RESUMEN
The aim of this study was to evaluate the involvement of both B1 and B2 kinins receptors (B1R and B2R) in the fibroblast proliferation induced by the cytokine tumour necrosis factor (TNF) attempting to establish an in vitro model of wound healing. Murine fibroblasts L-929 were cultivated in 24 wells plaque until total confluence (DMEM (Vitrocell®); 5% fetal bovine serum, 5% CO2, 37⯰C) and then submitted to the scratch assay. The cells were treated with PBS, TNF (2â¯ng/mL) and/or mr-TNF antibody (200⯵g/mL), or PDTC. The cells received the second set of treatment (3â¯h later): PBS; 1⯵M HOE-140; 1⯵M des-Arg9-Leu8-BK (DALBK) or 100⯵M PDTC. TNF was able to increase the cell proliferation when compared with the group treated with PBS. The co-treatment with the TNF antibody completely reversed the TNF effect. The TNF-proliferative effect was blocked by B1 (DALBK) and B2 (HOE-140) kinin receptor antagonists administered separately or along, suggesting the involvement of both receptors in the TNF mechanism of action. Furthermore, the treatment with a NF-ĸB inhibitor PDTC completely blocked the cell proliferation. The TNF cell proliferation was incremented with BK (1⯵M) treatment, and its effect was totally reversed by HOE-140 treatment. No effect was observed for TNF plus DABK. Eventually, TNF treatment was able to increase TNF level in the growing medium; however, this increase was suppressed by BK treatment. These results suggest that TNF induces cell proliferation and the induced signalling cascade has the B2R participation. All these events seem to be totally dependent on the NF-ĸB activation. These inflammatory mediators can improve the wound healing in the resolution of inflammation.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Cininas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Cininas/genética , RatonesRESUMEN
BACKGROUND: Envenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Bmv (1 µg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT. CONCLUSION/SIGNIFICANCE: These data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.
Asunto(s)
Analgésicos/efectos adversos , Citocinas/metabolismo , Hiperalgesia/terapia , Cininas/metabolismo , Terapia por Luz de Baja Intensidad , Neuronas/efectos de los fármacos , Mordeduras de Serpientes/terapia , Venenos de Serpiente/efectos adversos , Analgésicos/administración & dosificación , Animales , Bothrops , Citocinas/genética , Femenino , Humanos , Hiperalgesia/etiología , Hiperalgesia/genética , Hiperalgesia/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Cininas/genética , Masculino , Ratones , Mordeduras de Serpientes/etiología , Mordeduras de Serpientes/genética , Mordeduras de Serpientes/metabolismo , Venenos de Serpiente/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes.
Asunto(s)
Área Hipotalámica Lateral/metabolismo , Cininas/deficiencia , Proteínas del Tejido Nervioso/biosíntesis , Obesidad/genética , Obesidad/metabolismo , Animales , Peso Corporal/fisiología , Ingestión de Energía/fisiología , Inmunohistoquímica , Hibridación in Situ , Cininas/genética , Cininas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/metabolismo , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin--a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Anuros/genética , Piel/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Anuros/clasificación , Anuros/metabolismo , Secuencia de Bases , Bradiquinina/química , Bradiquinina/genética , Bradiquinina/metabolismo , Biblioteca de Genes , Cininas/química , Cininas/genética , Cininas/metabolismo , Datos de Secuencia Molecular , Preservación Biológica , Proteoma/genética , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Piel/química , América del Sur , Transcripción GenéticaRESUMEN
Bradykinin and Lys-bradykinin are potent peptide mediators implicated in several physiopathological effects in mammals. They act through activation of G-protein-coupled constitutive B(2) or inducible kinin B(1) receptors linked to signaling pathways involving increased intracellular Ca(++) concentrations and/or release of mediators including arachidonic acid metabolites, NO and EDHF. In the cardiovascular system, the kallikrein-kinin system exerts a fine control of vascular smooth muscle tone and arterial blood pressure, and plays a significant cardioprotective effect. This has been lately confirmed in experimental studies employing transgenic mice overexpressing human tissue kallikrein and animals with knockout of kinin B(1) and B(2) receptor gene. Disturbances in this system are associated with arterial hypertension, myocardial ischaemia and other clinical complications. Inhibitors of kininase II (angiotensin-converting enzyme) have been prescribed successfully to patients with cardiovascular diseases, but there is still a great interest in developing drugs or pharmacological strategies that augment the activity of kininogen-kallikrein-kinin system in pathological conditions. Delivery of adenovirus vector containing the human tissue kallikrein gene (gene kallikrein therapy) has emerged as a great potential to satisfy these conditions. This review provides a summary of plasma and tissue kallikrein-kinin system, focusing on the pharmacological properties, kinin receptors and drugs reported to interfere with their actions. The modulatory effects of the kallikrein-kinin system on cardiovascular system, particularly in regulating smooth muscle tone and arterial blood pressure and in preventing myocardium ischaemia have also been explored in the review.