RESUMEN
REASONS FOR PERFORMING STUDY: In sepsis models, mitogen-activated protein kinases (MAPKs) are reported to incite inflammatory injury to tissues and are purported to be a therapeutic target. OBJECTIVES: To assess MAPK signalling in lamellae in sepsis-related laminitis (SRL) at different time points after induction of laminitis via carbohydrate overload, and to determine the effect of regional deep hypothermia (RDH) on MAPK signalling. STUDY DESIGN: In vitro study using archived tissue samples. METHODS: Lamellar concentrations of MAPKs were assessed in archived lamellar samples from 2 studies: 1) the starch gruel model of SRL with 3 groups (n = 6/group) of horses (control, onset of fever [DEV] Obel Grade 1 lameness [OG1]); and 2) from limbs maintained at ambient (AMB) and hypothermic (ICE) temperatures (n = 6/group) in animals given a bolus of oligofructose. Immunoblotting and immunolocalisation were used to assess lamellar concentrations and cellular localisation of total and activated (phosphorylated) forms of p38 MAPK, extracellular-regulated kinase (ERK) 1/2, and stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK) 1/2. RESULTS: Lamellar samples had statistically significant increased concentrations of activated ERK 1/2 at the onset of OG1 laminitis (vs. control) in the starch gruel model, but showed no significant change between ICE and AMB limbs in the RDH model. Phospho-SAPK/JNK 1/2 exhibited a similar significant increase in the OG1 samples, but was also increased in ICE (vs. AMB) limbs. No statistically significant changes in lamellar p38 MAPK concentrations were noted. CONCLUSIONS: Increased concentrations of activated ERK 1/2 and SAPK/JNK in the acute stages of SRL indicate a possible role of these signalling proteins in lamellar injury. Signalling related to ERK 1/2 and SAPK/JNK 1/2 pathways should be further investigated to determine if these play a detrimental role in laminitis and may be therapeutic targets to be manipulated independently of RDH.
Asunto(s)
Enfermedades del Pie/veterinaria , Pezuñas y Garras/metabolismo , Enfermedades de los Caballos/inducido químicamente , Inflamación/veterinaria , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Frío , Carbohidratos de la Dieta/efectos adversos , Epidermis , Enfermedades del Pie/inducido químicamente , Enfermedades del Pie/metabolismo , Pezuñas y Garras/patología , Enfermedades de los Caballos/metabolismo , Caballos , Inflamación/inducido químicamente , Inflamación/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genéticaRESUMEN
REASONS FOR PERFORMING STUDY: Hypoxia-inducible factor-1α (HIF-1A) is an important protein in the regulation/induction of many genes in the cellular and tissue response to hypoxia and a central mediator in inflammatory signalling. As both hypoxia and inflammatory events are purported to occur in the lamellar epidermis in sepsis-related laminitis in the equid, HIF-1A may play a central role in this disease process. OBJECTIVESS: To assess the regulation of HIF-1A and HIF-1A-related genes in the equine keratinocyte in vitro and in the lamellar tissue of horses with sepsis-related laminitis. STUDY DESIGN: In vivo and in vitro experiments. METHODS: Real-time quantitative PCR (RT-qPCR) and immunoblotting were performed to assess the mRNA and protein concentrations of HIF-1A and the mRNA concentrations of HIF-1A-related genes in cultured equine keratinocytes and in lamellar samples from black walnut extract (BWE)- and carbohydrate overload (CHO)-induced laminitis. Hypoxia-inducible factor-1α was further localised via indirect immunofluorescence in frozen lamellar tissue sections. RESULTS: Hypoxia-inducible factor-1α appears to be regulated primarily at the post transcriptional level in the cultured equine keratinocyte, resulting in increased HIF-1A in response to hypoxia but not to lipopolysaccharide exposure. Hypoxia-inducible factor-1α is present at high concentrations in the normal equine lamina, and is increased in Obel grade 1 (OG1) stage laminitis in the CHO model of laminitis. Equine lamellar mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase, but not glucose transporter 1, are increased in the BWE and CHO models of laminitis. CONCLUSIONS AND POTENTIAL RELEVANCE: These data indicate that the normal equine lamellae are profoundly hypoxic in comparison with other tissues. The increased mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase 2 in equine keratinocytes exposed to hypoxia and lipopolysaccharide, and in lamellar tissue from BWE and CHO models of sepsis-related laminitis, suggest that the marked lamellar inflammatory gene expression in sepsis-related laminitis may be due to an interaction of constitutively high lamellar keratinocyte HIF-1A signalling with inflammatory signalling, possibly induced by circulating inflammatory mediators.