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Chylomicrons can regulate hepatic gene expression1
ABSTRACTS FROM THE SOCIETY OF BLACK ACADEMIC SURGEONS Ninth Annual Meeting, March 18 –20, 1999, Louisville, Kentucky CHYLOMICRONS CAN REGULATE HEPATIC GENE EXPRESSION
PEROXYNITRITE (PN) EFFECTS ON SMALL INTESTINAL ENTEROCYTES (IEC-6)
Authors:
Authors:
ZL Kumwenda, MD WJ Welch, PhD HW Harris, MD San Francisco, CA
Purpose: Chylomicrons (CM) can bind endotoxin (LPS) and protect against lethality in rodents by increasing the clearance of this toxic macromolecule by the liver. We have previously shown that once cleared by hepatocytes, these CM-LPS complexes attenuate the hepatocellular response to cytokines by inhibiting the activation of NF-kB, a transcription factor critical for activation of numerous proinflammatory genes. We postulated that CM-LPS complexes exert a selective rather than a global inhibitory effect on hepatocellular gene expression. Methods: To study this question, cultured rat hepatocytes were pretreated with LPS, CM, or CM-LPS complexes for 2 hours. After an overnight recovery period, the pretreated cells were stimulated in various ways to determine the relative integrity of hepatocellular gene expression. Specifically, cells were stimulated either by exposure to a mixture of proinflammatory cytokines (TNF-␣, IL-1, IFN-␥) or forskolin (a stimulator of cAMP response element binding protein, CREB) or a heat shock (43°C ⫻ 30 minutes). Gene expression was examined via gel shift assay and Western blot analysis.
JS Upperman, MD P Boyle R Hoffmann, PhD S Watkins, PhD HR Ford, MD Pittsburgh, PA
Purpose: Our laboratory has shown that intestinal barrier failure following necrotizing enterocolitis or LPS peritonitis is associated with upregulation of inducible nitric oxide synthase, 3-nitrotyrosine residues (the PN footprints), and apoptosis in the ileum. The mechanism of PN-mediated gut barrier failure remains elusive. This study examines PN-mediated epithelial cell injury in vitro. Methods: We incubated IEC-6 cells with 12.5 M PN or phosphatebuffered saline (PBS) or media for 60 minutes followed by a recovery period of 6, 12, 18, or 24 hours in culture media. Apoptosis was measured by fluorescentactivated cell sorting using the propidium iodide method and electron microscopy. We also tested the ability of pretreatment with 3-aminobenzamide (3AB) (1 mM),
Results: The CM-LPS complexes attenuated activation of NF-kB (Fig. 1), yet had no effect on the activation of CREB (Fig. 2) or the heat shock response (Fig. 3). Summary: Chylomicron-LPS complexes exert a selective antiinflammatory effect on hepatocytes and may yield a new strategy for treating gram negative sepsis.
FIGURE 2. CREB.
FIGURE 1. NF-kB.
The Society of Black Academic Surgeons expresses its appreciation to Dr. Walter Pories and the editorial staff of Current Surgery for their interest in the Society and the publication of these abstracts.
382
CURRENT SURGERY
•
FIGURE 3. hsp 72 Western.
© 2000 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/99/$20.00 PII S0149-7944(99)00113-0
PEROXYNITRITE (PN) EFFECTS ON SMALL INTESTINAL ENTEROCYTES (IEC-6)
Authors:
Authors:
ZL Kumwenda, MD WJ Welch, PhD HW Harris, MD San Francisco, CA
Purpose: Chylomicrons (CM) can bind endotoxin (LPS) and protect against lethality in rodents by increasing the clearance of this toxic macromolecule by the liver. We have previously shown that once cleared by hepatocytes, these CM-LPS complexes attenuate the hepatocellular response to cytokines by inhibiting the activation of NF-kB, a transcription factor critical for activation of numerous proinflammatory genes. We postulated that CM-LPS complexes exert a selective rather than a global inhibitory effect on hepatocellular gene expression. Methods: To study this question, cultured rat hepatocytes were pretreated with LPS, CM, or CM-LPS complexes for 2 hours. After an overnight recovery period, the pretreated cells were stimulated in various ways to determine the relative integrity of hepatocellular gene expression. Specifically, cells were stimulated either by exposure to a mixture of proinflammatory cytokines (TNF-␣, IL-1, IFN-␥) or forskolin (a stimulator of cAMP response element binding protein, CREB) or a heat shock (43°C ⫻ 30 minutes). Gene expression was examined via gel shift assay and Western blot analysis.
JS Upperman, MD P Boyle R Hoffmann, PhD S Watkins, PhD HR Ford, MD Pittsburgh, PA
Purpose: Our laboratory has shown that intestinal barrier failure following necrotizing enterocolitis or LPS peritonitis is associated with upregulation of inducible nitric oxide synthase, 3-nitrotyrosine residues (the PN footprints), and apoptosis in the ileum. The mechanism of PN-mediated gut barrier failure remains elusive. This study examines PN-mediated epithelial cell injury in vitro. Methods: We incubated IEC-6 cells with 12.5 M PN or phosphatebuffered saline (PBS) or media for 60 minutes followed by a recovery period of 6, 12, 18, or 24 hours in culture media. Apoptosis was measured by fluorescentactivated cell sorting using the propidium iodide method and electron microscopy. We also tested the ability of pretreatment with 3-aminobenzamide (3AB) (1 mM),
Results: The CM-LPS complexes attenuated activation of NF-kB (Fig. 1), yet had no effect on the activation of CREB (Fig. 2) or the heat shock response (Fig. 3). Summary: Chylomicron-LPS complexes exert a selective antiinflammatory effect on hepatocytes and may yield a new strategy for treating gram negative sepsis.
FIGURE 2. CREB.
FIGURE 1. NF-kB.
The Society of Black Academic Surgeons expresses its appreciation to Dr. Walter Pories and the editorial staff of Current Surgery for their interest in the Society and the publication of these abstracts.
382
CURRENT SURGERY
•
FIGURE 3. hsp 72 Western.
© 2000 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/99/$20.00 PII S0149-7944(99)00113-0