathological immune responses in cases of dysregulation. Here, we review the general features of NETosis as well as the generation of autoantigens and damage-associated molecular patterns by NETosis in autoimmune diseases. This review discusses the pathogenic role of NETosis in rheumatoid arthritis and systemic lupus erythematosus, where neutrophils may play a key role in the pathogenesis of these diseases, and suggest the possibility of neutrophil extracellular traps as biomarkers and therapeutic targets for the treatment of autoimmune diseases.