Document Type

Thesis - University Access Only

Award Date

2010

Degree Name

Master of Science (MS)

Department / School

Animal Science

Abstract

Endotoxemia is often manifested by an overproduction of circulating proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a) in response to lipopolysaccharide (LPS). Genistein is a non-steroidal tyrosine kinase inhibitor that has been shown to decrease the release of TNF-a from circulating monocytes in response to LPS. However, genistein also has estrogenic properties that have been shown in some species to reduce LPS stimulated TNF-a production. To further examine if genistein could be used to reduce LPS stimulated TNF-a release in the horse and to examine if the estrogenic properties of genistein are involved in reducing LPS stimulated TNF-a release, and mRNA production of other proinflammatory cytokines in the horse, the following experiments were performed. Blood was collected from mature healthy geldings and an enriched population of peripheral blood mononuclear cells (PBMC) was isolated. Peripheral blood mononuclear cells were treated with varying concentrations of genistein and 100 pg/mL LPS (E.coli O55:B5). The supernatants were collected by centrifugation and frozen at -80°C until assayed for TNF-a by ELISA. Differences in supernatant concentrations of TNF-a were determined using the Proc Mixed procedure of SAS. Supernatant concentrations of TNF-a in tubes containing no LPS were not different (P>0.05) among replicates and were at the detection limit of the assay. Supernatant concentrations of TNF-a decreased (P< 0.05) as genistein concentrations increased. Tubes containing 1 and 10 μM genistein produced less (P< 0.05) TNF-a, where as 1 pM, 1 nM, and 10 nM treatments in response to LPS were not different than LPS alone (P>0.05). These data suggest that genistein may be beneficial in reducing TNF-a release in response to LPS in the horse. In a subsequent experiment, blood was collected from mature healthy geldings and an enriched PBMC population was isolated. Peripheral blood mononuclear cells were treated with various concentrations of estradiol (E2), genistein, and an estrogen receptor antagonist, ICI-182, 780, (ICI) with and without I 00 pg/mL LPS (E. coli O55:B5). The supernatants were collected by centrifugation and frozen at -80°C until assayed for TNF-a by ELISA. Differences in supernatant concentrations of TNF-a were determined using the Proc Mixed procedure of SAS. Supernatant concentrations of TNFa in tubes containing no LPS were not different (P>0.05) among triplicates and were at the detection limit of the assay. Supernatant concentrations of TNF-a were not different (P>0.05) in tubes containing 0.1 pM to 10 nM E2+LPS vs LPS. Supernatant concentrations of TNF-a were increased (P< 0.05) in tubes containing I nM to 10 μM ICI+LPS vs LPS. Supernatant concentrations of TNF-a were not different (P>0.05) in tubes containing ICI+E2+LPS vs LPS. Supernatant concentrations of TNF-a were decreased (P< 0.05) in tubes containing ICI+genistein+LPS vs LPS. These preliminary data suggest that estradiol does not inhibit LPS stimulated TNF-a in the horse. However, genistein does not appear to work through estrogen receptors to decrease LPS stimulated TNF-a production in horse. Peripheral blood mononuclear cells were collected and total RNA was isolated. F RNA was reverse transcribed into cDNA for further analysis. Primers for eTNF-a, eIL-1 p, eIL 6, eIL-10, and equine P-Actin were designed using the software supplied by Integrated DNA Technologies. Proinflammatory cytokines TNF-a, IL-IP, IL-6, and ILIO were quantified relative to the amount of P-Actin in cDNA. The proinflammatory cytokine TNF-a after 6 h LPS stimulation increased expression (P< 0.05) when compared to untreated PBMC, but treatment with genistein plus LPS decreased expression (P< 0.05) when compared to LPS treated PBMC. At 12 hours there was a decrease in TNF-a expression (P< 0.05) when PBMC treated with genistein were compared to untreated PBMC. Untreated PBMC had a decrease in TNF-a expression (P< 0.05) when PBMC incubated for 24 hours was compared to PBMC incubated for 12 hours. The LPS treatment had a decrease in TNF-a expression (P< 0.05) at 12 hand 24 h incubation times compared to 6 h incubation. No differences were detected (P>0.05) at all other time points or treatments for TNF-a. The proinflammatory cytokine, IL- Ip, increased in expression (P< 0.05) at 6 h when LPS treated PBMC were compared to PBMC. There is also an increase in IL-1 p expression (P< 0.05) when PBMC treated with genistein plus LPS were compared to untreated PBMC at 6 and 12 h. Untreated PBMC decreased IL-IP expression (P< 0.05) at 12 and 24 h when compared to 6 h, LPS treated PBMC decreased IL- Ip expression (P< 0.05) at all times, 6, 12, and 24 h, when compared to each other, and genistein plus LPS treated PBMC expression for IL-1 ~ decreased (P< 0.05) when 24 h was compared to 6 h, and when 24 h was compared to 12 h. No differences were detected (P>0.05) in all other comparisons. Expression oflL-6 increased (P< 0.05) in PBMC treated with LPS when compared to untreated PBMC at 6 and 12 h. Genistein plus LPS treated PBMC decreased IL-6 expression (P< 0.05) when compared to LPS treated PBMC at 6 h. Untreated PBMC expression oflL-6 decreased (P< 0.05) at 12 and 24 h when compared to 6 h, LPS treated PBMC decreased IL-6 expression (P< 0.05) when all time points, 6, 12, and 24 h, were compared to each other, and PBMC treated with genistein plus LPS decreased IL-6 expression (P< 0.05) at 12 and 24 h when compared to 6 h. No differences were detected (P>0.05) in all other treatments. Expression for IL-10 was increased (P< 0.05) when LPS treated PBMC were compared to untreated PBMC at 6, 12, and 24 h. At 6 and 24 h, IL-10 expression increased (P< 0.05) when cells treated with genistein plus LPS were compared to untreated PBMC. No differences were detected (P>0.05) in all other treatments. These data provide evidence that genistein does play a role in the expression of proinflammatory cytokines. However, genistein may not inhibit the production of all the major proinflammatory cytokines.

Keywords: Genistein, LPS, Cytokines, Equine

Library of Congress Subject Headings

Tumor necrosis factor

Genistein

Endotoxins

Estrogen -- Physiological effect

Cytokines

Inflammation

Format

application/pdf

Number of Pages

104

Publisher

South Dakota State University

Download

Share

COinS