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

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 1alpha(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.

Fig. 1.

Smooth muscle actin (SMA) accumulation in rat precision-cut liver slices (PCLS) treated with 25 mM ethanol and/or 4-methylpyrazole (4-MP). Rat PCLS from all treatment groups and times were lysed with RIPA as described in materials and methods. Lysates were analyzed by SDS-PAGE and immunoblotting as described. Accumulation of SMA at each time point was normalized to time 0 (t₀). Tubulin was used as a loading control. Representative bands are shown below the graph. C, control; E, ethanol; EP, ethanol + 4-MP; numbers represent hours of treatment. #P < 0.05 compared with t₀; *P < 0.05 compared with corresponding controls; **P < 0.001 compared with corresponding control; n = 6 different rat experiments.

Fig. 2.

Collagen accumulation in PCLS treated with 25 mM ethanol and/or 4-MP up to 96 h. Rat PCLS were treated as described in materials and methods. Every 24 h, slices were lysed with RIPA as described and subjected to SDS-PAGE and immunoblotting. Collagen accumulation for each time point was normalized to t₀. Tubulin was used as a loading control. Representative bands corresponding to collagen and tubulin for each time point and condition are shown below the graph. *P < 0.05; n = 5 different rat experiments.

Fig. 3.

A: Sirius red stain of PCLS. PCLS were incubated in the absence (C) or presence of 25 mM ethanol (E) or ethanol + 0.5 mM 4-MP (EP) up to 72 h. At each point, PLCS were rinsed, fixed with buffered formalin, and embedded in paraffin. Sections (4 μm thick) were stained with Sirius red. Slides were analyzed with a Nikon Eclipse 80i at 10× power and Nis-Elements 3.0 software (Nikon, Melville, NY). Photographs are representative of 4 separate animal experiments. B: quantification of Sirius red staining of PCLS. PCLS were incubated in the absence or presence of 25 mM ethanol or ethanol + 0.5 mM 4-MP up to 72 h. Samples were stained for Sirius red and photographed. Pictures were deconvoluted, subjected to threshold analysis, and quantified by use of MBF-ImageJ software. PCLS slides from 4 separate animals using 10 random fields per slide were analyzed. *P < 0.01 compared with control or ethanol + 4-MP.

Fig. 4.

Total cellular GSH levels. Rat PCLS were treated with ethanol in the absence or presence of 4-MP as described in materials and methods. Every 24 h, slices were analyzed for total cellular GSH by using a kit according to manufacturer's instructions. #P < 0.001 compared with corresponding control; *P < 0.05 compared with corresponding control; n = 5 different rat experiments.

Fig. 5.

Thiobarbituric acid-reactive substance (TBARS) analysis of rat PCLS after ethanol treatment. Rat PCLS were treated with ethanol in the absence or presence of 4-MP as described in materials and methods. Every 24 h, slices were analyzed for TBARS by using a kit according to manufacturer's instructions. **P < 0.05 compared with corresponding controls; *P < 0.005 compared with corresponding control; #P < 0.05 compared with t₀; n = 6 different rat experiments.

Fig. 6.

IL-6 secretion in media (A) and mRNA levels (B) from rat PCLS treated with 25 mM ethanol and/or 4-MP. A: media from all treatment groups and times were analyzed by using an ELISA to IL-6 as described in materials and methods. B: slices from all treatment groups and times were used for RT-PCR by using primers to IL-6 as described in materials and methods. #P < 0.05 compared with corresponding controls; *P < 0.005 compared with corresponding controls; **P < 0.0005 compared with corresponding control; n = 6 different rat experiments.