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1.
FASEB J ; 35(11): e21956, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34605573

RESUMO

MicroRNAs are key regulators of the cardiac response to injury. MiR-100 has recently been suggested to be involved in different forms of heart failure, but functional studies are lacking. In the present study, we examined the impact of transgenic miR-100 overexpression on cardiac structure and function during physiological aging and pathological pressure-overload-induced heart failure in mice after transverse aortic constriction surgery. MiR-100 was moderately upregulated after induction of pressure overload in mice. While in our transgenic model the cardiomyocyte-specific overexpression of miR-100 did not result in an obvious cardiac phenotype in unchallenged mice, the transgenic mouse strain exhibited less left ventricular dilatation and a higher ejection fraction than wildtype animals, demonstrating an attenuation of maladaptive cardiac remodeling by miR-100. Cardiac transcriptome analysis identified a repression of several regulatory genes related to cardiac metabolism, lipid peroxidation, and production of reactive oxygen species (ROS) by miR-100 overexpression, possibly mediating the observed functional effects. While the modulation of ROS-production seemed to be indirectly affected by miR-100 via Alox5-and Nox4-downregulation, we demonstrated that miR-100 induced a direct repression of the scavenger protein CD36 in murine hearts resulting in a decreased uptake of long-chain fatty acids and an alteration of mitochondrial respiratory function with an enhanced glycolytic state. In summary, we identified miR-100 as a modulator of cardiac metabolism and ROS production without an apparent cardiac phenotype at baseline but a protective effect under conditions of pressure-overload-induced cardiac stress, providing new insight into the mechanisms of heart failure.


Assuntos
Antígenos CD36/metabolismo , Insuficiência Cardíaca/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Animais , Antígenos CD36/genética , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Células HEK293 , Insuficiência Cardíaca/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , NADPH Oxidase 4/genética , Ratos , Volume Sistólico/genética , Transfecção , Remodelação Ventricular/genética
2.
J Immunol ; 207(8): 2118-2128, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34507947

RESUMO

Sepsis is a life-threatening organ dysfunction caused by a dysfunctional host response to infection. Neutrophils play a protective role by releasing antibacterial proteins or by phagocytizing bacteria. However, excess neutrophils can induce tissue damage. Recently, a novel intercellular communication pathway involving extracellular vesicles (EVs) has garnered considerable attention. However, whether EVs secreted by macrophages mediate neutrophil recruitment to infected sites has yet to be studied. In this study, we assessed the chemotactic effect of EVs isolated from mouse Raw264.7 macrophages on mouse neutrophils and found that CXCL2 was highly expressed in these EVs. By regulating CXCL2 in Raw264.7 macrophages, we found that CXCL2 on macrophage EVs recruited neutrophils in vitro and in vivo. The CXCL2 EVs activated the CXCR2/PKC/NOX4 pathway and induced tissue damage. This study provides information regarding the mechanisms underlying neutrophil recruitment to tissues and proposes innovative strategies and targets for the treatment of sepsis.


Assuntos
Quimiocina CXCL2/metabolismo , Vesículas Extracelulares/metabolismo , Macrófagos/imunologia , NADPH Oxidase 4/metabolismo , Neutrófilos/imunologia , Proteína Quinase C/metabolismo , Sepse/imunologia , Animais , Ceco/cirurgia , Modelos Animais de Doenças , Doenças do Sistema Imunitário , Transtornos Leucocíticos , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo , Transdução de Sinais
3.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576259

RESUMO

BACKGROUND: The ischemia-reperfusion injury (IRI) of neuronal tissue, such as the brain and retina, leads to possible cell death and loss of function. Current treatment options are limited, but preliminary observations suggest a protective effect of hydrogen sulfide (H2S). However, the dosage, timing, and mechanism of inhaled H2S treatment after IRI requires further exploration. METHODS: We investigated possible neuroprotective effects of inhaled H2S by inducing retinal ischemia-reperfusion injury in rats for the duration of 1 h (120 mmHg), followed by the administration of hydrogen sulfide (H2S) for 1 h at different time points (0, 1.5, and 3 h after the initiation of reperfusion) and at different H2S concentrations (120, 80, and 40 ppm). We quantified the H2S effect by conducting retinal ganglion cell counts in fluorogold-labeled animals 7 days after IRI. The retinal tissue was harvested after 24 h for molecular analysis, including qPCR and Western blotting. Apoptotic and inflammatory mediators, transcription factors, and markers for oxidative stress were investigated. Histological analyses of the retina and the detection of inflammatory cytokines in serum assays were also performed. RESULTS: The effects of inhaled H2S were most evident at a concentration of 80 ppm administered 1.5 h after IRI. H2S treatment increased the expression of anti-apoptotic Bcl-2, decreased pro-apoptotic Bax expression, reduced the release of the inflammatory cytokines IL-1ß and TNF-α, attenuated NF-κB p65, and enhanced Akt phosphorylation. H2S also downregulated NOX4 and cystathionine ß-synthase. Histological analyses illustrated a reduction in TNF-α in retinal ganglion cells and lower serum levels of TNF-α in H2S-treated animals after IRI. CONCLUSION: After neuronal IRI, H2S mediates neuroprotection in a time- and dose-dependent manner. The H2S treatment modulated transcription factor NF-κB activation and reduced retinal inflammation.


Assuntos
Sulfeto de Hidrogênio/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Retina/efeitos dos fármacos , Animais , Apoptose , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Inflamação , Masculino , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Fatores de Tempo
4.
Elife ; 102021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528872

RESUMO

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.


Assuntos
Células Epiteliais Alveolares , Senescência Celular/fisiologia , Células-Tronco Mesenquimais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/fisiologia , Animais , Células Cultivadas , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Organoides/citologia , Organoides/metabolismo , Estresse Oxidativo
5.
Cell Physiol Biochem ; 55(4): 489-504, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34416105

RESUMO

BACKGROUND/AIMS: Diaphragm dysfunction with increased reactive oxygen species (ROS) occurs within 72 hrs post-myocardial infarction (MI) in mice and may contribute to loss of inspiratory maximal pressure and endurance in patients. METHODS: We used wild-type (WT) and whole-body Nox4 knockout (Nox4KO) mice to measure diaphragm bundle force in vitro with a force transducer, mitochondrial respiration in isolated fiber bundles with an O2 sensor, mitochondrial ROS by fluorescence, mRNA (RT-PCR) and protein (immunoblot), and fiber size by histology 72 hrs post-MI. RESULTS: MI decreased diaphragm fiber cross-sectional area (CSA) (~15%, p = 0.015) and maximal specific force (10%, p = 0.005), and increased actin carbonylation (5-10%, p = 0.007) in both WT and Nox4KO. Interestingly, MI did not affect diaphragm mRNA abundance of MAFbx/atrogin-1 and MuRF-1 but Nox4KO decreased it by 20-50% (p < 0.01). Regarding the mitochondria, MI and Nox4KO decreased the protein abundance of citrate synthase and subunits of electron transport system (ETS) complexes and increased mitochondrial O2 flux (JO2) and H2O2 emission (JH2O2) normalized to citrate synthase. Mitochondrial electron leak (JH2O2/JO2) in the presence of ADP was lower in Nox4KO and not changed by MI. CONCLUSION: Our study shows that the early phase post-MI causes diaphragm atrophy, contractile dysfunction, sarcomeric actin oxidation, and decreases citrate synthase and subunits of mitochondrial ETS complexes. These factors are potential causes of loss of inspiratory muscle strength and endurance in patients, which likely contribute to the pathophysiology in the early phase post-MI. Whole-body Nox4KO did not prevent the diaphragm abnormalities induced 72 hrs post-MI, suggesting that systemic pharmacological inhibition of Nox4 will not benefit patients in the early phase post-MI.


Assuntos
Diafragma/enzimologia , Mitocôndrias Musculares/enzimologia , Contração Muscular , Atrofia Muscular/enzimologia , Infarto do Miocárdio/enzimologia , NADPH Oxidase 4/deficiência , Animais , Diafragma/patologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , NADPH Oxidase 4/metabolismo
6.
Life Sci ; 284: 119895, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34450166

RESUMO

AIMS: Dipeptidyl peptidase-4 inhibitor (DPP4i), a new antidiabetic agent, is reported to affect the progression of chronic liver diseases. The study aims to investigate the effects of DPP4i on contractile response, splanchnic hemodynamics, and portal pressure in cirrhotic rats. MATERIALS AND METHODS: A rat model of carbon tetrachloride-induced cirrhosis was used in this study. Sixteen rats with cirrhosis were treated with DDP4i sitagliptin for 5 consecutive days. Portal and systemic pressures and portal blood flow were measured. Mesenteric arterioles were isolated, and concentration-response curves to norepinephrine (NE) were evaluated. The expression of NADPH oxidase (Nox)1, Nox2, Nox4, and soluble epoxide hydrolase (sEH) were detected. Reactive oxygen species (ROS) and epoxyeicosatrienoic acid (EET) levels in mesenteric arteries were also measured. KEY FINDINGS: In cirrhotic rats, sitagliptin significantly reduced portal blood flow and portal pressure without effects on systemic pressure and reversed the decreased response of mesenteric arterioles to NE in an endothelium-dependent manner. Sitagliptin suppressed the increased Nox4 expression and ROS production. In vitro studies showed that Nox4 inhibitor enhanced arteriolar response to NE and reduced hydrogen peroxide (H2O2) level in cirrhotic rats. Sitagliptin also reduced EET levels and increased sEH expression of mesenteric vessels. Pre-incubation with sEH inhibitor in vitro reversed sitagliptin-induced augmentation of response to NE in cirrhotic rats. SIGNIFICANCE: DPP4 inhibition by sitagliptin in vivo has beneficial effects on portal hypertension in cirrhotic rats through normalizing arterial hypocontractility. DDP4 inhibitor may be a novel strategy in the treatment of patients with cirrhosis and portal hypertension.


Assuntos
Artérias/fisiopatologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Hipertensão Portal/complicações , Hipertensão Portal/tratamento farmacológico , Cirrose Hepática/complicações , Cirrose Hepática/fisiopatologia , Vasoconstrição , Animais , Artérias/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/farmacologia , Hemodinâmica/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Hipertensão Portal/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , NADPH Oxidase 4/metabolismo , Norepinefrina/farmacologia , Ratos Sprague-Dawley , Fosfato de Sitagliptina/farmacologia , Regulação para Cima/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos
7.
Cells ; 10(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34440842

RESUMO

This study aimed to investigate the mechanistic pathway of Naja atra (Taiwan cobra) cardiotoxin 1 (CTX1)-induced death of leukemia cell lines U937 and HL-60. CTX1 increased cytoplasmic Ca2+ and reactive oxygen species (ROS) production, leading to the death of U937 cells. It was found that Ca2+-induced NOX4 upregulation promoted ROS-mediated p38 MAPK phosphorylation, which consequently induced c-Jun and ATF-2 phosphorylation. Using siRNA knockdown, activated c-Jun and ATF-2 were demonstrated to regulate the expression of Fas and FasL, respectively. Suppression of Ca2+-mediated NOX4 expression or ROS-mediated p38 MAPK activation increased the survival of U937 cells exposed to CTX1. FADD depletion abolished CTX1-induced cell death, caspase-8 activation, and t-Bid production, supporting the correlation between the Fas death pathway and CTX1-mediated cytotoxicity. Among the tested N. atra CTX isotoxins, only CTX1 induced Fas and FasL expression. Chemical modification studies revealed that intact Met residues were essential for the activity of CTX1 to upregulate Fas and FasL expression. Taken together, the data in this study indicate that CTX1 induces c-Jun-mediated Fas and ATF-2-mediated FasL transcription by the Ca2+/NOX4/ROS/p38 MAPK axis, thereby activating the Fas death pathway in U937 cells. Furthermore, CTX1 activates Fas/FasL death signaling in the leukemia cell line HL-60.


Assuntos
Cardiotoxinas/farmacologia , Proteína Ligante Fas/metabolismo , Naja naja/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor fas/metabolismo , Fator 2 Ativador da Transcrição/antagonistas & inibidores , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Proteína de Domínio de Morte Associada a Fas/antagonistas & inibidores , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia/metabolismo , Leucemia/patologia , NADPH Oxidase 4/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Cells ; 10(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209278

RESUMO

Development of resistance to therapy in ovarian cancer is a major hinderance for therapeutic efficacy; however, new mechanisms of the resistance remain to be elucidated. NADPH oxidase 4 (NOX4) is responsible for higher NADPH activity to increase reactive oxygen species (ROS) production. In this study, we showed that higher levels of NOX4 were detected in a large portion of human ovarian cancer samples. To understand the molecular mechanism of the NOX4 upregulation, we showed that NOX4 expression was induced by HIF-1α and growth factor such as IGF-1. Furthermore, our results indicated that NOX4 played a pivotal role in chemotherapy and radiotherapy resistance in ovarian cancer cells. We also demonstrated that NOX4 knockdown increased sensitivity of targeted therapy and radiotherapy through decreased expression of HER3 (ERBB3) and NF-κB p65. Taken together, we identified a new HIF-1α/NOX4 signal pathway which induced drug and radiation resistance in ovarian cancer. The finding may provide a new option to overcome the therapeutic resistance of ovarian cancer in the future.


Assuntos
Resistencia a Medicamentos Antineoplásicos , NADPH Oxidase 4/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Afatinib/farmacologia , Processamento Alternativo/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Modelos Biológicos , NADPH Oxidase 4/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Intervalo Livre de Progressão , Fator de Transcrição RelA/metabolismo , Transcrição Genética/efeitos dos fármacos , Trastuzumab/farmacologia
9.
PLoS One ; 16(7): e0253777, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270583

RESUMO

Dupuytren's disease (DD) is a fibroproliferative disorder of the palmar aponeurosis, which is characterized by a compound myofibrogenesis and evidenced by an increased expression of α-smooth muscle actin (α-SMA). In Dupuytren's tissue, higher levels of reactive oxygen species (ROS) are documented, stimulating the proliferation and differentiation of myofibroblasts. Our preliminary study demonstrates that α-SMA-expression is significantly inhibited by blue light irradiation in DD. The objective of this study was to investigate the beneficial effect of blue light irradiation and to elucidate the influence of ROS on myofibrogenesis in the pathogenesis of DD. Therefore, an in-vitro model of human DD fibroblasts was used. DD fibroblasts and control fibroblasts isolated from carpal tunnel syndrome (CTS) were daily irradiated with 40 J/cm2 (λ = 453 nm, 38 mW/cm2). Protein expression of ROS-modulating enzymes (Catalase, NOX4, SOD1, MnSOD) and α-SMA were determined, and additionally analysed after a pharmacological inhibition of the TGF-ß1-signaling with SB431542. Furthermore, the protein expression of α-SMA as surrogate parameter for myofibrogenesis was evaluated after applying different concentrations of long-lasting ROS. It could be determined that the beneficial blue light irradiation, which inhibited myofibrogenesis, is mediated by a significant inhibition of catalase protein expression. This effect should be accompanied with an increased intracellular ROS level. Proof of evidence was an H2O2-application on DD fibroblasts, also leading to a decreased myofibrogenesis. Furthermore, it could be demonstrated that endogenous MnSOD was significantly downregulated in resting DD fibroblasts. If DD fibroblasts were treated with the pharmacological inhibitor SB431542, myofibrogenesis was inhibited, but MnSOD expression was simultaneously elevated, which ought to affect ROS level by raising intracellular H2O2 amount. Blue light irradiation as well as the pharmacological action of SB431542 in consequence mediates their beneficial effect on disturbed myofibrogenesis in DD by further increasing ROS level. The present study demonstrates the importance of intracellular ROS homeostasis in DD and illuminates the beneficial effect of blue light as a promising therapy option for DD.


Assuntos
Catalase/metabolismo , Contratura de Dupuytren/metabolismo , NADPH Oxidase 4/metabolismo , Superóxido Dismutase-1/metabolismo , Raios Ultravioleta , Actinas/metabolismo , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade
10.
Aging (Albany NY) ; 13(14): 18606-18619, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326272

RESUMO

This study focused on the relationship between extracellular-regulated kinase (ERK) and obesity-induced increases in neuropathic pain. We fed rats a high-fat diet to establish the obesity model, and rats were given surgery to establish the chronic compression of the dorsal root ganglia (CCD) model. U0126 was applied to inhibit ERK, and metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) was applied to cause AMP-activated protein kinase (AMPK) activation. Paw withdrawal mechanical threshold (PWMT) were calculated to indicate the level of neuropathic pain. The data indicated that compared with normal CCD rats, the PWMT of obese CCD rats were decreased, accompanied with an increase of ERK phosphorylation, NAD(P)H oxidase 4 (NOX4) protein expression, oxidative stress and inflammatory level in the L4 to L5 spinal cord and dorsal root ganglia (DRG). Administration of U0126 could partially elevate the PWMT and reduce the protein expression of NOX4 and the above pathological changes in obese CCD rats. In vitro, ERK phosphorylation, NOX4 protein expression increased significantly in DRG neurons under the stimulation of palmitic acid (PA), accompanied with increased secretion of inflammatory factors, oxidative stress and apoptosis level, while U0126 partially attenuated the PA-induced upregulation of NOX4 and other pathological changes. In the rescue experiment, overexpression of NOX4 abolished the above protective effect of U0126 on DRG neurons in high-fat environment. Next, we explore upstream mechanisms. Metformin gavage significantly reduced neuropathic pain in obese CCD rats. For the mechanisms, activating AMPK with metformin (obese CCD rats) or AICAR (DRG neurons in a high-fat environment) not only inhibited the ERK-NOX4 pathway, but also improved oxidative stress and inflammation caused by high-fat. In conclusion, the AMPK-ERK-NOX4 pathway may has a pivotal role in mediating obesity-induced increases in neuropathic pain.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Gânglios Espinais , Sistema de Sinalização das MAP Quinases , NADPH Oxidase 4/metabolismo , Neuralgia/etiologia , Obesidade/complicações , Medula Espinal , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Apoptose , Butadienos/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hipoglicemiantes/farmacologia , Inflamação , Masculino , Metformina/farmacologia , Neuralgia/metabolismo , Nitrilas/farmacologia , Obesidade/metabolismo , Estresse Oxidativo , Limiar da Dor , Fosforilação , Ratos Wistar , Ribonucleotídeos/farmacologia , Medula Espinal/metabolismo , Medula Espinal/patologia
11.
Life Sci ; 284: 119664, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34090859

RESUMO

AIM: Present study focuses on the effect of daidzein in an experimental model of diabetic cardiomyopathy in rats. MATERIALS AND METHODS: Diabetes was induced in male Sprague Dawley rats by a single intraperitoneal injection of STZ at dose 55 mg/kg. Daidzein treatment was started after six weeks of diabetes induction. Animals received daidzein at a dose of 25, 50, and 100 mg/kg orally for the next four weeks. KEY FINDINGS: Diabetic control animals showed significant prolongation in QT interval, PR interval, and R wave amplitude as compared to normal control animals. Treatment with daidzein at dose 100 mg/kg significantly normalized the QT interval, PR interval, and R wave amplitude. A significant reduction in QRS duration was observed in diabetic animals. Treatment with daidzein significantly improved the QRS duration after treatment. Hemodynamic parameters like systolic pressure (SBP), diastolic pressure (DBP) and mean atrial pressure (MAP) were found to be significantly decreased in diabetic animals. Treatment with daidzein at dose 100 mg/kg significantly improved the SBP, DBP, and MAP. Daidzein treatment prevented the loss of cardiac marker enzyme from heart tissue and also increased the level of AMPK and SIRT1 in plasma. Protein expression of NOX-4 and RAC-1 was also found to be reduced in cardiac tissue of daidzein treated animals. Daidzein treatment improved oxidative defense mechanism and reduced cardiac tissue necrosis and fibrosis. SIGNIFICANCE: From the results, it can be concluded that daidzein mitigates the progression of diabetic cardiomyopathy by inhibiting NOX-4 induced oxidative stress in cardiac tissue.


Assuntos
Diabetes Mellitus Experimental/patologia , Isoflavonas/farmacologia , Miocárdio/patologia , Adenilato Quinase/metabolismo , Animais , Antioxidantes/metabolismo , Biomarcadores/sangue , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Cardiomegalia/sangue , Cardiomegalia/complicações , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/diagnóstico por imagem , Diabetes Mellitus Experimental/fisiopatologia , Eletrocardiografia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Isoflavonas/química , Lipídeos/sangue , Masculino , Miocárdio/enzimologia , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Sirtuína 1/metabolismo , Troponina I/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
12.
Sci Rep ; 11(1): 11970, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099836

RESUMO

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia-reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.


Assuntos
Antioxidantes/química , Inibidores Enzimáticos/química , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxirredução , Estresse Oxidativo , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
13.
J Biol Chem ; 297(1): 100810, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023385

RESUMO

Pulmonary fibrosis is a progressive lung disease often occurring secondary to environmental exposure. Asbestos exposure is an important environmental mediator of lung fibrosis and remains a significant cause of disease despite strict regulations to limit exposure. Lung macrophages play an integral role in the pathogenesis of fibrosis induced by asbestos (asbestosis), in part by generating reactive oxygen species (ROS) and promoting resistance to apoptosis. However, the mechanism by which macrophages acquire apoptosis resistance is not known. Here, we confirm that macrophages isolated from asbestosis subjects are resistant to apoptosis and show they are associated with enhanced mitochondrial content of NADPH oxidase 4 (NOX4), which generates mitochondrial ROS generation. Similar results were seen in chrysotile-exposed WT mice, while macrophages from Nox4-/- mice showed increased apoptosis. NOX4 regulated apoptosis resistance by activating Akt1-mediated Bcl-2-associated death phosphorylation. Demonstrating the importance of NOX4-mediated apoptosis resistance in fibrotic remodeling, mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages exhibited increased apoptosis and were protected from pulmonary fibrosis. Moreover, resolution occurred when Nox4 was deleted in monocyte-derived macrophages in mice with established fibrosis. These observations suggest that NOX4 regulates apoptosis resistance in monocyte-derived macrophages and contributes to the pathogenesis of pulmonary fibrosis. Targeting NOX4-mediated apoptosis resistance in monocyte-derived macrophages may provide a novel therapeutic target to protect against the development and/or progression of pulmonary fibrosis.


Assuntos
Apoptose , Progressão da Doença , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/patologia , Macrófagos/enzimologia , Macrófagos/patologia , NADPH Oxidase 4/metabolismo , Animais , Linhagem Celular , Feminino , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Biológicos , Monócitos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo
14.
J Cell Mol Med ; 25(11): 5269-5282, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33955666

RESUMO

The blood-brain barrier (BBB) damage is a momentous pathological process of ischaemic stroke. NADPH oxidases 4 (NOX4) boosts BBB damage after ischaemic stroke and its expression can be influenced by microRNAs. This study aimed to probe into whether miR-92b influenced the BBB damage after ischaemic stroke by regulating NOX4 expression. Here, miR-92b expression was lessened in the ischaemic brains of rats and oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). In middle cerebral artery occlusion (MCAo) rats, miR-92b overexpression relieved the ameliorated neurological function and protected the BBB integrity. In vitro model, miR-92b overexpression raised the viability and lessened the permeability of OGD-induced BMECs. miR-92b targeted NOX4 and regulated the viability and permeability of OGD-induced BMECs by negatively modulating NOX4 expression. The transcription factor Foxo1 bound to the miR-92b promoter and restrained its expression. Foxo1 expression was induced by OGD-induction and its knockdown abolished the effects of OGD on miR-92b and NOX4 expressions, cell viability and permeability of BMECs. In general, our findings expounded that Foxo1-induced lessening miR-92b boosted BBB damage after ischaemic stroke by raising NOX4 expression.


Assuntos
Barreira Hematoencefálica/patologia , Isquemia Encefálica/fisiopatologia , AVC Isquêmico/fisiopatologia , MicroRNAs/antagonistas & inibidores , NADPH Oxidase 4/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Apoptose , Barreira Hematoencefálica/metabolismo , Regulação da Expressão Gênica , Masculino , MicroRNAs/genética , NADPH Oxidase 4/genética , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley
15.
Oxid Med Cell Longev ; 2021: 5511881, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859775

RESUMO

Diabetic nephropathy is a microvascular complication induced by diabetes, and methylglyoxal (MGO) is a reactive carbonyl species causing oxidative stress that contributes to the induction of inflammatory response in kidney cells. Cudrania tricuspidata (CT), cultivated in Northeast Asia, has been used as traditional medicine for treating various diseases, including neuritis, liver damage, and cancer. In this study, we determined whether a CT root extract (CTRE) can prevent MGO-induced reactive oxygen species (ROS) production and inflammation and assessed underlying mechanisms using a kidney epithelial cell line, HK-2. We observed that CTRE inhibited MGO-induced ROS production. Additionally, CTRE ameliorated the activation of MGO-induced inflammatory signaling pathways such as p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-JUN N-terminal kinase (JNK). Consistent with these results, expressions of p-nuclear factor-kappa B (NFκB) and inflammatory cytokines, tumor necrosis factor-α, interleukin- (IL-) 1ß, and IL-6, were decreased when compared with MGO-only exposed HK-2 cells. CTRE alleviated the MGO-induced decrease in nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and antioxidant enzyme mRNA expressions. MGO induced the expression of NADPH oxidase 4 (NOX4); CTRE pretreatment inhibited this induction. Further studies revealed that the NOX4 expression was inhibited owing to the suppression of MGO-induced protein kinase C (PKC) activation following CTRE treatment. Collectively, our data suggest that CTRE attenuates MGO-induced inflammation and oxidative stress via inhibition of PKC activation and NOX4 expression, as well as upregulating the Nrf2-antioxidant enzyme pathway in HK-2 cells.


Assuntos
Inflamação/prevenção & controle , Rim/efeitos dos fármacos , Moraceae/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Aldeído Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , NADPH Oxidase 4/metabolismo , Extratos Vegetais/química , Raízes de Plantas/química , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Oxid Med Cell Longev ; 2021: 6668971, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33824697

RESUMO

Background: Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD). NADPH oxidase 4- (NOX4-) mediated reactive oxygen species (ROS) production plays a crucial role in cell differentiation and extracellular matrix (ECM) synthesis in ASM remodeling. However, the precise mechanisms underpinning its pathogenic roles remain elusive. Methods: The expression of NOX4 and TGF-ß 1 in the airway of the lung was measured in COPD patients and the control group. Cigarette smoke- (CS-) induced emphysema mice were generated, and the alteration of α-SMA, NOX4, TGF-ß 1, and collagen I was accessed. The changes of the expression of ECM markers, NOX4, components of TGF-ß/Smad, and MAPK/Akt signaling in human bronchial smooth muscle cells (HBSMCs) were ascertained for delineating mechanisms of NOX4-mediated ROS production on cell differentiation and remodeling in human ASM cells. Results: An increased abundance of NOX4 and TGF-ß 1 proteins in the epithelial cells and ASM of lung was observed in COPD patients compared with the control group. Additionally, an increased abundance expression of NOX4 and α-SMA was observed in the lungs of the CS-induced emphysema mouse model. TGF-ß 1 displayed abilities to increase the oxidative burden and collagen I production, along with enhanced phosphorylation of ERK, p38MAPK, and p-Akt473 in HBSMCs. These effects of TGF-ß 1 could be inhibited by the ROS scavenger N-acetylcysteine (NAC), siRNA-mediated knockdown of Smad3 and NOX4, and pharmacological inhibitors SB203580 (p38MAPK inhibitor) and LY294002 (Akt inhibitor). Conclusions: NOX4-mediated ROS production alters TGF-ß 1-induced cell differentiation and collagen I protein synthesis in HBSMCs in part through the p38MAPK/Akt signaling pathway in a Smad-dependent manner.


Assuntos
Colágeno/metabolismo , Miócitos de Músculo Liso/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Idoso , Animais , Brônquios/citologia , Brônquios/metabolismo , Diferenciação Celular , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Mol Biol Rep ; 48(3): 2365-2375, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33792826

RESUMO

Epithelial-mesenchymal transition (EMT), transforming growth factor ß(TGF-ß) and reactive oxygen species(ROS) plays a central role in cancer metastasis. Moreover, nicotinamide adenine dinucleotide phosphate 4(NOX4) is one of the main sources of ROS in lung cancer cells suggesting that NOX4 is associated with tumor cell migration. NF-κB(Nuclear factor-Kappa-B) is known to regulate ROS-mediated EMT process by activating Snail transcription factor in A549 cells. The purpose of this study was to explore the relationship between NF-κB and NOX4 in ROS production during TGF-ß induced EMT process. Several fractions have been pooled to evaluates the EMT process on lung cancer cells through real-time PCR, Western Blot and flow cytometry with DCFH-DA probe etc. Cells proliferation and migration activities were monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and wound healing assay respectively. The result showed that TGF-ß induction decreased the expression of E-cadherin, increased the Vimentin and the EMT transcription factor Snail in A549 cells. DPI (Diphenyleneiodonium chloride, an inhibitor of NOX4) inhibited the NOX4 expression and reduced ROS production induced by TGF-ß, but didn't affect the activation of NF-κB induced by TGF-ß (P > 0.05). BAY11-7082 (an inhibitor of NF-κB) inhibited the NF-κB (p65) expression and prevented the increase of NOX4 expression and ROS production induced by TGF-ß (P < 0.001), which has also verified reduced TGF-ß induced cell migration by inhibiting the EMT process, and also reduced cell proliferation of A549 cells (P < 0.001). The current research confirmed the TGF-ß mediated EMT process via NF-κB/NOX4/ROS signaling pathway, NF-κB and NOX4 are likely to be the potential therapeutic targets for lung cancer metastasis.


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Movimento Celular , Proliferação de Células , Humanos
18.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836590

RESUMO

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2 By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.


Assuntos
Carcinógenos/toxicidade , NADPH Oxidase 4/genética , Neoplasias/induzido quimicamente , Animais , Núcleo Celular/metabolismo , Citosol/metabolismo , Dano ao DNA , Instabilidade Genômica , Camundongos , NADPH Oxidase 4/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Oxirredução , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais
19.
Arch Biochem Biophys ; 704: 108875, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33891961

RESUMO

Previous reports indicate that the central nervous system (CNS) is a target of air pollution, causing tissue damage and functional alterations. Oxidative stress and neuroinflammation have been pointed out as possible mechanisms mediating these effects. The aim of this work was to study the chronic effects of urban air pollution on mice brain cortex, focusing on oxidative stress markers, and mitochondrial function. Male 8-week-old BALB/c mice were exposed to filtered air (FA, control) or urban air (UA) inside whole-body exposure chambers, located in a highly polluted area of Buenos Aires city, for up to 4 weeks. Glutathione levels, assessed as GSH/GSSG ratio, were decreased after 1 and 2 weeks of exposure to UA (45% and 25% respectively vs. FA; p < 0.05). A 38% increase in lipid peroxidation was found after 1 week of UA exposure (p < 0.05). Regarding protein oxidation, carbonyl content was significantly increased at week 2 in UA-exposed mice, compared to FA-group, and an even higher increment was found after 4 weeks of exposure (week 2: 40% p < 0.05, week 4: 54% p < 0.001). NADPH oxidase (NOX) and glutathione peroxidase (GPx) activities were augmented at all the studied time points, while superoxide dismutase (Cu,Zn-SOD cytosolic isoform) and glutathione reductase (GR) activities were increased only after 4 weeks of UA exposure (p < 0.05). The increased NOX activity was accompanied with higher expression levels of NOX2 regulatory subunit p47phox, and NOX4 (p < 0.05). Also, UA mice showed impaired mitochondrial function due to a 50% reduction in O2 consumption in active state respiration (p < 0.05), a 29% decrease in mitochondrial inner membrane potential (p < 0.05), a 65% decrease in ATP production rate (p < 0.01) and a 30% increase in H2O2 production (p < 0.01). Moreover, respiratory complexes I-III and II-III activities were decreased in UA group (30% and 36% respectively vs. FA; p < 0.05). UA exposed mice showed alterations in mitochondrial function, increased oxidant production evidenced by NOX activation, macromolecules damage and the onset of the enzymatic antioxidant system. These data indicate that oxidative stress and impaired mitochondrial function may play a key role in CNS damage mechanisms triggered by air pollution.


Assuntos
Poluentes Atmosféricos/toxicidade , Poluição do Ar/efeitos adversos , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
Cells ; 10(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916471

RESUMO

Noise-induced hearing loss (NIHL) is a common inner ear disease but has complex pathological mechanisms, one of which is increased oxidative stress in the cochlea. The high-mobility group box 1 (HMGB1) protein acts as an inflammatory mediator and shows different activities with redox modifications linked to the generation of reactive oxygen species (ROS). We aimed to investigate whether manipulation of cochlear HMGB1 during noise exposure could prevent noise-induced oxidative stress and hearing loss. Sixty CBA/CaJ mice were divided into two groups. An intraperitoneal injection of anti-HMGB1 antibodies was administered to the experimental group; the control group was injected with saline. Thirty minutes later, all mice were subjected to white noise exposure. Subsequent cochlear damage, including auditory threshold shifts, hair cell loss, expression of cochlear HMGB1, and free radical activity, was then evaluated. The levels of HMGB1 and 4-hydroxynonenal (4-HNE), as respective markers of reactive nitrogen species (RNS) and ROS formation, showed slight increases on post-exposure day 1 and achieved their highest levels on post-exposure day 4. After noise exposure, the antibody-treated mice showed markedly less ROS formation and lower expression of NADPH oxidase 4 (NOX4), nitrotyrosine, inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) than the saline-treated control mice. A significant amelioration was also observed in the threshold shifts of the auditory brainstem response and the loss of outer hair cells in the antibody-treated versus the saline-treated mice. Our results suggest that inhibition of HMGB1 by neutralization with anti-HMGB1 antibodies prior to noise exposure effectively attenuated oxidative stress and subsequent inflammation. This procedure could therefore have potential as a therapy for NIHL.


Assuntos
Cóclea/metabolismo , Cóclea/patologia , Proteína HMGB1/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Provocada por Ruído/patologia , Inflamação/patologia , Estresse Oxidativo , Aldeídos/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Camundongos Endogâmicos CBA , NADPH Oxidase 4/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Regulação para Cima/genética
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