RESUMEN
Though the past few years have witnessed exciting achievements in targeted and immunotherapeutic treatments of all breast cancer subtypes, yet the decline in breast cancer mortality has been slowed, urging the need for further expanding options of high-quality treatments. Prostaglandin D2 synthase (PTGDS)/prostaglandin D2 (PGD2) play important roles in a variety of cancer types and show tissue-specificity, however, there are limited relevant reports in breast cancer. Therefore, the aims of the present study were to investigate the effects of PTGDS/PGD2 in breast cancer by large-scale bioinformatic analysis and in vitro experiments conducted on human breast cancer cell lines. Results of our study indicated that patients with high levels of PTGDS expression showed a reduced potential of tumor proliferation. PGD2 treatment significantly inhibited the proliferation and migration of breast cancer cells, which was mediated by the reduced expression of TWIST2. Overexpression of TWIST2 reversed the inhibitory effects of PGD2 on breast cancer cell proliferation. These results provided the novel evidence that PTGDS may play a significant role in modulating breast cancer growth, with implications for its potential use in treating breast cancer.
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Neoplasias de la Mama/patología , Proliferación Celular/fisiología , Oxidorreductasas Intramoleculares/biosíntesis , Lipocalinas/biosíntesis , Prostaglandina D2/biosíntesis , Proteína Relacionada con Twist 2/biosíntesis , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular , Humanos , Transducción de Señal/fisiologíaRESUMEN
The human ABO blood group system is of great importance in blood transfusion and organ transplantation. ABO transcription is known to be regulated by a constitutive promoter in a CpG island and regions for regulation of cell-specific expression such as the downstream + 22.6-kb site for epithelial cells and a site in intron 1 for erythroid cells. Here we investigated whether the + 22.6-kb site might play a role in transcriptional regulation of the gene encoding odorant binding protein 2B (OBP2B), which is located on the centromere side 43.4 kb from the + 22.6-kb site. In the gastric cancer cell line KATOIII, quantitative PCR analysis demonstrated significantly reduced amounts of OBP2B and ABO transcripts in mutant cells with biallelic deletions of the site created using the CRISPR/Cas9 system, relative to those in the wild-type cells, and Western blotting demonstrated a corresponding reduction of OBP2B protein in the mutant cells. Moreover, single-molecule fluorescence in situ hybridization assays indicated that the amounts of both transcripts were correlated in individual cells. These findings suggest that OBP2B could be co-regulated by the + 22.6-kb site of ABO.
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Sistema del Grupo Sanguíneo ABO , Células Epiteliales/metabolismo , Lipocalinas/biosíntesis , Línea Celular Tumoral , Islas de CpG , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Hibridación in Situ , Hibridación Fluorescente in Situ , Intrones , Lipocalinas/genética , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , RNA-Seq , Espectrometría de Fluorescencia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.
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Oxidorreductasas Intramoleculares/biosíntesis , Lipocalinas/biosíntesis , Activación de Macrófagos/fisiología , Regeneración Nerviosa/fisiología , Neuropatía Ciática/enzimología , Animales , Femenino , Oxidorreductasas Intramoleculares/genética , Lipocalinas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/patología , Neuropatía Ciática/genética , Neuropatía Ciática/patologíaRESUMEN
RATIONALE: Fluid shear stress (FSS) maintains NOS-3 (endothelial NO synthase) expression. Homozygosity for the C variant of the T-786C single-nucleotide polymorphism of the NOS3 gene, which solely exists in humans, renders the gene less sensitive to FSS, resulting in a reduced endothelial cell (EC) capacity to generate NO. Decreased bioavailability of NO in the arterial vessel wall facilitates atherosclerosis. Consequently, individuals homozygous for the C variant have an increased risk for coronary heart disease (CHD). OBJECTIVE: At least 2 compensatory mechanisms seem to minimize the deleterious effects of this single-nucleotide polymorphism in affected individuals, one of which is characterized herein. METHODS AND RESULTS: Human genotyped umbilical vein ECs and THP-1 monocytes were used to investigate the role of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in vitro. Its concentration in plasma samples from genotyped patients with CHD and age-matched CHD-free controls was determined using quantitative ultraperformance LC-MS/MS. Exposure of human ECs to FSS effectively reduced monocyte transmigration particularly through monolayers of CC-genotype ECs. Primarily in CC-genotype ECs, FSS elicited a marked rise in COX (cyclooxygenase)-2 and L-PGDS (lipocalin-type prostaglandin D synthase) expression, which appeared to be NO sensitive, and provoked a significant release of 15d-PGJ2 over baseline. Exogenous 15d-PGJ2 significantly reduced monocyte transmigration and exerted a pronounced anti-inflammatory effect on the transmigrated monocytes by downregulating, for example, transcription of the IL (interleukin)-1ß gene (IL1B). Reporter gene analyses verified that this effect is due to binding of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) to 2 AREs (antioxidant response elements) in the proximal IL1B promoter. In patients with CHD, 15d-PGJ2 plasma levels were significantly upregulated compared with age-matched CHD-free controls, suggesting that this powerful anti-inflammatory prostanoid is part of an endogenous defence mechanism to counteract CHD. CONCLUSIONS: Despite a reduced capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype through an enhanced FSS-dependent release of 15d-PGJ2.
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Células Endoteliales/metabolismo , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico/sangre , Polimorfismo de Nucleótido Simple , Prostaglandina D2/análogos & derivados , Adaptación Fisiológica , Anciano , Anciano de 80 o más Años , Enfermedad Coronaria/sangre , Enfermedad Coronaria/genética , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Inducción Enzimática , Femenino , Genes Reporteros , Predisposición Genética a la Enfermedad , Hemorreología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/genética , Lipocalinas/biosíntesis , Lipocalinas/genética , Masculino , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/fisiología , Óxido Nítrico Sintasa de Tipo III/genética , Prostaglandina D2/biosíntesis , Prostaglandina D2/sangre , Prostaglandina D2/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Células THP-1RESUMEN
AIMS: Cachexia is a severe consequence of cancer. Although cancer-induced heart atrophy leads to cardiac dysfunction and heart failure (HF), biomarkers for their diagnosis have not been identified. Neutrophil gelatinase-associated lipocalin (NGAL) is an aldosterone-responsive gene increased in HF. We studied NGAL and its association with aldosterone levels in a model of cancer cachexia-induced cardiomyopathy. METHODS AND RESULTS: Rats were injected with Yoshida 108 AH-130 hepatoma cells to induce tumour. Cachectic rats were treated daily, for 16 days, with placebo or with 5 or 50 mg/kg/day of spironolactone. Cardiac function was analysed by echocardiography at baseline and at Day 11. Weight loss and atrophy of lean body and fat mass of cachectic rats were significantly attenuated by spironolactone. Cardiac dysfunction of tumour-bearing rats was improved by spironolactone. Plasma aldosterone was up-regulated from 337 ± 7 pg/mL in sham animals to 591 ± 31 pg/mL in the cachectic rats (P < 0.001 vs. sham). Treatment with 50 or 5 mg/kg/day of spironolactone reduced plasma aldosterone to 396 ± 22 and 391 ± 25 pg/mL (P < 0.01 vs. placebo). Plasma levels of NGAL were also increased in cachectic rats (1.462 ± 0.3603 µg/mL) than in controls (0.0936 ± 6 µg/mL, P < 0.001). Spironolactone treatment (50 mg/kg/day) significantly reduced cardiac mRNA and protein NGAL levels (P < 0.05 and P < 0.001 vs. placebo, respectively). NGAL mRNA and protein levels were overexpressed in cachectic animal hearts treated with placebo, compared with control (P < 0.05 and P < 0.01 vs. sham). Spironolactone treatment at 50 mg/kg/day reduced significantly cardiac NGAL (P < 0.05 and P < 0.001 vs. placebo). CONCLUSIONS: Cancer cachexia induced increased levels of aldosterone and NGAL, contributing to worsening cardiac damage in cancer cachexia-induced cardiomyopathy. Spironolactone treatment may greatly attenuate cardiac dysfunction and lean mass atrophy associated with cancer cachexia.
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Proteínas de Fase Aguda , Caquexia , Cardiomiopatías , Regulación Neoplásica de la Expresión Génica , Lipocalinas , Miocardio , Neoplasias Experimentales , Proteínas Proto-Oncogénicas , Animales , Masculino , Ratas , Proteínas de Fase Aguda/biosíntesis , Proteínas de Fase Aguda/genética , Western Blotting , Caquexia/complicaciones , Caquexia/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Lipocalina 2 , Lipocalinas/biosíntesis , Lipocalinas/genética , Miocardio/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Distribución Aleatoria , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Neoplásico/genéticaRESUMEN
BACKGROUND: We sought to confirm that neutrophil gelatinase-associated lipocalin (NGAL) protects against apoptosis during endotoxemia. METHODS: Endotoxemia was induced in rats with lipopolysaccharide (LPS; 3.5 mg/kg) and serum creatinine (SCr), urinary NGAL (uNGAL), renal histopathology confirmed acute kidney injury (AKI). Renal caspase 3 and NGAL were assayed with immunohistochemistry 6 h later. A HK-2 cell model was used in which NGAL and caspase 3 mRNA were evaluated by qRT-PCR within 6 h after LPS (50 µM) treatment, and correlations were studied. NGAL and caspase 3 mRNA expression were measured after delivering NGAL siRNA in HK-2 cells and apoptosis was measured with TUNEL and flow cytometry. RESULTS: SCr and uNGAL were significantly increased after LPS treatment and renal morphology data indicated AKI and renal tubular epithelial cell apoptosis. Caspase 3 and NGAL were predominantly expressed in the tubular epithelial cells and there was a correlation between caspase 3 and NGAL protein (r = 0.663, p = 0.01). In vitro, there was a strong correlation between caspase 3 and NGAL mRNA in LPS-injured HK-2 cells within 24 h (r = 0.448, p < 0.05). Suppressing the NGAL gene in HK-2 cells increased caspase 3 mRNA 4.5-fold and apoptosis increased 1.5-fold after LPS treatment. CONCLUSIONS: NGAL is associated with caspase 3 in renal tubular cells with endotoxin-induced kidney injury, and may regulate its expression and inhibit apoptosis.
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Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Proteínas de Fase Aguda/biosíntesis , Apoptosis/fisiología , Túbulos Renales/metabolismo , Lipocalinas/biosíntesis , Lipopolisacáridos/toxicidad , Proteínas Proto-Oncogénicas/biosíntesis , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Humanos , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Lipocalina 2 , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies. In this study, we report the prokaryotic expression, purification, and refolding of recombinant hLCN6. Purified hLCN6 protein was used to generate monoclonal antibody (mAb) against this protein using conventional hybridoma techniques. The sensitivity and specificity of the anti-hLCN6 mAb were determined based on their activities in enzyme-linked immunosorbent assay and Western blotting analysis using various human tissues. The results showed that the antibody induced by recombinant hLCN6 protein had high sensitivity and specificity. Taken together, the recombinant hLCN6 protein and mAb against this protein obtained from our study provided useful tools for further exploration of the biological functions and molecular mechanism, as well as pathological significance of LCN6 in human.
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Anticuerpos Monoclonales de Origen Murino/química , Lipocalinas/biosíntesis , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/aislamiento & purificación , Especificidad de Anticuerpos , Western Blotting , Epidídimo/metabolismo , Escherichia coli , Femenino , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Lipocalinas/química , Lipocalinas/inmunología , Masculino , Ratones Endogámicos BALB C , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
STUDY QUESTION: Can supplementation of medium with prolactin (PRL), epidermal growth factor (EGF) and 4-hydroxyestradiol (4-OH-E2) prior to embryo transfer improve implantation potential in mouse blastocysts derived from IVF? SUMMARY ANSWER: Combined treatment with PRL, EGF and 4-OH-E2 improves mouse blastocyst implantation rates, while alone, each factor is ineffective. WHAT IS KNOWN ALREADY: Blastocyst dormancy during delayed implantation caused by ovariectomy is maintained by continued progesterone treatment in mice, and estrogen injection rapidly activates blastocysts to implantation-induced status in vivo. While the expression of many proteins is upregulated in implantation-induced blastocysts, selective proteolysis by proteasomes, such as estrogen receptor α (ESR1), occurs in implantation-induced blastocysts to achieve implantation-competent status. It is worth evaluating the proteins expressed during these periods to identify humoral factors that might improve the implantation potential of IVF-derived blastocysts because the poor quality of embryos obtained by IVF is one of the major causes of implantation failure. STUDY DESIGN, SIZE, DURATION: Superovulated oocytes from ICR mice were fertilized with spermatozoa and then cultured in vitro in potassium simplex optimized medium (KSOM) without phenol red (KSOM-P) for 90-96 h. Blastocysts were treated with PRL (10 or 20 mIU/mL), EGF (5 or 10 ng/mL) or 4-OH-E2 (1 or 10 nM) in KSOM-P for 24 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Levels of breast cancer 1 (BRCA1), EGF receptor (EGFR, also known as ERBB1), ERBB4, tubulointerstitial nephritis antigen-like 1 (TINAGL1) and ESR1 protein were examined with immunohistochemical analysis using immunofluorescence methods and confocal laser scanning microscopy. For embryo transfer, six blastocysts were suspended in HEPES-buffered KSOM-P medium and transferred into the uteri of recipient mice on the morning of Day 4 (0900-1000 h) of pseudopregnancy (Day 1 = vaginal plug). The number of implantation sites was then recorded on Day 6 using the blue dye method. MAIN RESULTS AND THE ROLE OF CHANCE: PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the trophectoderm (TE). While PRL treatment resulted in an increase in EGFR, EGF increased both EGFR and ERBB4 in the blastocyst TE. TINAGL1 in the TE was enhanced by 4-OH-E2, which also increased localization of this protein to the basement membrane. Treatment with PRL, EGF or 4-OH-E2 alone did not improve blastocyst implantation rates. Combined treatment with PRL, EGF and 4-OH-E2 resulted in increased levels of EGFR, ERBB4, TINAGL1 and BRCA1 in the TE, whereas ESR1 was not upregulated in the treated blastocysts. Furthermore, combined treatment with PRL, EGF and 4-OH-E2 improved blastocyst implantation rates versus control (P = 0.009). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our studies were carried out in a mouse model, and the conclusions were drawn from limited results obtained from one species. Whether the increase in EGFR, ERBB4 and TINAGL1 protein in the TE improves implantation potential of blastocysts needs to be further studied experimentally by assessing other expressed proteins. The influence of combined supplementation in vitro of PRL, EGF and 4-OH-E2 on implantation also requires further examination and optimization in human blastocysts before it can be considered for clinical use in ART. WIDER IMPLICATIONS OF THE FINDINGS: Enhanced implantation potential by combined treatment with PRL, EGF and 4-OH-E2 appears to result in the upregulation of at least two distinct mechanisms, namely signaling via EGF receptors and basement membrane formation during the peri-implantation period in mice. While PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the TE, treatment with each alone did not improve blastocyst implantation. Therefore, BRCA1 protein appears to be unnecessary for the attachment reaction in blastocysts in mice Combined supplementation of PRL, EGF and 4-OH-E2 might also be of relevance for embryo transfer of human IVF-derived blastocysts for ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the JSPS KAKENHI [Grant numbers 22580316 and 25450390 (to H.M.)] and the Joint Research Project of Japan-U.S. Cooperative Science Program (to H.M.). The authors have no conflict of interest to declare.
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Blastocisto/efectos de los fármacos , Implantación del Embrión/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Estrógenos de Catecol/farmacología , Prolactina/farmacología , Animales , Proteína BRCA1 , Blastocisto/metabolismo , Medios de Cultivo , Interacciones Farmacológicas , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Fertilización In Vitro , Genes BRCA1 , Genes erbB-1 , Lipocalinas/biosíntesis , Lipocalinas/genética , Ratones , Ratones Endogámicos ICR , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Receptor ErbB-4/genética , Técnicas de Cultivo de Tejidos , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Prostaglandins (PGs), particularly prostaglandin D2 (PGD2), E2 (PGE2), and I2 (PGI2), are considered to play a role in migraine pain. In humans, infusion of PGD2 causes lesser headache as compared to infusion of PGE2 and PGI2. Follow-up studies in rats have shown that infusion of PGE2 and PGI2 dilate the middle meningeal artery (MMA), and mRNA for PGE2 and PGI2 receptors is present in rat trigeminovascular system (TVS) and in the brain structures associated with pain. In the present study, we have characterized the dilatory effect of PGD2 on rat MMA and studied the relative mRNA expression of PGD2 receptors and lipocalin-type of PGD2 synthase (L-PGDS). METHOD: Rat closed-cranial window (CCW) model was used to study the effect of the DP1 receptor antagonist, MK-0524, on PGD2-induced vasodilation of middle meningeal artery. The qPCR technique was used for mRNA expression analysis. RESULTS: PGD2 infusion evoked a dose-dependent dilation of the rat MMA. The calculated mean pED50 value was 5.23±0.10 and Emax was 103±18% (n=5). MK-0524 significantly (â¼61%, p<0.05) blocked the PGD2-induced dilation of MMA. mRNA for the DP1, DP2 and L-PGDS were expressed differentially in all tested tissues. DP1 receptor mRNA was expressed maximally in trigeminal ganglion (TG) and in cervical dorsal root ganglion (DRG). CONCLUSIONS: High expression of DP1 mRNA in the TG and DRG suggest that PGD2 might play a role in migraine pathophysiology. Activation of the DP1 receptor in MMA was mainly responsible for vasodilation induced by PGD2 infusion.
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Oxidorreductasas Intramoleculares/genética , Lipocalinas/genética , Arterias Meníngeas/efectos de los fármacos , Dolor/genética , Prostaglandina D2/farmacología , ARN Mensajero/genética , Receptores de Prostaglandina/genética , Animales , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Oxidorreductasas Intramoleculares/biosíntesis , Lipocalinas/biosíntesis , Arterias Meníngeas/metabolismo , Dolor/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Prostaglandina/biosíntesis , Transcriptoma , Núcleos del Trigémino/irrigación sanguínea , Núcleos del Trigémino/efectos de los fármacos , Núcleos del Trigémino/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
The role of lipocalin prostaglandin D2 synthase (L-PGDS) in brain ischemia has not been fully clarified to date. Vagus nerve stimulation (VNS) protects against cerebral ischemia/reperfusion (I/R) injury, but the mechanisms involved need further exploration. This study investigated the role of L-PGDS in cerebral I/R and whether this process was involved in the mechanism of VNS-mediated neuroprotection. Male Sprague-Dawley rats were pretreated with a lentiviral vector (LV) through intracerebroventricular injection, followed by middle cerebral artery occlusion (MCAO) and VNS treatment. The expression of L-PGDS in the peri-infarct cortex was examined. The localization of L-PGDS was determined using double immunofluorescence staining. Neurologic scores, infarct volume and neuronal apoptosis were evaluated at 24 h after reperfusion. The expression of apoptosis-related molecules was measured by western blot analysis. The expression of L-PGDS in the peri-infarct cortex increased at 12 h, reached a peak at 24 h after reperfusion, and lasted up to 3 days. VNS treatment further enhanced the expression of L-PGDS following ischemic stroke. L-PGDS was mainly expressed in neurons in the peri-infarct cortex. I/R rats treated with VNS showed better neurological deficit scores, reduced infarct volume, and decreased neuronal apoptosis as indicated by the decreased levels of Bax and cleaved caspase-3 as well as increased levels of Bcl-2. Strikingly, the beneficial effects of VNS were weakened after L-PGDS down-regulation. In general, our results suggest that L-PGDS is a potential mediator of VNS-induced neuroprotection against I/R injury.
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Apoptosis/fisiología , Isquemia Encefálica/metabolismo , Oxidorreductasas Intramoleculares/biosíntesis , Lipocalinas/biosíntesis , Accidente Cerebrovascular/metabolismo , Estimulación del Nervio Vago , Animales , Isquemia Encefálica/prevención & control , Modelos Animales de Enfermedad , Oxidorreductasas Intramoleculares/uso terapéutico , Lipocalinas/uso terapéutico , Masculino , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/prevención & control , Estimulación del Nervio Vago/métodosRESUMEN
Obstructive sleep apnea (OSA) is a common disorder characterized by chronic intermittent hypoxia (CIH). Excessive daytime sleepiness (EDS) is one of severe complications frequently associated with OSA. Lipocalin-type prostaglandin synthase (L-PGDS) is potentially responsible for the production of prostaglandin D2 (PGD2) which is an endogenous sleep inducer. To date, whether the content of PGD2 and PGDS is related to intermittent hypoxia has never been reported. The aim of this study was to compare the content of PGD2 and L-PGDS in rats' brains with and without intermittent hypoxia. Adult male Wistar rats (n = 48; 8-10 weeks) were averagely divided into two groups. One was control group, and the other group was exposed to IH (12 h/day for 6 weeks). In each group there are four time-points including 0, 2, 4 and 6 weeks, and six rats were killed and studied at each time-point. At the end of 0, 2, 4 and 6 weeks, the concentrations of PGD2 in brains were measured by LC-MS/MS. In addition, the expressions of L-PGDS protein and mRNA in brains were investigated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed the concentrations of PGD2 in CIH rat brains were higher than those in control groups from the second week. At the end of 6 weeks, the concentrations of PGD2 in CIH and control groups were 11.1 and 5.9 ng/g, respectively. The levels of L-PGDS protein and mRNA followed the same trend during the whole 6 weeks. The results will provide a new idea to explore that patients with OSA are always accompanied by excessive daytime sleepiness.
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Encéfalo/metabolismo , Regulación de la Expresión Génica , Hipoxia/fisiopatología , Oxidorreductasas Intramoleculares/biosíntesis , Lipocalinas/biosíntesis , Prostaglandina D2/biosíntesis , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Perfilación de la Expresión Génica , Masculino , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sueño/fisiología , Espectrometría de Masas en TándemRESUMEN
Diabetic nephropathy is a serious microvascular complication and one of the main causes of end-stage renal disease. L-Glutamine (LG) is naturally occurring amino acids with antidiabetic and antioxidant potential. The aim of present investigation was to evaluate the potential of LG against streptozotocin (STZ)-induced diabetic nephropathy (DN) in laboratory rats. DN was induced in male Wistar rats (200-220 g) by intraperitoneal administration of STZ (55 mg/kg). Animals were treated orally with either distilled water (10 mg/kg) or LG (250, 500, and 1000 mg/kg) or Sitagliptin (5 mg/kg). Various biochemical, molecular, and histological (hematoxylin-eosin and Masson's trichrome stain) parameters were assessed. Administration of LG (500 and 1000 mg/kg) significantly inhibited (p < .05) STZ-induced alterations in serum and urine biochemistry (urine creatinine, uric acid, albumin, and BUN). It also significantly increased creatinine clearance rate. STZ induced increase in renal oxidonitrosative stress was significantly decreased (p < .05) by LG (500 and 1000 mg/kg) treatment. Upregulated renal KIM-1, NGAL, TGF-ß1, and collagen-1 mRNA expression after STZ administration was significantly inhibited (p < .05) by LG (500 and 1000 mg/kg) treatment. Correlation analysis also revealed that antidiabetic potential of LG attenuates STZ-induced elevated renal KIM-1, NGAL, TGF-ß1, and collagen-1 mRNA expression. Histopathological alteration induced by STZ in renal tissue was ameliorated by LG treatment. In conclusion, results of present investigation suggest that treatment with LG ameliorated STZ-induced DN via the inhibition of oxidonitrosative stress as well as downregulation of KIM-1, NGAL, TGF-ß1, and collagen-1 mRNA expressions.
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Proteínas de Fase Aguda/genética , Moléculas de Adhesión Celular/genética , Colágeno Tipo I/genética , Nefropatías Diabéticas/tratamiento farmacológico , Regulación de la Expresión Génica , Glutamina/farmacología , Lipocalinas/genética , Proteínas Proto-Oncogénicas/genética , Factor de Crecimiento Transformador beta1/genética , Proteínas de Fase Aguda/biosíntesis , Animales , Moléculas de Adhesión Celular/biosíntesis , Colágeno Tipo I/biosíntesis , Diabetes Mellitus Experimental , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Lipocalina 2 , Lipocalinas/biosíntesis , Masculino , Proteínas Proto-Oncogénicas/biosíntesis , ARN/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
INTRODUCTION: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxiï¬ed by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats. MATERIALS AND METHODS: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were ï¬xed in paraformaldehyde and cut in 20 micrometer slices by cryostat. RESULTS: The expression of NGAL signiï¬cantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene. DISCUSSION: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.
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Proteínas de Fase Aguda/biosíntesis , Lipocalinas/biosíntesis , Regeneración Nerviosa/genética , Nervios Periféricos/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Nervio Ciático/metabolismo , Proteínas de Fase Aguda/genética , Animales , Depuradores de Radicales Libres/metabolismo , Regulación de la Expresión Génica , Humanos , Lipocalina 2 , Lipocalinas/genética , Nervios Periféricos/patología , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Nervio Ciático/lesiones , Nervio Ciático/fisiopatologíaRESUMEN
Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some ß-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.
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Antiinfecciosos/química , Células Epiteliales/microbiología , Epitelio/microbiología , Tracto Gastrointestinal/microbiología , Interleucina-17/metabolismo , Interleucinas/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/patología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Epitelio/inmunología , Epitelio/metabolismo , Gastritis/microbiología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Humanos , Inflamación , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucina-8/biosíntesis , Interleucina-8/metabolismo , Interleucinas/biosíntesis , Interleucinas/genética , Complejo de Antígeno L1 de Leucocito/biosíntesis , Complejo de Antígeno L1 de Leucocito/metabolismo , Lipocalinas/biosíntesis , Lipocalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Estómago/microbiología , Interleucina-22RESUMEN
Hypoxia-induced cardiomyocyte apoptosis contributes significantly to the development of numerous cardiac diseases, such as ischemic heart disease, heart failure, etc. Promoting cell survival by inhibiting apoptosis is one of the available strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. Previous studies have been demonstrated that miR-138 and lipocalin-2 (Lcn2) play important roles in cardiomyocyte apoptosis and survival. We presently determined whether Lcn2 is a target gene of miR-138 involved in hypoxia-induced cardiomyocyte apoptosis. Firstly, mimics of miR-138 were transfected into HL-1 cells to investigate its effect on cell apoptosis. Using 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-FITC/PI flow cytometer assays, over-expression of miR-138 significantly enhanced the cell growth and significantly attenuated the cell apoptosis in hypoxic conditions. Dual-luciferase reporter gene and western blot results confirmed Lcn2 was a direct target of miR-138. Then, the recombinant plasmid, pcDNA3.1/Lcn2 was transfected into the HL-1 cells that over-expressed miR-138. We further observed that the over-expression of Lcn2 diminished the protection of miR-138 over-expression from hypoxia-induced cell survival and apoptosis. In conclusion, our study demonstrated that up-regulation of miR-138 inhibits the hypoxia-induced cardiomyocyte apoptosis via down-regulating the pro-apoptotic gene expression of Lcn2.
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Proteínas de Fase Aguda/antagonistas & inhibidores , Proteínas de Fase Aguda/biosíntesis , Apoptosis , Hipoxia/patología , Lipocalinas/antagonistas & inhibidores , Lipocalinas/biosíntesis , MicroARNs/metabolismo , Miocitos Cardíacos/fisiología , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/biosíntesis , Animales , Western Blotting , Supervivencia Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Genes Reporteros , Lipocalina 2 , Luciferasas/análisis , Luciferasas/genética , RatonesRESUMEN
Tumor initiating cancer stem-like cells (TICSCs) have recently become the object of intensive study. Human-Lipocalin-2 (hLCN2) acts as a biomarker for cancers. The aim of the present study was to explore new insights regarding the potential role of LCN2 in inducing epithelial to mesenchymal transition (EMT) by transfecting LCN2 into CD133+-A549-TICSCs and its cross-talk with the NF-κB signaling pathway in adenocarcinoma of the lung. Furthermore, EMT was confirmed by transcriptomic analysis, immunoblotting and immunocyto/histochemical analyses. Tumorigenesis and metastasis were confirmed by molecular therapeutics tracer 2DG infrared optical probe in BALB/cSIc-nude mice. It was observed that the CD133+-expressing-LCN2-A549 TICSCs population increased in adenocarcinoma of the lung compared to the normal lung tissue. The expressions of genes involved in stemness, adhesion, motility and drug efflux was higher in these cells than in their non-LCN2 expressing counterparts. The present study revealed that elevated expression of LCN2 significantly induced metastasis via EMT. Overexpression of LCN2 significantly increased stemness and tumor metastasis by modulating NF-κB cellular signaling. BRM270, a novel inhibitor of NF-κB plays a significant role in the EMT reversal. BRM270, a naturaceutical induces cell shrinkage, karyorrhexis and programmed cell death (PCD) which were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. Also, 2DG guided in vivo model revealed that BRRM270 significantly (P<0.0003) reduced tumor metastasis and increased percent survival in real-time with complete resection. An elaborate study on the novel concept with respect to linking of naturaceutics as selective and potential anticancer agent that eliminates the elevated LCN2 induced EMT and tumor dissemination through cooperation with the NF-κB signaling as the baseline data for the planning of new therapeutic strategies was conducted for the first time. Our results also illustrate a molecular mechanistic approach for 2DG-guided molecular imaging-based cancer therapy using BRM270 as a novel cancer therapeutic drug to enhance the effect of doxorubicin (Dox)-resistant LCN2 induced metastasis of solid tumors in nude mice.
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Proteínas de Fase Aguda/genética , Adenocarcinoma/genética , Carcinogénesis/genética , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Lipocalinas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Fase Aguda/biosíntesis , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lipocalina 2 , Lipocalinas/biosíntesis , Neoplasias Pulmonares/patología , Ratones , FN-kappa B/genética , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To examine the significance of the Neutrophil gelatinase-associated lipocalin (NGAL) in diagnosing head and neck squamous cell carcinoma (HNSCC) and predicting regional metastasis. We first used GEO dataset to analyze the NGAL gene expression in HNSCC. Then, we summarized the characteristics of patients retrospectively selected in clinic. Expression of NGAL protein in human HNSCC tumor, lymph node and normal samples were analyzed using immunohistochemistry. Next, we further investigated the NGAL expression in a tissue microassay to analyze the relationship between NGAL protein expression and TNM stage. Finally, we tested the NGAL protein expression in head and neck cancer cell lines. Analysis of GEO dataset concluded that NGAL gene expression in HNSCC was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL gene expression between T-stage and N-stage (P>0.05). NGAL protein expression in tumor was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL protein expression between metastasis group and non-metastasis group (P>0.05). Expression of NGAL protein was not correlated with TNM stage of HNSCC. Aggressive HNSCC cell lines have lower NGAL protein expression. Our data demonstrated that the expression of NGAL protein was correlated with tumorigenesis of HNSCC, but not with regional metastasis. It may serve as a novel biomarker for prognostic evaluation of patients with HNSCC.
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Proteínas de Fase Aguda/biosíntesis , Carcinogénesis/patología , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Lipocalinas/biosíntesis , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Western Blotting , Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Regulación hacia Abajo , Femenino , Neoplasias de Cabeza y Cuello/genética , Humanos , Inmunohistoquímica , Lipocalina 2 , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello , Análisis de Matrices Tisulares , Transcriptoma , Adulto JovenRESUMEN
Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.
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Ácidos Araquidónicos/metabolismo , Metilación de ADN/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Metilación de ADN/efectos de los fármacos , Decitabina , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Oxidorreductasas Intramoleculares/biosíntesis , Riñón/citología , Riñón/enzimología , Lipocalinas/biosíntesis , Hígado/citología , Hígado/enzimología , Masculino , Metabolómica , Ratones , Tromboxano-A Sintasa/biosíntesisRESUMEN
PURPOSE: The current study was done to determine the role of lipocalin-2 (LCN2) in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model. METHODS: The EAON was induced by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG35-55) peptide in mice. The LCN2 expression was examined in the optic nerve after MOG peptide injection. Degree of demyelination, inflammatory infiltration, glial activation, and expression profile of inflammatory mediators in the optic nerve were compared between LCN2 knockout (KO) animals and wild-type littermates by histological analysis and real-time PCR following EAON induction. Plasma levels of LCN2 in patients with optic neuritis were measured by ELISA. RESULTS: The expression of LCN2 was notably increased in the optic nerve after EAON induction. Expression of LCN2 was colocalized with reactive astrocytes. A significant reduction of demyelination, inflammatory infiltration, and gliosis was demonstrated in the optic nerve of LCN2 KO mice. The LCN2 KO mice also showed markedly reduced gene expression associated with the M1-polarized glia phenotype and toll-like receptor signaling in the optic nerve. The LCN2 levels in plasma were significantly higher in optic neuritis patients (71.6 ± 10.6 ng/mL) compared to healthy controls (37.4 ± 9.1 ng/mL, P = 0.0284). CONCLUSIONS: In this study, we demonstrated a significant induction of LCN2 expression in astrocytes of the optic nerve following EAON induction. Our results imply that astrocyte-derived LCN2 may have a pivotal role in the development of demyelinating optic neuritis, and LCN2 can be a therapeutic target to alleviate immune and inflammatory damage in the optic nerve.