Identification and In Vivo Characterization of Inflammatory Biomarkers in Swine.

摘要

Non-steroidal anti-inflammatory drugs (NSAID) are a family of chemicals that function to reduce pain, fever, and inflammation, and are commonly used in people and animals for this purpose. Currently there are no NSAIDs approved for the management of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. Flunixin meglumine is a common NSAID utilized in swine although it is not an FDA-approved anti-inflammatory. A swine model based on inflammation biomarkers could prove very useful in the drug approval process.;This study, which was composed of in vitro and in vivo investigations, was designed to achieve the following objectives: 1) to identify and verify candidate markers representing LPS-induced inflammation in swine, 2) to verify expression patterns of in vitro candidate markers in vivo, and 3) to determine biomarker utility through simultaneous LPS- stimulation and Flunixin meglumine treatment of swine.;The in vitro study investigated the differential expression of genes altered in response to LPS- induced inflammation. Unstimulated whole blood from swine was diluted with tissue culture media, stimulated with LPS, and RNA was extracted at the following time points 0h, 1h, 3h, 24h, and 48 h. A DNA microarray was utilized as a screen to determine potential biomarkers, focusing on the genes that exhibited the greatest degree of differential expression. Following analysis, fourteen genes with significantly altered expression (10 up- and 4 down-regulated) were chosen for verification via quantitative RT-PCR (qRT-PCR). The qRT-PCR analysis confirmed the differential expression of all 14 genes chosen via the microarray analyses. Specifically, genes such as SAA, G-CSF, and IL-10 were up-regulated, while CD4 was down-regulated. All of the genes were altered by 24 or 48 h post-stimulation.;The in vivo model consisted of Flunixin meglumine- treated and/or LPS- stimulated swine. Five of the genes from the in vitro data (MCP-1, SAA2, CD4, CD1, and Caspase 1) were investigated in this system. The swine that received the LPS stimulation only, generally followed the expression patterns demonstrated for these genes in the in vitro model. The swine that received both Flunixin meglumine and LPS demonstrated a reduced effect in comparison to the LPS-stimulated swine. These results, in conjunction with the in vitro, results demonstrate that two out of five genes (SAA2 and CD1) can serve as biomarkers of inflammation as well as indicators of NSAID efficacy.