Laminar Oxidant Stress and Antioxidant Gene Expression in the Black Walnut Extract Model of Equine Laminitis
T.L. Westerman; A. Pettigrew; J.K. Belknap
Due to the central role of oxidant stress in organ injury in human sepsis, we investigated both the presence of laminar oxidative injury, and the protective "antioxidant response" of the laminar tissue in the black walnut extract (BWE) model of equine laminitis. Laminar oxidative injury was assessed in the forms of lipid peroxidation and protein carbonylation. Lipid peroxidation of tissue was evaluated via 4-hydroxy-2-nonenal (4-HNE) immunochemistry (slot blot), whereas protein carbonylation was assessed via ELISA. Laminar tissue antioxidant response was assessed by analyzing mRNA concentrations of critical isoforms of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). Archived snap frozen tissue samples (laminae, skin, lung and liver) were used from the BWE model at two time points post-BWE administration (3H [onset of leucopenia], n=5, and 12H [onset of lameness], n=5); these samples were compared to control group samples (3HC [3H post water administration], n=5, and 12HC [12H post water administration], n=5). Real time quantitative PCR (RT-qPCR) was performed to assess mRNA concentrations of SOD isoforms (SOD 1-3), cytosolic glutathione peroxidase (cGPx) and phospholipid hydroperoxide GPx (phGPx, important in protection from membrane lipid peroxidation). Significant increases in 4-HNE concentrations were evident in laminar tissue at both 3H and 12H time points post-BWE (compared to control groups), whereas no increase in 4-HNE was present in the lung, liver or skin samples. No changes in laminar protein carbonylation were present between principal and control groups. Marked increases in the expression of laminar SOD-2 (mitochondrial SOD) was present at both the 3H and 12H time points (p<0.05). Laminar cGPX mRNA concentration did not change significantly, but laminar phGPx mRNA concentration increased at the 12H time point (p<0.05). Increased laminar 4-HNE concentration with no increase in protein carbonylation possibly indicate acute oxidant stress in the form of lipid peroxidation to the laminar cells, but not the end stage oxidative injury characterized by protein carbonylation. phGPx, an enzyme which blocks membrane lipid peroxidation, undergoes slow induction in the laminae. This late induction in the presence of a rapid onset of oxidant stress may contribute to the marked lipid peroxidation in affected laminae. The increased laminar SOD-2 (mitochondrial SOD) mRNA concentrations indicate a similar pattern of SOD-2 expression as previously reported in organ injury in rodent models of sepsis and human sepsis patients. Increased SOD-2 mRNA in light of a recently reported lack of laminar SOD activity at the 3H time point in the BWE model suggests either a post-transcriptional inhibition, or possibly a delayed increase in laminar SOD concentration and activity. These results indicate that oxidant stress may contribute to the development of acute laminitis and should be further studied to determine the potential of oxidant stress as a therapeutic target in equine laminitis.