RESUMEN
Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling.
Asunto(s)
Ácido Araquidónico/metabolismo , Núcleo Celular/metabolismo , Inflamación/metabolismo , Mecanotransducción Celular , Actinas/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Calcio/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Leucocitos/metabolismo , Lámina Nuclear/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Pez CebraRESUMEN
Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.
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Enfermedades de los Peces/inmunología , Infiltración Neutrófila/inmunología , Transducción de Señal/inmunología , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Enfermedades de los Peces/microbiología , Inmunidad Innata/inmunología , Larva/inmunología , Larva/microbiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fosfolipasas A2 Citosólicas/inmunología , Fosfolipasas A2 Citosólicas/metabolismo , Pez Cebra/genética , Pez Cebra/microbiología , Proteínas de Pez Cebra/inmunología , Proteínas de Pez Cebra/metabolismoRESUMEN
Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.
Asunto(s)
Eugenol , Embolia Pulmonar , Humanos , Ratones , Animales , Eugenol/farmacología , Eugenol/uso terapéutico , Eugenol/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos Animales de Enfermedad , Activación Plaquetaria , Agregación Plaquetaria , Plaquetas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Tromboxano A2/metabolismo , Colágeno/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
Increased prevalence of cancer in obese individuals is involved with dyslipidemia- induced chronic inflammation and immune suppression. Although apolipoprotein C-III (ApoC3)-transgenic mice (ApoC3TG mice) or poloxamer 407 (P407)-treated mice had hyperlipidemia, CD8+ T cells with upregulated antitumor activities were observed in ApoC3TG mice, and decreased CD8+ T cell activities were observed in P407-treated mice. Increased ApoC3 expression in hepatocellular carcinoma was associated with increased infiltration of CD8+ T cells and predicted survival. Recombinant ApoC3 had no direct effects on CD8+ T cells. The upregulation of CD8+ T cells in ApoC3TG mice was due to cross-talk with context cells, as indicated by metabolic changes and RNA sequencing results. In contrast to dendritic cells, the macrophages of ApoC3TG mice (macrophagesTG) displayed an activated phenotype and increased IL-1ß, TNF-α, and IL-6 production. Coculture with macrophagesTG increased CD8+ T cell function, and the adoptive transfer of macrophagesTG suppressed tumor progression in vivo. Furthermore, spleen tyrosine kinase (Syk) activation induced by TLR2/TLR4 cross-linking after ApoC3 ligation promoted cellular phospholipase A2 (cPLA2) activation, which in turn activated NADPH oxidase 2 (NOX2) to promote an alternative mode of inflammasome activation. Meanwhile, mitochondrial ROS produced by increased oxidative phosphorylation of free fatty acids facilitated the classical inflammasome activation, which exerted an auxiliary effect on inflammasome activation of macrophagesTG. Collectively, the increased antitumor activity of CD8+ T cells was mediated by the ApoC3-stimulated inflammasome activation of macrophages, and the mimetic ApoC3 peptides that can bind TLR2/4 could be a future strategy to target liver cancer.
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Inflamasomas , Neoplasias , Ratones , Animales , Inflamasomas/metabolismo , Apolipoproteína C-III/metabolismo , Apolipoproteína C-III/farmacología , Linfocitos T CD8-positivos/metabolismo , Receptor Toll-Like 2/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolipasas A2 Citosólicas/farmacología , Ratones Endogámicos C57BLRESUMEN
Spinal cord injury (SCI) is a devastating injury that may result in permanent motor impairment. The active ingredients of medications are unable to reach the affected area due to the bloodâbrain barrier. Elamipretide (SS-31) is a new and innovative aromatic cationic peptide. Because of its alternating aromatic and cationic groups, it freely crosses the bloodâbrain barrier. It is also believed to decrease inflammation and protect against a variety of neurological illnesses. This study explored the therapeutic value of SS-31 in functional recovery after SCI and its possible underlying mechanism. A spinal cord contusion injury model as well as the Basso Mouse Scale, footprint assessment, and inclined plane test were employed to assess how well individuals could function following SCI. The area of glial scarring, the number of dendrites, and the number of synapses after SCI were confirmed by HE, Masson, MAP2, and Syn staining. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays were employed to examine the expression levels of pyroptosis-, autophagy-, lysosomal membrane permeabilization (LMP)- and MAPK signalling-related proteins. The outcomes showed that SS-31 inhibited pyroptosis, enhanced autophagy and attenuated LMP in SCI. Mechanistically, we applied AAV vectors to upregulate Pla2g4A in vivo and found that SS-31 enhanced autophagy and attenuated pyroptosis and LMP by inhibiting phosphorylation of cPLA2. Ultimately, we applied asiatic acid (a p38-MAPK agonist) to test whether SS-31 regulated cPLA2 partially through the MAPK-P38 signalling pathway. Our group is the first to suggest that SS-31 promotes functional recovery partially by inhibiting cPLA2-mediated autophagy impairment and preventing LMP and pyroptosis after SCI, which may have potential clinical application value.
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Piroptosis , Traumatismos de la Médula Espinal , Ratones , Animales , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Lisosomas/metabolismo , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
BACKGROUND: Ventilator-induced lung injury (VILI) is a clinical complication of mechanical ventilation observed in patients with acute respiratory distress syndrome. It is characterized by inflammation mediated by inflammatory cells and their secreted mediators. METHODS: To investigate the mechanisms underlying VILI, a C57BL/6J mouse model was induced using high tidal volume (HTV) mechanical ventilation. Mice were pretreated with Clodronate liposomes to deplete alveolar macrophages or administered normal bone marrow-derived macrophages or Group V phospholipase A2 (gVPLA2) intratracheally to inhibit bone marrow-derived macrophages. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected to assess lung injury and measure Ca2 + concentration, gVPLA2, downstream phosphorylated cytoplasmic phospholipase A2 (p-cPLA2), prostaglandin E2 (PGE2), protein expression related to mitochondrial dynamics and mitochondrial damage. Cellular experiments were performed to complement the animal studies. RESULTS: Depletion of alveolar macrophages attenuated HTV-induced lung injury and reduced gVPLA2 levels in alveolar lavage fluid. Similarly, inhibition of alveolar macrophage-derived gVPLA2 had a similar effect. Activation of the cPLA2/PGE2/Ca2 + pathway in alveolar epithelial cells by gVPLA2 derived from alveolar macrophages led to disturbances in mitochondrial dynamics and mitochondrial dysfunction. The findings from cellular experiments were consistent with those of animal experiments. CONCLUSIONS: HTV mechanical ventilation induces the secretion of gVPLA2 by alveolar macrophages, which activates the cPLA2/PGE2/Ca2 + pathway, resulting in mitochondrial dysfunction. These findings provide insights into the pathogenesis of VILI and may contribute to the development of therapeutic strategies for preventing or treating VILI.
Asunto(s)
Enfermedades Mitocondriales , Lesión Pulmonar Inducida por Ventilación Mecánica , Humanos , Ratones , Animales , Macrófagos Alveolares/metabolismo , Dinoprostona/metabolismo , Dinoprostona/uso terapéutico , Ratones Endogámicos C57BL , Pulmón , Líquido del Lavado Bronquioalveolar , Fosfolipasas A2/metabolismo , Fosfolipasas A2/uso terapéutico , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
Epidemiological studies have demonstrated that particulate matter (PM) can induce or exacerbate hypertension. High relative humidity has been associated with elevated blood pressure in certain regions. However, the coupling effect of humidity and PM on elevated blood pressure and the underlying mechanisms remain unknown. Herein, we aimed to explore the effects of exposure to PM and/or high relative humidity on hypertension, as well as elucidate underlying mechanisms. Male C57/BL6 mice were intraperitoneally administered NG-nitro-L-arginine methyl ester (L-NAME) to establish a hypertensive mouse model. The hypertensive mice were exposed to PM (0.15 mg/kg/day) and/or different relative humidities (45/90%) for eight weeks. Histopathological changes, systolic blood pressure (SBP), endothelial-derived contracting factors (thromboxane B2 [TXB2], Prostaglandin F2α [PGF2α], endothelin-1 [ET-1], and angiotensin II [Ang II]), and relaxing factors (prostaglandin I2 [PGI2] and nitric oxide [NO]) were measured to assess the effects of PM exposure and humidity on hypertension in mice. Levels of transient receptor potential vanilloid 4 (TRPV4), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX2) were measured to explore their potential mechanisms. Herein, exposure to 90% relative humidity or PM alone had a slight but insignificant effect on hypertension. However, pathological changes and elevated blood pressure were markedly exacerbated following exposure to PM and 90% relative humidity. Levels of PGF2α, TXB2, and ET-1 were significantly increased, whereas the PGI2 level was substantially decreased. HC-067047-mediated blockade of TRPV4 suppressed TRPV4, cPLA2, and COX2 expression and effectively alleviated the increased blood pressure induced by exposure to PM and 90% relative humidity. These results indicate that 90% relative humidity and PM can activate the TRPV4-cPLA2-COX2 ion channel in the aorta, altering the endothelial-derived contracting and relaxing factors and enhancing blood pressure in hypertensive mice.
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Antineoplásicos , Hipertensión , Animales , Masculino , Ratones , Antineoplásicos/farmacología , Presión Sanguínea , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Humedad , Hipertensión/inducido químicamente , Óxido Nítrico/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/farmacología , Canales Catiónicos TRPV/uso terapéutico , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.
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Trombina , Tromboxanos , Animales , Ratones , Plaquetas , Fibrinolíticos/farmacología , Agregación Plaquetaria , Trombina/farmacología , Tromboxano A2 , Umbeliferonas/farmacología , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
Glaucoma is a neurodegenerative disease that leads to blindness, and lowering intraocular pressure (IOP) is very important in glaucoma treatment. The trabecular meshwork is responsible for aqueous humor outflow, and the accumulation of fibronectin in trabecular meshwork is known to cause ocular hypertension. We have already shown that Piezo1 activation has an IOP lowering effect in mice and suppresses fibronectin expression level in human trabecular meshwork cells (HTMC). In this study, we report the mechanism of the reduction of fibronectin caused by Piezo1 activation. Activation of Piezo1 in HTMC showed increased expression of matrix metalloproteinase-2 (MMP-2) and cyclooxygenase (COX)-2, and decreased fibronectin expression. In addition, Piezo1 activation enhanced phosphorylation of cytosolic phospholipase A2 (cPLA2), and inhibitors targeting cPLA2 and COX-2 suppressed Yoda 1, a Piezo1 agonist, induced fibronectin reduction. These results indicate that the arachidonic acid cascade underlies this reaction, and, in support of this hypothesis, activation of Piezo1 promoted secretion of prostaglandin F2α (PGF2α) in HTMC. These results indicate that the activation of Piezo1 in HTMC promotes the degrading of fibronectin by promoting the arachidonic acid cascade and increasing the expression of PGF2α and MMP-2.
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Ácido Araquidónico , Dinoprost , Fibronectinas , Glaucoma , Enfermedades Neurodegenerativas , Hipertensión Ocular , Animales , Humor Acuoso/metabolismo , Ácido Araquidónico/metabolismo , Dinoprost/metabolismo , Fibronectinas/metabolismo , Glaucoma/metabolismo , Presión Intraocular , Canales Iónicos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Enfermedades Neurodegenerativas/metabolismo , Hipertensión Ocular/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolipasas A2 Citosólicas/farmacología , Malla Trabecular/metabolismoRESUMEN
Escherichia coli is the most common Gram-negative bacillary organism causing neonatal meningitis. Escherichia coli meningitis remains an important cause of mortality and morbidity, but the pathogenesis of E. coli penetration of the blood-brain barrier remains incompletely understood. Escherichia coli entry into the brain occurs in the meningeal and cortex capillaries, not in the choroid plexus, and exploits epidermal growth factor receptor (EGFR) and cysteinyl leukotrienes (CysLTs) for invasion of the blood-brain barrier. The present study examined whether EGFR and CysLTs are inter-related in their contribution to E. coli invasion of the blood-brain barrier and whether counteracting EGFR and CysLTs is a beneficial adjunct to antibiotic therapy of E. coli meningitis. We showed that (a) meningitis isolates of E. coli exploit EGFR and CysLTs for invasion of the blood-brain barrier, (b) the contribution of EGFR is upstream of that of CysLTs, and (c) counteracting EGFR and CysLTs as an adjunctive therapy improved the outcome (survival, neuronal injury and memory impairment) of animals with E. coli meningitis. These findings suggest that investigation of host factors contributing to E. coli invasion of the blood-brain barrier will help in enhancing the pathogenesis and development of new therapeutic targets for E. coli meningitis in the era of increasing resistance to conventional antibiotics.
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Acetatos/uso terapéutico , Barrera Hematoencefálica/microbiología , Ciclopropanos/uso terapéutico , Cisteína/metabolismo , Receptores ErbB/metabolismo , Escherichia coli/patogenicidad , Gefitinib/uso terapéutico , Leucotrienos/metabolismo , Meningitis por Escherichia coli/microbiología , Quinolinas/uso terapéutico , Sulfuros/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Barrera Hematoencefálica/fisiopatología , Encéfalo/irrigación sanguínea , Ceftriaxona/uso terapéutico , Células Cultivadas , Quimioterapia Combinada , Células Endoteliales , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Recién Nacido , Antagonistas de Leucotrieno/uso terapéutico , Masculino , Meningitis por Escherichia coli/tratamiento farmacológico , Ratones , Permeabilidad , Fosfolipasas A2 Citosólicas/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
BACKGROUND: Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450 mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. METHODS AND RESULTS: Young adult male rats sustaining upper-moderate BACs (~150 mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1 week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. CONCLUSIONS: The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.
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Antiinflamatorios/farmacología , Etanol/farmacología , Etanolaminas/metabolismo , Fosfolipasas A2 Calcio-Independiente/farmacología , Fosfolipasas A2 Citosólicas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Fosfolipasas A2/metabolismo , RatasRESUMEN
RATIONALE: The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A2, which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A2 are formed after the concerted actions of cPLA2α (cytosolic phospholipase A2) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF1α (PGI-M) and 11-dehydro-TXB2 (TX-M) have been taken as biomarkers of prostacyclin and thromboxane A2 formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. OBJECTIVE: We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA2α, causing almost complete loss of prostacyclin and thromboxane A2, who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA2α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A2 in the circulation. METHODS AND RESULTS: Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F1α) and thromboxane A2 (measured as TXB2), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A2 remained negligible. CONCLUSIONS: These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromboxane A2 by platelets in the general circulation. Previous work relying on urinary metabolites of prostacyclin and thromboxane A2 as markers of whole-body endothelial and platelet function now requires reevaluation.
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6-Cetoprostaglandina F1 alfa/análogos & derivados , Aloinjertos/metabolismo , Trasplante de Riñón , Riñón/metabolismo , Mutación con Pérdida de Función , Fosfolipasas A2 Citosólicas/genética , Tromboxano B2/análogos & derivados , 6-Cetoprostaglandina F1 alfa/metabolismo , 6-Cetoprostaglandina F1 alfa/orina , Biomarcadores/orina , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Fenotipo , Fosfolipasas A2 Citosólicas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tromboxano B2/metabolismo , Tromboxano B2/orinaRESUMEN
Phospholipase A2 (PLA2 ) is involved in eicosanoid release, and F2 -isoprostanes (F2 -IsoPs), as free radical-generated eicosanoids released by PLA2 , are indicators of oxidative stress in different human conditions. This study investigated the interplay between cytosolic PLA2 (cPLA2 ), F2 -IsoPs and sperm features in male infertility, when the involvement of oxidative stress has been reported. Semen evaluation was performed following WHO guidelines, sperm ultrastructure was detected by transmission electron microscopy indicating a fertility index, and the percentages of sperm immaturity, apoptosis and necrosis. In sperm cells and seminal plasma, cPLA2 levels were determined by immunological method, whereas F2 -IsoPs by mass spectrometry. Sperm concentration, morphology, vitality and fertility index values were significantly lower in infertile groups compared with fertile men. An increase in sperm apoptosis and necrosis (p < .01), apoptosis (p < .01) and immaturity (p < .001) was detected in leucocytospermia, idiopathic infertility and varicocele, respectively. Seminal cPLA2 showed the highest value in varicocele group (p < .05), whereas seminal F2 -IsoPs increased in varicocele (p < .001) and leucocytospermia (p < .05) groups. In the whole population, F2 -IsoP and cPLA2 levels were positively correlated (p < .05). On the contrary, F2 -IsoPs and cPLA2 were not significantly different when investigated in sperm cells. Our data indicate that fatty acid oxidation/metabolism plays a role in different male reproductive pathological conditions.
Asunto(s)
F2-Isoprostanos/metabolismo , Infertilidad Masculina/enzimología , Fosfolipasas A2 Citosólicas/metabolismo , Análisis de Semen , Varicocele/enzimología , Adulto , Estudios de Casos y Controles , Humanos , MasculinoRESUMEN
During radiotherapy, an inflammatory response might be induced by activating various enzymes involved in membrane lipid metabolism. The eicosanoid pathway associated with cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COXs), and lipoxygenases (LOXs) can be induced by radiation, and many lipid metabolites might contribute to cancer-associated inflammation, cell proliferation, and cell survival in cancer. The lipid metabolites are also involved in the establishment of the tumor-associated microenvironment through promotion of angiogenesis and formation of vascular network. These biological activities of lipid metabolites are responsible for malignant progression with the acquisition of radioresistance, leading to unsatisfactory outcome of cancer radiotherapy. Many efforts have been made to identify the mechanisms associated with bioactive lipid metabolites and radiation signaling that lead to radioresistance and to develop potent radiosensitizers to improve therapeutic efficacy. Beneficial outcomes would be achieved by targeting the enzymes, such as cPLA2, COXs, and LOXs, responsible for arachidonic acid metabolism and cancer-associated inflammation during cancer radiotherapy. The current study demonstrated a brief review for the radioresistant effects of bioactive lipid metabolites and their enzymes in cancer and the radiosensitizing effects of inhibitors for the enzymes on cancer therapy.
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Ácidos Araquidónicos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Metabolismo de los Lípidos/efectos de la radiación , Neoplasias/metabolismo , Neoplasias/radioterapia , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Biomarcadores , Ensayos Clínicos como Asunto , Terapia Combinada , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Susceptibilidad a Enfermedades , Activación Enzimática/efectos de la radiación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Neoplasias/enzimología , Neoplasias/genética , Fosfolipasas A2 Citosólicas/genética , Fosfolipasas A2 Citosólicas/metabolismo , Pronóstico , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Resultado del TratamientoRESUMEN
The proliferation and adipogenesis of preadipocytes played important roles in the development of adipose tissue and contributed much to the processes of obesity. On the other hand, lipopolysaccharide (LPS), also known as endotoxin, is a key outer membrane component of gram-negative bacteria in the gut microbiota, and has a dominant role in linking inflammation to high-fat diet-induced metabolic syndrome. Studies suggested the potential roles of LPS in hepatic steatosis and in obese mice models. However, the molecular mechanisms underlying LPS-regulated obesity remained largely unknown. Here we reported that LPS stimulated expression of cyosolic phospholipase A2 (cPLA2), one of inflammation regulators of obesity, in the preadipocytes. Pretreatment the inhibitors of JAK2, STAT3, STAT5 or AMPK significantly reduced LPS-increased mRNA and protein expression of cPLA2 together with phosphorylation of JAK2, STAT3, STAT5 and AMPK, separately. Similarly, transfection of siRNA against JAK2 or AMPK abolished expression of cPLA2 and phosphorylation of JAK2 or AMPK together with downregulated expression of JAK2 and AMPK protein. LPS enhanced activation of STAT3 and STAT5 via JAK2-dependent manner in the preadipocytes. Transfection of JAK2 or AMPK siRNA further proofed the independence of JAK2 and AMPK in LPS-treated preadipocytes. In addition, LPS-increased DNA synthesis, cell numbers and cell viability of preadipocytes were attenuated by AACOCF3, AG490, BML-275, cPLA2 siRNA, JAK2 siRNA or AMPK siRNA. Attenuation JAK2/STAT or AMPK-dependent cPLA2 expression reduced LPS-mediated adipogenesis of preadipocytes. Stimulation of arachidonic acid or AMPK activator, A-769662, increased cell numbers and cell viability and promoted differentiation of preadipocytes. Collectively, these results indicated that LPS increased preadipocytes proliferation and adipogenesis via JAK/STAT and AMPK-dependent cPLA2 expression. The mechanisms of LPS-stimulated cPLA2 expression may be a link between bacteria and obesity and provides the molecular basis for preventing metabolic syndrome or hyperplasic obesity.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos , Adipogénesis/efectos de los fármacos , Janus Quinasa 2/metabolismo , Lipopolisacáridos/farmacología , Fosfolipasas A2 Citosólicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endotoxinas/farmacología , RatonesRESUMEN
BACKGROUND The aim of this study was to elucidate the involvement of cPLA2-AA-COX-2 pathway factors and their potential role in lung cancer early diagnosis and prognosis. MATERIAL AND METHODS We selected 80 lung cancer patients as the cancer group, and 30 normal patients were selected as the normal group. Serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 were measured, and mRNA levels of COX-2, cPLA2, COX-1, and mPGES in serum were determined. Spearman's P-test was used to analyze the correlation between expression of PGI2 and mPGES in serum and the clinical characteristics of these lung cancer patients. The factors affecting the prognosis lung cancer were analyzed by COX regression model. RESULTS The serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 in the cancer patient group were significantly higher (p<0.05) than in the normal group; after treatment, the serum contents of these factors were significantly decreased (p<0.05). However, distant metastasis had a significant effect on serum contents of mPGES and PGI2 (p<0.05), but not on the other factors. The mRNA levels of COX-2, cPLA2, COX-1, and mPGES in cancer patients were significantly higher than in normal patients. In addition, the 5-year survival rate of patients with high expression of mPGES and/or PGI2 was lower than that of the low expression group. Cox regression analysis showed that the expression of mPGES and PGI2 had statistical significance in predicting the prognosis of lung cancer. CONCLUSIONS The cPLA2-AA-COX-2 pathway is closely associated with lung cancer. These findings are important for clinical diagnosis of lung cancer.
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Ácido Araquidónico/metabolismo , Ciclooxigenasa 2/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Adulto , Anciano , Ácido Araquidónico/sangre , Ácido Araquidónico/genética , Ciclooxigenasa 2/sangre , Ciclooxigenasa 2/genética , Citosol/metabolismo , Dinoprostona/metabolismo , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Isoenzimas , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Fosfolipasas A2 Citosólicas/sangre , Fosfolipasas A2 Citosólicas/genética , Pronóstico , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/genéticaRESUMEN
Obesity is associated with metabolic endotoxemia, reactive oxygen species (ROS), chronic inflammation, and obese kidney fibrosis. Although the fat-intestine-kidney axis has been documented, the pathomechanism and therapeutic targets of obese kidney fibrosis remain unelucidated. To mimic obese humans with metabolic endotoxemia, high-fat-diet-fed mice (HF group) were injected with lipopolysaccharide (LPS) to yield the obese kidney fibrosis-metabolic endotoxemia mouse model (HL group). Therapeutic effects of ROS, cytosolic phospholipases A2 (cPLA2) and cyclooxygenase-2 (COX-2) inhibitors were analyzed with a quantitative comparison of immunohistochemistry stains and morphometric approach in the tubulointerstitium of different groups. Compared with basal and HF groups, the HL group exhibited the most prominent obese kidney fibrosis, tubular epithelial lipid vacuoles, and lymphocyte infiltration in the tubulointerstitium. Furthermore, inhibitors of nonspecific ROS, cPLA2 and COX-2 ameliorated the above renal damages. Notably, the ROS-inhibitor-treated group ameliorated not only oxidative injury but also the expression of cPLA2 and COX-2, indicating that ROS functions as the upstream signaling molecule in the inflammatory cascade of obese kidney fibrosis. ROS acts as a key messenger in the signaling transduction of obese kidney fibrosis, activating downstream cPLA2 and COX-2. The given antioxidant treatment ameliorates obese kidney fibrosis resulting from a combined high-fat diet and LPS-ROS could serve as a potential therapeutic target of obese kidney fibrosis with metabolic endotoxemia.
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Ciclooxigenasa 2/genética , Endotoxemia/complicaciones , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Obesidad/complicaciones , Fosfolipasas A2 Citosólicas/genética , Especies Reactivas de Oxígeno/metabolismo , Animales , Biomarcadores , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Metabolismo de los Lípidos , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Terapia Molecular Dirigida , Estrés Oxidativo , Fosfolipasas A2 Citosólicas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A2 specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A2. The differences between each enzyme's specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme's specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A2 for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A2, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A2 favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol.
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Fosfolipasas A2 Calcio-Independiente/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolipasas A2 Secretoras/metabolismo , Fosfolípidos/metabolismo , Sitios de Unión , Dominio Catalítico , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Fosfolipasas A2 Calcio-Independiente/química , Fosfolipasas A2 Citosólicas/química , Fosfolipasas A2 Secretoras/química , Fosfolípidos/química , Especificidad por SustratoRESUMEN
Acute nephron reduction such as after living kidney donation may increase the risk of hypertension. Uninephrectomy induces major hemodynamic changes in the remaining kidney, resulting in rapid increase of single-nephron glomerular filtration rate (GFR) and fluid delivery in the distal nephron. Decreased sodium (Na) fractional reabsorption after the distal tubule has been reported after uninephrectomy in animals preserving volume homeostasis. In the present study, we thought to specifically explore the effect of unilateral nephrectomy on epithelial Na channel (ENaC) subunit expression in mice. We show that γ-ENaC subunit surface expression was specifically downregulated after uninephrectomy, whereas the expression of the aldosterone-sensitive α-ENaC and α1-Na-K-ATPase subunits as well as of kidney-specific Na-K-Cl cotransporter isoform and Na-Cl cotransporter were not significantly altered. Because acute nephron reduction induces a rapid increase of single-nephron GFR, resulting in a higher tubular fluid flow, we speculated that local mechanical factors such as fluid shear stress (FSS) were involved in Na reabsorption regulation after uninephrectomy. We further explore such hypothesis in an in vitro model of FSS applied on highly differentiated collecting duct principal cells. We found that FSS specifically downregulates ß-ENaC and γ-ENaC subunits at the transcriptional level through an unidentified heat-insensitive paracrine basolateral factor. The primary cilium as a potential mechanosensor was not required. In contrast, protein kinase A and calcium-sensitive cytosolic phospholipase A2 were involved, but we could not demonstrate a role for cyclooxygenase or epoxygenase metabolites.
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Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Mecanotransducción Celular , Nefrectomía , Reabsorción Renal , Sodio/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Células Epiteliales/patología , Canales Epiteliales de Sodio/genética , Túbulos Renales Colectores/patología , Masculino , Ratones Endogámicos C57BL , Comunicación Paracrina , Fosfolipasas A2 Citosólicas/metabolismo , Transducción de Señal , Estrés Mecánico , Transcripción GenéticaRESUMEN
Colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third in males. In this work, we aim to investigate the possible anti-cancer effects of interferon-gamma (IFN-γ) in CRC cells. We observed that IFN-γ induced mitochondria-derived reactive oxygen species (ROS) production in a time-dependent manner in SW480 and HCT116â¯cell lines. The IFN-γ-induced mitochondrial ROS generation was dependent on the activation of cytosolic phospholipase A2 (cPLA2). In addition, a mitochondria-targeted antioxidant SS31 and/or cPLA2 inhibitor AACOCF3 abolished the IFN-γ-induced ROS production and subsequent autophagy and apoptosis. Moreover, suppression of autophagy by CQ was able to reduce IFN-γ-induced cell apoptosis. Beclin-1 gene silencing resulted in caspase-3 inactivation, decreased Bax/Bcl-2 ratio and less population of apoptotic cells. Collectively, our results suggested that IFN-γ induces autophagy-associated apoptosis in CRC cells via inducing cPLA2-dependent mitochondrial ROS production.