RESUMEN

This study evaluated the antihyperalgesic and anti-inflammatory effects of percutaneous vagus nerve electrical stimulation (pVNS) by comparing the effects of alternating and random frequencies in an animal model of persistent inflammatory hyperalgesia. The model was induced by Freund's complete adjuvant (CFA) intraplantar (i.pl.) injection. Mice were treated with different protocols of time (10, 20, or 30 min), ear laterality (right, left or both), and frequency (alternating or random). Mechanical hyperalgesia was evaluated, and some groups received i.pl. WRW4 (FPR2/ALX antagonist) to determine the involvement. Edema, paw surface temperature, and spontaneous locomotor activity were evaluated. Interleukin-1ß, IL-6, IL-10, and IL4 levels were verified by enzyme-linked immunosorbent assay. AnxA1, FPR2/ALX, neutrophil, M1 and M2 phenotype macrophage, and apoptotic cells markers were identified using western blotting. The antihyperalgesic effect pVNS with alternating and random frequency effect is depending on the type of frequency, time, and ear treated. The pVNS random frequency in the left ear for 10 min had a longer lasting antihyperalgesic effect, superior to classical stimulation using alternating frequency and the FPR2/ALX receptor was involved in this effect. There was a reduction in the levels of pro-inflammatory cytokines and an increase in the immunocontent of AnxA1 and CD86 in mice paw. pVNS with a random frequency in the left ear for 10 min showed to be optimal for inducing an antihyperalgesic effect. Thus, the random frequency was more effective than the alternating frequency. Therefore, pVNS may be an important adjunctive treatment for persistent inflammatory pain.

Asunto(s)

Anexina A1 , Animales , Ratones , Anexina A1/química , Anexina A1/genética , Anexina A1/metabolismo , Estimulación Eléctrica , Hiperalgesia/complicaciones , Hiperalgesia/terapia , Hiperalgesia/metabolismo , Inflamación/complicaciones , Inflamación/metabolismo , Dolor , Receptores de Formil Péptido , Nervio Vago/metabolismo

RESUMEN

AIMS: In this study we evaluated the effect of pharmacological treatment with AnxA1-derived peptide Ac2-26 in an experimental model of toxicity induced by cisplatin. MAIN METHODS: Male rats were divided into Sham (control), Cisplatin (received intraperitoneal injections of 10 mg/kg/day of cisplatin for 3 days) and Ac2-26 (received intraperitoneal injections of 1 mg/kg/day of peptide, 15 min before cisplatin) groups. KEY FINDINGS: After 6 h of the last dose of cisplatin, an acute inflammatory response was observed characterized by a marked increase in the number of neutrophils and GM-CSF, IL-ß, IL-6, IL-10 and TNF-α plasma levels. These findings were associated with increased AnxA1 protein levels in liver and kidneys, as well as positive AnxA1/Fpr2 circulating leukocytes. Treatment with Ac2-26 produced higher levels of GM-CSF, corroborating the high numbers of neutrophils, and the anti-inflammatory cytokine IL-4. Ac2-26 preserved the morphology of liver structures and increased Fpr1 expression, preventing the damage caused by cisplatin. In the kidneys, Ac2-26 caused downregulation of renal Fpr1 and Fpr2 levels and abrogated the increased levels of the CLU and KIM-1 biomarkers of kidney damage induced by cisplatin. However, no effect of peptide treatment was detected in cisplatin-induced kidney morphology injury. SIGNIFICANCE: Despite activation of the anti-inflammatory AnxA1/Fpr axis during cisplatin administration, treatment with Ac2-26 did not efficiently prevent its deleterious effects on the liver and kidneys.

Asunto(s)

Anexina A1 , Animales , Anexina A1/química , Anexina A1/metabolismo , Anexina A1/farmacología , Antiinflamatorios/farmacología , Cisplatino/metabolismo , Cisplatino/toxicidad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Masculino , Péptidos/química , Ratas

RESUMEN

Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.

Asunto(s)

Anexina A1/metabolismo , Conjuntivitis Alérgica/metabolismo , Receptores de Formil Péptido/metabolismo , Animales , Anexina A1/química , Quimiocinas/metabolismo , Conjuntivitis Alérgica/inmunología , Conjuntivitis Alérgica/patología , Regulación hacia Abajo/efectos de los fármacos , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Ovalbúmina/inmunología , Fragmentos de Péptidos/farmacología

RESUMEN

The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2-26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2-26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2-26 peptide, in a concentration of 1.7µM and 33.8µM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2-26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2-26 peptide at 33.8µM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2-26 peptide as an innovative therapy for the treatment of ocular inflammation.

Asunto(s)

Anexina A1/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Péptidos/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Aciltransferasas/genética , Aciltransferasas/metabolismo , Anexina A1/química , Anexina A1/metabolismo , Antiinflamatorios/farmacología , Western Blotting , Línea Celular , Movimiento Celular/genética , Proliferación Celular/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Oftalmopatías/genética , Oftalmopatías/prevención & control , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Inflamación/genética , Inflamación/prevención & control , Lipopolisacáridos/farmacología , Péptidos/química , Péptidos/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Annexin A1 (AnxA1) is an endogenous glucocorticoid regulated protein that modulates anti-inflammatory process and its therapeutic potential has recently been recognized in a range of systemic inflammatory disorders. The effect of the N-terminal peptide Ac2-26 of AnxA1 on the toxic activities of Bothrops moojeni crude venom (CV) and its myotoxin II (MjTX-II) were evaluated using a peritonitis rat model. Peritonitis was induced by the intraperitoneal injection of either CV or MjTX-II, a Lys-49 phospholipase A2. Fifteen minutes after the injection, the rats were treated with either Ac2-26 or PBS. Four hours later, the CV and MjTX-II-induced peritonitis were characterized by neutrophilia (in the peritoneal exudate, blood and mesentery) and increased number of mesenteric degranulated mast cells and macrophages. At 24 hours post-injection, the local inflammatory response was attenuated in the CV-induced peritonitis while the MjTX-II group exhibited neutrophilia (peritoneal exudates and blood). Ac2-26 treatment prevented the influx of neutrophils in MjTX-II-induced peritonitis and diminished the proportion of mesenteric degranulated mast cells and macrophages in CV-induced peritonitis. Additionally, CV and MjTX-II promoted increased levels of IL-1ß and IL-6 in the peritoneal exudates which were significantly reduced after Ac2-26 treatment. At 4 and 24 hours, the endogenous expression of AnxA1 was upregulated in the mesenteric neutrophils (CV and MjTX-II groups) and mast cells (CV group). In the kidneys, CV and MjTX-II administrations were associated with an increased number of macrophages and morphological alterations in the juxtamedullary nephrons in proximal and distal tubules. Ac2-26 promoted significant recovery of the juxtamedullary structures, decreased the number of macrophages and diminished the AnxA1 in epithelial cells from distal tubules and renal capsules. Our results show that Ac2-26 treatment significantly attenuates local and systemic inflammatory processes and indicate this peptide as a potential target for the development of new therapeutic strategies for the snakebite envenomation treatment.

Asunto(s)

Anexina A1/química , Venenos de Crotálidos/toxicidad , Inflamación/prevención & control , Lisina/química , Péptidos/farmacología , Fosfolipasas A2/toxicidad , Animales , Anexina A1/farmacología , Bothrops , Modelos Animales de Enfermedad , Masculino , Imitación Molecular , Fosfolipasas A2/química , Ratas , Ratas Wistar

RESUMEN

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1(2-26) (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.

Asunto(s)

Anexina A1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Anexina A1/química , Carcinoma de Células Escamosas/inmunología , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Neoplasias Laríngeas/inmunología , Masculino , Mastocitos/citología , Mastocitos/efectos de los fármacos , Metaloproteasas/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Prostaglandinas/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.

Asunto(s)

Anexina A1/genética , Antiinflamatorios/farmacología , Péptidos/farmacología , Uveítis/genética , Animales , Anexina A1/administración & dosificación , Anexina A1/química , Anexina A1/metabolismo , Anexina A1/farmacología , Antiinflamatorios/administración & dosificación , Humor Acuoso/citología , Humor Acuoso/inmunología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Endotoxinas/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , FN-kappa B/metabolismo , Infiltración Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Oligopéptidos/farmacología , Péptidos/administración & dosificación , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Ratas , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Uveítis/inducido químicamente , Uveítis/inmunología

RESUMEN

Recent studies have revealed that binding of annexin I to phospholipids induces the formation of a second phospholipid binding site. It is shown that the N terminus on the concave side of membrane-bound annexin I is cleaved much faster by trypsin or cathepsin than the N terminus of the free protein. The reactivity of the unique disulfide bond located near the concave face was similarly increased by membrane binding. These results demonstrate that Ca(2+)-dependent membrane binding induces a conformational change on the concave side of the annexin I molecule and support the notion that this face of the molecule may contribute to the formation of the secondary membrane-binding site.

Asunto(s)

Anexina A1/química , Liposomas , Fosfatidilserinas , Conformación Proteica , Anexina A1/metabolismo , Sitios de Unión , Cloruro de Calcio/farmacología , Cinética , Fosfatidiletanolaminas , Conformación Proteica/efectos de los fármacos , Tripsina/metabolismo