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1.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502552

RESUMO

Immune response control is critical as excessive cytokine production can be detrimental and damage the host. Interleukin-10 (Il-10), an anti-inflammatory cytokine produced primarily by macrophages, is a key regulator that counteracts and controls excessive inflammatory response. Il-10 expression is regulated through the transcription factor c-Maf. Another regulator of Il-10 production is p35, an activator of the cyclin-dependent kinase 5 (Cdk5), which decreases Il-10 production in macrophages, thus increasing inflammation. However, Cdk5 regulation of c-Maf and the involvement of Il-10 production in macrophages has not yet been investigated. We used in vitro primary bone marrow-derived macrophages (BMDMs) lacking Cdk5, stimulated them with lipopolysaccharid (LPS) and observed increased levels of c-Maf and Il-10. In an in vivo mouse model of LPS-induced endotoxemia, mice lacking Cdk5 in macrophages showed increased levels of c-Maf and elevated levels of Il-10 in lungs as well as in plasma, resulting in ameliorated survival. Taken together, we identified Cdk5 as a potential novel regulator of Il-10 production through c-Maf in macrophages under inflammatory conditions. Our results suggest that inhibition of Cdk5 enhances the c-Maf-Il-10 axis and thus potentiates improvement of anti-inflammatory therapy.


Assuntos
Quinase 5 Dependente de Ciclina/genética , Endotoxemia/genética , Inflamação/genética , Interleucina-10/genética , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , Animais , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Regulação da Expressão Gênica , Inflamação/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-maf/metabolismo
2.
Sci Rep ; 11(1): 17772, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493741

RESUMO

We investigated the impact of aerobic exercise (AE) on multiple organ dysfunction syndrome (MODS), aortic injury, pathoglycemia, and death during sepsis. ICR mice were randomized into four groups: Control (Con), Lipopolysaccharide (LPS), Exercise (Ex), and Exercise + LPS (Ex + LPS) groups. Mice were trained with low-intensity for 4 weeks. LPS and Ex + LPS mice received 5 mg/kg LPS intraperitoneally for induction of sepsis. Histopathological micrographs showed the organ morphology and damage. This study examined the effects of AE on LPS-induced changes in systemic inflammation, pulmonary inflammation, lung permeability, and bronchoalveolar lavage fluid (BALF) cell count, oxidative stress-related indicators in the lung, blood glucose levels, plasma lactate levels, serum insulin levels, plasma high-mobility group box 1 (HMGB1) levels, glucose transporter 1 (Glut1) and HMGB1, silent information regulator 1 (Sirt-1), and nuclear factor erythroid 2-related factor 2 (Nrf-2) mRNA expression levels in lung tissue. AE improved sepsis-associated multiple organ dysfunction syndrome (MODS), aortic injury, hypoglycemia, and death. AE prominently decreased pulmonary inflammation, pulmonary edema, and modulated redox balance during sepsis. AE prominently decreased neutrophil content in organ. AE prominently downregulated CXCL-1, CXCL-8, IL-6, TNF-α, Glu1, and HMGB1 mRNA expression but activated IL-1RN, IL-10, Sirt-1, and Nrf-2 mRNA expression in the lung during sepsis. AE decreased the serum levels of lactate and HMGB1 but increased blood glucose levels and serum insulin levels during sepsis. A 4-week AE improves sepsis-associated MODS, aortic injury, pathoglycemia, and death. AE impairs LPS-induced lactate and HMGB1 release partly because AE increases serum insulin levels and decreases the levels of Glut1. AE is a novel therapeutic strategy for sepsis targeting aerobic glycolysis.


Assuntos
Endotoxemia/terapia , Exercício Físico , Glicólise/fisiologia , Insuficiência de Múltiplos Órgãos/prevenção & controle , Condicionamento Físico Animal/fisiologia , Animais , Glicemia/análise , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/análise , Endotoxemia/induzido quimicamente , Endotoxemia/complicações , Transportador de Glucose Tipo 1/sangue , Proteína HMGB1/sangue , Humanos , Imunidade Inata , Insulina/sangue , Lactatos/sangue , Lipopolissacarídeos/toxicidade , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/patologia , Neutrófilos/patologia , Estresse Oxidativo , Distribuição Aleatória , Vísceras/patologia
3.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445223

RESUMO

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1ß, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1ß was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.


Assuntos
Benzoxazóis/farmacologia , Encéfalo/metabolismo , Endotoxemia , Isoquinolinas/farmacologia , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Piperazinas/farmacologia , Piperidinas/farmacologia , Receptores de Ácidos Lisofosfatídicos , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Microglia/patologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200709

RESUMO

Sepsis is characterized by multiple-organ dysfunction caused by the dysregulated host response to infection. Until now, however, the role of the Wnt signaling has not been fully characterized in multiple organs during sepsis. This study assessed the suppressive effect of a Wnt signaling inhibitor, Wnt-C59, in the kidney, lung, and liver of lipopolysaccharide-induced endotoxemic mice, serving as an animal model of sepsis. We found that Wnt-C59 elevated the survival rate of these mice and decreased their plasma levels of proinflammatory cytokines and organ-damage biomarkers, such as BUN, ALT, and AST. The Wnt/ß-catenin and NF-κB pathways were stimulated and proinflammatory cytokines were upregulated in the kidney, lung, and liver of endotoxemic mice. Wnt-C59, as a Wnt signaling inhibitor, inhibited the Wnt/ß-catenin pathway, and its interaction with the NF-κB pathway, which resulted in the inhibition of NF-κB activity and proinflammatory cytokine expression. In multiple organs of endotoxemic mice, Wnt-C59 significantly reduced the ß-catenin level and interaction with NF-κB. Our findings suggest that the anti-endotoxemic effect of Wnt-C59 is mediated via reducing the interaction between ß-catenin and NF-κB, consequently suppressing the associated cytokine upregulation in multiple organs. Thus, Wnt-C59 may be useful for the suppression of the multiple-organ dysfunction during sepsis.


Assuntos
Benzenoacetamidas/farmacologia , Citocinas/metabolismo , Endotoxemia/tratamento farmacológico , Lipopolissacarídeos/toxicidade , NF-kappa B/antagonistas & inibidores , Piridinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , Animais , Citocinas/genética , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Domínios e Motivos de Interação entre Proteínas , beta Catenina/metabolismo
5.
J Food Biochem ; 45(7): e13805, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096077

RESUMO

High mobility group box 1 (HMGB1) is a well-defined mediator involved in the pathophysiologic response to endotoxemia and sepsis. However, the mechanisms and therapeutic agents that could prevent its release are not fully elucidated. Here, the present study demonstrates that the ginseng leaf extract (GLE) regulates lipopolysaccharide (LPS)-triggered release of HMGB1 in macrophages and endotoxemic animal model. Treatment of RAW264.7 macrophages with GLE significantly inhibited the release of HMGB1 stimulated by LPS. GLE also suppressed the generation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) in a dose-dependent manner. These effects of GLE were accompanied by inhibition of HMGB1 release stimulated by LPS, indicating a potential mechanism by which GLE regulates HMGB1 release through NO signaling. Furthermore, induction of suppressor of cytokine signaling 1 by GLE-mediated GLE-dependent suppression of HMGB1 release and NO/iNOS induction by inhibiting Janus kinase 2/signal transducer and activator of transcription 1 signal in RAW 264.7 cells exposed to LPS. Finally, administration of the GLE ameliorated the survival rate of LPS-injected endotoxemic mice in a NO-dependent manner. Thus, GLE may block the LPS-stimulated release of HMGB1 by regulating cellular signal networks, thereby providing a therapeutic strategy for endotoxemia as a functional food. PRACTICAL APPLICATIONS: High mobility group box 1 (HMGB1) is released into the extracellular milieu when immune cells are exposed to pathogen-related molecules such as lipopolysaccharide (LPS), in which it acts as a critical mediator of lethality in sepsis and endotoxemia. The extract of ginseng leaf, which is a part that can be easily thrown away, ameliorated the survival rate of endotoxemic mice by inhibiting HMGB1 secretion in a NO-dependent manner. Thus, this study suggests that ginseng leaf can be used as a functional food by resolving the immune responses in the pathology of endotoxemia.


Assuntos
Endotoxemia , Proteína HMGB1 , Panax , Animais , Endotoxemia/induzido quimicamente , Endotoxemia/tratamento farmacológico , Camundongos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Células RAW 264.7
6.
Sci Rep ; 11(1): 10834, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035380

RESUMO

Fluid resuscitation following severe inflammation-induced hypoperfusion is critical for the restoration of hemodynamics and the prevention of multiorgan dysfunction syndrome during septic shock. Fluid resuscitation with commercially available crystalloid and colloid solutions only provides transient benefits, followed by fluid extravasation and tissue edema through the inflamed endothelium. The increased molecular weight (M.W.) of polymerized human serum albumin (PolyHSA) can limit fluid extravasation, leading to restoration of hemodynamics. In this prospective study, we evaluated how fluid resuscitation with PolyHSA impacts the hemodynamic and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model. Additionally, we evaluated fluid resuscitation with PolyHSA in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Resuscitation with PolyHSA attenuated the immune response and improved the maintenance of systemic hemodynamics and restoration of microcirculatory hemodynamics. This decrease in inflammatory immune response and maintenance of vascular wall shear stress likely contributes to the maintenance of vascular integrity following fluid resuscitation with PolyHSA. The sustained restoration of perfusion, decrease in pro-inflammatory immune response, and improved vascular integrity that results from the high M.W. of PolyHSA indicates that a PolyHSA based solution is a potential resuscitation fluid for endotoxic and septic shock.


Assuntos
Endotoxemia/tratamento farmacológico , Hidratação/métodos , Microcirculação/efeitos dos fármacos , Albumina Sérica Humana/administração & dosagem , Choque Séptico/tratamento farmacológico , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Masculino , Mesocricetus , Camundongos , Estudos Prospectivos , Albumina Sérica Humana/farmacologia , Choque Séptico/etiologia , Choque Séptico/imunologia
7.
Br J Anaesth ; 126(6): 1111-1118, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33896590

RESUMO

BACKGROUND: Dysregulation of coagulation occurs commonly in sepsis, ranging from mild coagulopathy with decreased platelets to disseminated intravascular coagulation (DIC). We investigated the effect of induced normothermia on coagulation during lipopolysaccharide (LPS)-induced endotoxaemia in healthy volunteers. METHODS: Twelve volunteers received an infusion of bacterial lipopolysaccharide (Escherichia coli; 2 ng kg-1) and were assigned to either induced normothermia or control. Induced normothermia to maintain core temperature at 37°C consisted of external surface cooling, cold i.v. fluids, and medication to reduce shivering (buspirone, clonidine, and magnesium sulphate). The primary outcome was the DIC score (International Society on Thrombosis and Haemostasis guideline). Prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, plasma von Willebrand factor (vWf), and rotational thromboelastometry (ROTEM) were measured before and 1, 3, 6, and 8 h after LPS infusion. Differences between groups were tested with a mixed effects model. RESULTS: In control subjects, lipopolysaccharide caused a fever, transiently decreased platelet levels and lowered activated partial thromboplastin time, while prolonging prothrombin time and increasing D-Dimer and vWf levels. Normothermia prevented the DIC-score exceeding 4, which occurred in 50% of control subjects. Normothermia also reduced the fall in platelet count by 67x109 L-1([95%CI:27-107]; p=0.002), aPTT (mean difference:3s [95%CI:1-5]; p=0.005) and lowered vWf levels by 89% ([95%CI:6-172]; p=0.03), compared to the fever group. ROTEM measurements were unaffected by lipopolysaccharide. CONCLUSION: In human endotoxaemia, induced normothermia decreases markers of endothelial activation and DIC. Maintaining normothermia may reduce coagulopathy in hyperinflammatory states.


Assuntos
Coagulação Sanguínea , Coagulação Intravascular Disseminada/prevenção & controle , Endotoxemia/terapia , Hipotermia Induzida , Adolescente , Adulto , Biomarcadores/sangue , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/diagnóstico , Coagulação Intravascular Disseminada/etiologia , Endotoxemia/sangue , Endotoxemia/induzido quimicamente , Endotoxemia/diagnóstico , Endotoxinas/administração & dosagem , Voluntários Saudáveis , Humanos , Hipotermia Induzida/efeitos adversos , Infusões Parenterais , Masculino , Fatores de Tempo , Adulto Jovem
8.
Sci Rep ; 11(1): 8377, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863923

RESUMO

Fisetin is a naturally occurring flavonoid that possesses several pharmacological benefits including anti-inflammatory activity. However, its precise anti-inflammatory mechanism is not clear. In the present study, we found that fisetin significantly inhibited the expression of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Additionally, fisetin attenuated LPS-induced mortality and abnormalities in zebrafish larvae and normalized the heart rate. Fisetin decreased the recruitment of macrophages and neutrophils to the LPS-microinjected inflammatory site in zebrafish larvae, concomitant with a significant downregulation of proinflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase-2a (COX-2a), IL-6, and TNF-α. Fisetin inhibited the nuclear localization of nuclear factor-kappa B (NF-κB), which reduced the expression of pro-inflammatory genes. Further, fisetin inactivated glycogen synthase kinase 3ß (GSK-3ß) via phosphorylation at Ser9, and inhibited the degradation of ß-catenin, which consequently promoted the localization of ß-catenin into the nucleus. The pharmacological inhibition of ß-catenin with FH535 reversed the fisetin-induced anti-inflammatory activity and restored NF-κB activity, which indicated that fisetin-mediated activation of ß-catenin results in the inhibition of LPS-induced NF-κB activity. In LPS-microinjected zebrafish larvae, FH535 promoted the migration of macrophages to the yolk sac and decreased resident neutrophil counts in the posterior blood island and induced high expression of iNOS and COX-2a, which was accompanied by the inhibition of fisetin-induced anti-inflammatory activity. Altogether, the current study confirmed that the dietary flavonoid, fisetin, inhibited LPS-induced inflammation and endotoxic shock through crosstalk between GSK-3ß/ß-catenin and the NF-κB signaling pathways.


Assuntos
Anti-Inflamatórios/farmacologia , Endotoxemia/prevenção & controle , Flavonóis/farmacologia , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peixe-Zebra , beta Catenina/genética
9.
Eur J Pharmacol ; 899: 173993, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33675782

RESUMO

Resveratrol has been reported to have beneficial effects on sepsis by regulating the inflammatory response. However, it remains unclear if resveratrol plays a role in the development of endotoxin tolerance. Treatment with resveratrol in macrophages stimulated with primary lipopolysaccharide (LPS) resulted in the increased production of TNF-α and IL-6 induced by a 2nd dose of LPS (by 74.5 ± 12.9% and 63.4 ± 12%, respectively, compared to untreated cells, P < 0.05). This effect was inhibited by compound C, an AMPK inhibitor, and STO609, a calcium/calmodulin-dependent protein kinase-kinase (CaMKK) inhibitor. Resveratrol diminished the expression of interleukin-1 receptor-associated kinase M (IRAK-M) and Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) by prolonging the exposure of cells to LPS (by 60.8 ± 16.3% and 70.3 ± 18.1%, respectively, compared to LPS only). The effect of resveratrol on the LPS-induced expression of IRAK-M and SHIP1 was inhibited by compound C or STO609. After a 2nd dose of LPS, resveratrol increased phosphorylation of ERK1/2, p38, and JNK in endotoxin tolerant macrophages. In vivo systemic administration of resveratrol prevented a significant increase in mortality rate by cecal ligation and puncture in LPS-induced endotoxin-tolerant mice. These results indicate that resveratrol induces AMPK activation through the Ca2+/CaMKKß pathway and suppresses the development of endotoxin tolerance by inhibiting LPS-induced expression of IRAK-M and SHIP1.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Endotoxemia/tratamento farmacológico , Fatores Imunológicos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Sinalização do Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/enzimologia , Ativação Enzimática , Mediadores da Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
10.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567713

RESUMO

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α-deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.


Assuntos
Diafragma/efeitos dos fármacos , Modelos Animais de Doenças , Endotoxemia/complicações , Heparina de Baixo Peso Molecular/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Animais , Endotoxemia/induzido quimicamente , Endotoxemia/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
11.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33573095

RESUMO

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.


Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Enterocolite/genética , Indometacina/efeitos adversos , Lúpus Eritematoso Sistêmico/genética , Receptores de IgG/genética , Animais , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/imunologia , Enterocolite/induzido quimicamente , Enterocolite/imunologia , Feminino , Deleção de Genes , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Receptores de IgG/imunologia
12.
Sci Rep ; 11(1): 610, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436885

RESUMO

Sepsis can lead to shock, multiple organ failure, and even death. Platelets play an active role in the pathogenesis of sepsis-induced multiple organ failure. Angiotensin (Ang)-(1-7), a biologically active peptide, counteracts various effects of Ang II and attenuates inflammatory responses, reactive oxygen species production, and apoptosis. We evaluated the effects of Ang-(1-7) on organ injury and platelet dysfunction in rats with endotoxaemia. We treated male Wistar rats with saline or lipopolysaccharide (LPS, 10 mg, intravenously) then Ang-(1-7) (1 mg/kg, intravenous infusion for 3 h beginning 30 min after LPS administration). We analysed several haemodynamic, biochemical, and inflammatory parameters, as well as platelet counts and aggregation. Ang-(1-7) improved hypotension and organ dysfunction, and attenuated plasma interleukin-6, chemokines and nitric oxide production in rats after LPS administration. The LPS-induced reduction in platelet aggregation, but not the decreased platelet count, was restored after Ang-(1-7) treatment. The protein expression of iNOS and IκB, but not phosphorylated ERK1/2 and p38, was diminished in Ang-(1-7)-treated LPS rats. The histological changes in liver and lung were significantly attenuated in Ang-(1-7)-treated LPS rats. Our results suggest that Ang-(1-7) ameliorates endotoxaemic-induced organ injury and platelet dysfunction, likely through the inhibition of the inflammatory response and nitric oxide production.


Assuntos
Angiotensina I/farmacologia , Plaquetas/efeitos dos fármacos , Endotoxemia/complicações , Hipotensão/prevenção & controle , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Insuficiência de Múltiplos Órgãos/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Animais , Plaquetas/patologia , Endotoxemia/induzido quimicamente , Hipotensão/etiologia , Hipotensão/patologia , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , Ratos , Ratos Wistar , Sepse/induzido quimicamente , Sepse/complicações , Vasodilatadores/farmacologia
13.
Methods Mol Biol ; 2261: 247-262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420994

RESUMO

The comprehensive analysis of serum cytokine levels can be challenging due to low sample volumes and time consuming when using single-target methods like enzyme-linked immunosorbent assay (ELISA). Bead-based detection systems allow the simultaneous detection of multiple analytes using minimal sample volumes. Here we describe the use of a multiplex cytokine, chemokine, and growth factor assay for mouse cytokines in a 96-well format. This assay is based on antibody-coupled fluorescent magnetic beads combined with biotinylated secondary detection antibody followed by fluorescent-tagged streptavidin in a sandwich-like composition. Final assay readout provides the concentrations of 23 different cytokines, chemokines, and growth factors in up to 76 samples.


Assuntos
Quimiocinas/sangue , Citocinas/sangue , Endotoxemia/sangue , Técnica Indireta de Fluorescência para Anticorpo , Fluorimunoensaio , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Proteômica , Animais , Biotinilação , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Lipopolissacarídeos , Camundongos Knockout , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/genética
14.
Nat Immunol ; 22(2): 154-165, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398185

RESUMO

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.


Assuntos
Alarminas/metabolismo , Endotoxemia/imunologia , Galectina 1/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alarminas/deficiência , Alarminas/genética , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Feminino , Galectina 1/sangue , Galectina 1/deficiência , Galectina 1/genética , Células HeLa , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos Comuns de Leucócito/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Necroptose , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Células RAW 264.7 , Sepse/sangue , Sepse/diagnóstico , Transdução de Sinais , Regulação para Cima
15.
Inflamm Res ; 70(2): 193-203, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33474594

RESUMO

OBJECTIVE: Intracellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein belonging to the immunoglobulin superfamily, plays a critical role in mediating cell-cell interaction and outside-in cell signaling during the immune response. ICAM-1 is expressed on the cell surface of several cell types including endothelial cells, epithelial cells, leucocytes, fibroblasts, and neutrophils. Despite ICAM-1 has been detected on macrophage, little is known about the function and mechanism of macrophage ICAM-1. METHODS: To investigate the role of lipopolysaccharide (LPS) in ICAM-1 regulation, both the protein and cell surface expression of ICAM-1 were measured. The phagocytosis of macrophage was evaluated by flow cytometry and Confocal microscopy. Small interfering RNA and neutralizing antibody of ICAM-1 were used to assess the effect of ICAM-1 on macrophage phagocytosis. TLR4 gene knockout mouse and cytoplasmic and mitochondrial ROS scavenger were used for the regulation of ICAM-1 expression. ROS was determined using flow cytometry. RESULTS: In this study, we reported that macrophage can be stimulated to increase both the protein and cell surface expression of ICAM-1 by LPS. Macrophage ICAM-1 expression was correlated with enhanced macrophage phagocytosis. We found that using ICAM-1 neutralizing antibody or ICAM-1 silencing to attenuate the function or expression of ICAM-1 could decrease LPS-induced macrophage phagocytosis. Furthermore, we found that knocking out of TLR4 led to inhibited cytoplasmic and mitochondrial ROS production, which in turn, attenuated ICAM-1 expression at both the protein and cell surface levels. CONCLUSION: This study demonstrates that the mechanism of ICAM-1-mediated macrophage phagocytosis is depending on TLR4-mediated ROS production and provides significant light on macrophage ICAM-1 in endotoxemia.


Assuntos
Endotoxemia/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Endotoxemia/induzido quimicamente , Molécula 1 de Adesão Intercelular/genética , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/imunologia , Superóxido Dismutase/imunologia , Receptor 4 Toll-Like/genética
16.
Eur J Immunol ; 51(2): 380-392, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33080044

RESUMO

Despite the availability of antibiotics, current therapies to treat sepsis are still ineffective and many clinical trials aimed at neutralizing specific inflammatory cytokines have failed, suggesting the urgent need for new treatments. Using two models of LPS-induced endotoxemia and cecal ligation and puncture (CLP)-induced sepsis, we investigated the effects of C1q/TNF-related protein 4(CTRP4) on septic lethality and sepsis-induced inflammation. The effects of CTRP4 on survival, inflammation, organ damage, and bacterial clearance were assessed. Here, we found that CTRP4 decreased the mortalities of mice and alleviated pathological lung injury in mice model. In vivo depletion and adoptive transfer studies showed CTRP4-expressing macrophages as the key cell type inhibiting LPS-induced septic shock. The mechanism associated with the CTRP4 deficiency involved promoting of TLR4 internalization and activation of downstream pathways that resulted in a lethal, prolonged proinflammatory cytokine storm. Treatment of macrophages with exogenous CTRP4 abrogated proinflammatory cytokine production. Our results showed CTRP4 regulates inflammatory response and could be a promising strategy to treat septic shock.


Assuntos
Adipocinas/metabolismo , Anti-Inflamatórios/metabolismo , Endotoxemia/metabolismo , Macrófagos/metabolismo , Choque Séptico/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Choque Séptico/induzido quimicamente
17.
Clin Exp Immunol ; 203(3): 433-447, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33232517

RESUMO

Sepsis is an intractable clinical syndrome characterized by organ dysfunction when the body over-responds to an infection. Sepsis has a high fatality rate and lacks effective treatment. Family with sequence similarity 96 member A (FAM96A) is an evolutionarily conserved protein with high expression in the immune system and is related to cytosolic iron assembly and tumour suppression; however, research has been rarely conducted on its immune functions. Our study found that Fam96a-/- mice significantly resisted lesions during sepsis simulated by caecal ligation and puncture (CLP) or endotoxicosis models. After a challenge with lipopolysaccharide (LPS) or infection, Fam96a-/- mice exhibited less organ damage, longer survival and better bacterial clearance with decreased levels of proinflammatory cytokines. While screening several subsets of immune cells, FAM96A-expressing macrophages as the key cell type inhibited sepsis development. In-vivo macrophage depletion or adoptive transfer experiments abrogated significant differences in the survival of sepsis between Fam96a-/- and wild-type mice. Results of the bone marrow-derived macrophage (BMDM) polarization experiment indicated that FAM96A deficiency promotes the transformation of uncommitted monocytes/macrophages (M0) into M2 macrophages, secreting fewer proinflammatory cytokines. FAM96A may mediate an immunometabolism shift - from oxidative phosphorylation (OXPHOS) to glycolysis - in macrophages during sepsis, mirrored by reactive oxygen species (ROS) and glucose uptake. These data demonstrate that FAM96A regulates inflammatory response and provide a novel genomic insight for sepsis treatment.


Assuntos
Proteínas de Transporte/genética , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Sepse/genética , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Citocinas/metabolismo , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/metabolismo , Análise de Sobrevida
18.
Am J Physiol Renal Physiol ; 320(3): F262-F272, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33356954

RESUMO

Mitochondrial damage in renal tubular epithelial cells (RTECs) is a hallmark of endotoxin-induced acute kidney injury (AKI). Forkhead box O1 (FOXO1) is responsible for regulating mitochondrial function and is involved in several kidney diseases. Here, we investigated the effect of FOXO1 on endotoxin-induced AKI and the related mechanism. In vivo, FOXO1 downregulation in mouse RTECs and mitochondrial damage were found in endotoxin-induced AKI. Overexpression of FOXO1 by kidney focal adeno-associated virus (AAV) delivery improved renal function and reduced mitochondrial damage. Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), a master regulator of mitochondrial biogenesis and function, was reduced in endotoxin-induced AKI, but the reduction was reversed by FOXO1 overexpression. In vitro, exposure to LPS led to a decline in HK-2 cell viability, mitochondrial fragmentation, and mitochondrial superoxide accumulation, as well as downregulation of FOXO1, PGC1-α, and mitochondrial complex I/V. Moreover, overexpression of FOXO1 in HK-2 cells increased HK-2 cell viability and PGC1-α expression, and it alleviated the mitochondrial injury and superoxide accumulation induced by LPS. Meanwhile, inhibition of FOXO1 in HK-2 cells by siRNA treatment decreased PGC1-α expression and HK-2 cell viability. Chromatin immunoprecipitation assays and PCR analysis confirmed that FOXO1 bound to the PGC1-α promoter in HK-2 cells. In conclusion, downregulation of FOXO1 in RTECs mediated endotoxin-induced AKI and mitochondrial damage. Overexpression of FOXO1 could improve renal injury and mitochondrial dysfunction, and this effect occurred at least in part as a result of PGC1-α signaling. FOXO1 might be a potential target for the prevention and treatment of endotoxin-induced AKI.


Assuntos
Injúria Renal Aguda/metabolismo , Endotoxemia/complicações , Células Epiteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Túbulos Renais/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Células Epiteliais/ultraestrutura , Proteína Forkhead Box O1/genética , Humanos , Túbulos Renais/ultraestrutura , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais
19.
BMC Vet Res ; 16(1): 422, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33148245

RESUMO

BACKGROUND: Paraoxonase-1 (PON-1) is an antioxidant enzyme, whose activity decreases during the acute phase response in many species. Little is known about PON-1 and its role as a negative acute phase protein during septic inflammation in horses, but promising findings about its utility in diagnosing SIRS and predicting the outcome in diseased horses, were recently highlighted. The objective of the study was to investigate the behaviour of PON-1 in horses after experimentally induced endotoxemia. To this aim, PON-1 activity was measured on 66 plasma samples collected from six clinically healthy mares, previously included in another study, before and at multiple time points between 12 and 240 h after intravenous infusion of Escherichia coli O55:B5 lipopolysaccharide (LPS). RESULTS: Compared with baseline values, a progressive transient decrease of PON-1 activity was observed starting from 24 h post-infusion, with lowest values observed between 3 to 7 days post-infusion, followed by a normalisation to pre-infusion levels the tenth day. CONCLUSIONS: The results of this study suggest that measurement and monitoring of PON-1 activity might be useful to evaluate progression and recovery from endotoxemia in horses. Further studies in horses with naturally occurring sepsis are warranted.


Assuntos
Arildialquilfosfatase/sangue , Endotoxemia/induzido quimicamente , Doenças dos Cavalos/diagnóstico , Animais , Endotoxemia/sangue , Endotoxemia/diagnóstico , Endotoxemia/enzimologia , Escherichia coli , Feminino , Doenças dos Cavalos/sangue , Doenças dos Cavalos/enzimologia , Cavalos , Lipopolissacarídeos/administração & dosagem
20.
Int J Mol Sci ; 21(21)2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33114200

RESUMO

Plant-extracted triterpenoids belong to a class of bioactive compounds with pleotropic functions, including antioxidant, anti-cancer, and anti-inflammatory effects. In this work, we investigated the anti-inflammatory and anti-oxidative activities of a semisynthetic derivative of 18ßH-glycyrrhetinic acid (18ßH-GA), soloxolone methyl (methyl 2-cyano-3,12-dioxo-18ßH-olean-9(11),1(2)-dien-30-oate, or SM) in vitro on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and in vivo in models of acute inflammation: LPS-induced endotoxemia and carrageenan-induced peritonitis. SM used at non-cytotoxic concentrations was found to attenuate the production of reactive oxygen species and nitric oxide (II) and increase the level of reduced glutathione production by LPS-stimulated RAW264.7 cells. Moreover, SM strongly suppressed the phagocytic and migration activity of activated macrophages. These effects were found to be associated with the stimulation of heme oxigenase-1 (HO-1) expression, as well as with the inhibition of nuclear factor-κB (NF-κB) and Akt phosphorylation. Surprisingly, it was found that SM significantly enhanced LPS-induced expression of the pro-inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells via activation of the c-Jun/Toll-like receptor 4 (TLR4) signaling axis. In vivo pre-exposure treatment with SM effectively inhibited the development of carrageenan-induced acute inflammation in the peritoneal cavity, but it did not improve LPS-induced inflammation in the endotoxemia model.


Assuntos
Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Endotoxemia/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Macrófagos/citologia , Triterpenos Pentacíclicos/administração & dosagem , Peritonite/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/metabolismo , Glutationa/metabolismo , Ácido Glicirretínico/química , Heme Oxigenase-1/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteínas de Membrana/genética , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/farmacologia , Peritonite/induzido quimicamente , Peritonite/genética , Peritonite/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
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