Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease - PubMed (original) (raw)

Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease

Norberto C Chavez-Tapia et al. BMC Gastroenterol. 2012.

Abstract

Background: In vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH.

Methods: HuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured.

Results: FFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species.

Conclusions: The exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.

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Figures

Figure 1

Figure 1

Dose dependent intracellular fat accumulation. Cell exposure to 600 and 1200 μM FFA for 24 h. Dose dependent intracellular fat accumulation evidenced by Nile Red staining assessed by fluorescence microscopy (A) and flow cytometry measured in 1 × 104 cells (B), M1 represents the percentage of events above the maximum value of fluorescence and M2 represents the percentage of events above the median value of fluorescence.

Figure 2

Figure 2

Inflammatory cytokines mRNA expression. Cells were cultured and treated with 600 and 1200 μM FFA for 24 h. mRNA expression of IL-6, IL-8 and TNF-alpha in HuH7 cells vs. control. * P < 0.05 versus control (0 μM).

Figure 3

Figure 3

Inflammatory cytokines protein production. Inflammatory cytokines present in the supernatant of HuH7 cells after 24 h of the treatment was significantly increased only for IL-8 at both experimental doses, TNF-alpha levels were unchanged, and the levels of IL-6 were lower than the assay sensitivity. * P < 0.05 versus control (0 μM).

Figure 4

Figure 4

Fibrogenic cytokines mRNA expression. Fibrogenic cytokines mRNA expression in HuH7 cells after 24 h exposure at 600 and 1200 μM FFA. * P < 0.05 versus control (0 μM).

Figure 5

Figure 5

Apoptosis induction secondary to fatty acids toxicity. FFA treatment increase apoptosis in hepatic cells (HuH7). There is a reduction in the live cells, and in consequence an increase number of cells in early-, late-apoptosis and necrosis with the FFA treatment. The results are presented as percentage compared to control and represent the mean ± SD, of at least three independent experiments (A). Representative dotplot graph of control and 1200 μM FFA treated cells stained with Annexin V and PI (B).* P < 0.05 versus control (0 μM).

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