Exposure of precision-cut rat liver slices to ethanol accelerates fibrogenesis

Courtney S. Schaffert, Michael J. Duryee, Robert G Bennett, Amy L. DeVeney, Dean J. Tuma, Peter Olinga, Karen C. Easterling, Geoffrey Milton Thiele, Lynell Warren Klassen

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1α(I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume299
Issue number3
DOIs
StatePublished - Sep 1 2010

Fingerprint

Ethanol
Liver
Myofibroblasts
Lipid Peroxidation
Oxidative Stress
Smooth Muscle
Actins
Interleukin-6
Collagen
Cytokines
Thiobarbituric Acid Reactive Substances
Therapeutics
Immunoblotting
Glutathione
Wistar Rats
Enzyme-Linked Immunosorbent Assay
Immunohistochemistry
Polymerase Chain Reaction
Wounds and Injuries

Keywords

  • Collagen
  • IL-6
  • Oxidative stress
  • Smooth muscle actin

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

Cite this

Exposure of precision-cut rat liver slices to ethanol accelerates fibrogenesis. / Schaffert, Courtney S.; Duryee, Michael J.; Bennett, Robert G; DeVeney, Amy L.; Tuma, Dean J.; Olinga, Peter; Easterling, Karen C.; Thiele, Geoffrey Milton; Klassen, Lynell Warren.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 299, No. 3, 01.09.2010.

Research output: Contribution to journalArticle

Schaffert, Courtney S. ; Duryee, Michael J. ; Bennett, Robert G ; DeVeney, Amy L. ; Tuma, Dean J. ; Olinga, Peter ; Easterling, Karen C. ; Thiele, Geoffrey Milton ; Klassen, Lynell Warren. / Exposure of precision-cut rat liver slices to ethanol accelerates fibrogenesis. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 2010 ; Vol. 299, No. 3.
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AB - Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1α(I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis.

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