Anti-inflammatory and anti-fibrotic profile of fish oil emulsions used in parenteral nutrition-associated liver disease

Abstract

Home parenteral nutrition (PN) is associated with many complications including severe hepatobiliary dysfunction. Commercial ω-6 fatty acid-soybean based-lipid emulsions in PN may mediate long term PN associate liver disease (PNALD) whereas ω-3-fish oil parenteral emulsions have shown to reverse PNALD in children. However, its clinical effectiveness in adults has been scarcely reported. In this work, we study the role of soybean and fish oil lipid commercial emulsions on inflammatory and profibrotic liver markers in adults with long term PNALD and in in vitro cellular models. Inflammatory and profibrotic markers were measured in serum of ten adults with long term PNALD and in culture supernatants of monocytes. Liver epithelial to mesenchymal transition (EMT) was induced by transforming growth factor beta 1 (TGFβ1) to evaluate in vitro liver fibrosis. Omegaven®, a 100% fish oil commercial emulsion, was infused during four months in two patients with severe long term PNALD reversing, at the first month, the inflammatory, profibrotic and clinical parameters of PNALD. The effect was maintained during the treatment course but impaired when conventional lipid emulsions were reintroduced. The other patients under chronic soybean oil-based PN showed elevated inflammatory and profibrotic parameters. In vitro human monocytes stimulated with lipopolysaccharide induced a strong inflammatory response that was suppressed by Omegaven®, but increased by soybean emulsions. In other experiments, TGFβ1 induced EMT that was suppressed by Omegaven® and enhanced by soybean oil lipid emulsions. Omegaven® improves clinical, anti-inflammatory and anti-fibrotic parameters in adults with long-term home PNALD.

Figure 1. Histological liver representation of parenteral nutrition-associated liver disease patients.

Hematoxilin & eosin and masson's trichrome staining of liver biopsies taken for diagnosis purposes. The images are representative of patients with non-alcoholic liver hepatic disease score (NAS) ≤6 and patients with NAS>6 who initiated Omegaven® treatment.

Figure 2. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced TGFβ1 and MMP-9 secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) TGFβ1 and (B) MMP-9 were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 3. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced IL-6 and TNFα secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) IL-6 and (B) TNFα were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 4. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced IL-8 and IL-1β secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) IL-8 and (B) IL-1β were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. One-way ANOVA was followed by the post hoc Bonferroni test. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 5. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced GM-CSF and IL-5 secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) GM-CSF and (B) IL-5 were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. One-way ANOVA was followed by the post hoc Bonferroni test. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 6. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced IL-4 and IL-10 secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) IL-4 and (B) IL-10 were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. One-way ANOVA was followed by the post hoc Bonferroni test. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 7. Effect of different commercial parenteral lipid emulsions on Lipopolysaccharide (LPS)-induced IL-2 and IL-12 secretion in human monocytes.

Human monocytes were isolated from healthy subjects and incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by LPS 1 µg/mL stimulation for additional 24 hours. (A) IL-2 and (B) IL-12 were measured in cell culture supernatants. The effect of lipid emulsions without stimulus was tested at 1/10 dilution. Results are expressed as means ± SEM of six independent experiments. One-way ANOVA was followed by the post hoc Bonferroni test. *p<0.05 related to the control group. #p<0.05 values below stimulus; ⊥p<0.05 values above the stimulus.

Figure 8. Effect of different lipid emulsions on the expression of myofibroblast markers.

Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at 1/100 dilutions for 72 hours. Representative visible morphology and immunofluorescences for alpha smooth muscle actin (αSMA) and collagen type I (col type I) are showed.

Figure 9. Omegaven® inhibits myofibroblast markers induced by TGFβ1.

Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours. (A) Visible morphology and immunofluorescence for alpha smooth muscle actin (αSMA) and collagen type I (col type I) distribution and expression. B) Expression of mRNA of αSMA and col type I. Scale bar: 10 µm. Results are expressed as means ± SEM of six independent experiments. *p<0.05 related to the control group. #p<0.05 related to the stimulus.

Figure 10. Omegaven® inhibits epithelial to mesenchymal transition induced by TGFβ1.

Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours (A and B) or 25 min (C). (A) Expression of mRNA of ZO-1 and (B) E-cadherin. (C) Phosphorylation of Samd3, ERK1/2 and Akt and nuclear expression of β-catenin. Representative western blot are showed and quantified in graphic bars. Results are expressed as means ± SEM of six independent experiments. *p<0.05 related to the control group. #p<0.05 related to the stimulus.

Figure 11. Omegaven® inhibits transcription factor an markers related with epithelial to mesenchymal transition induced by TGFβ1.

Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours. Expression of mRNA of Snail, Slug, and vimentin. Results are expressed as means ± SEM of six independent experiments. One-way ANOVA was followed by the post hoc Bonferroni test. *p<0.05 related to the control group. #p<0.05 related to the stimulus.