Article Figures & Data
Figures
- Fig. 1.
Arachidonic acid metabolism-related gene expression levels in the liver from mice fed an HFD. Scheme of the main arachidonic acid bioactive products (A). Heat map generated from NGS data of mRNA profiling of genes involved in arachidonic acid metabolism. Blue and red colors indicate downregulation and upregulation in the HFD group, respectively (B). Spearman correlation matrices within the HFD group, between hepatic mRNA expression levels of EET-related enzymes and those of canonical genes involved in inflammation and fibrogenesis (C). CYP2C8 mRNA expression level in liver biopsies from NAFLD and non-NAFLD patients. n = 5/group. Data are means ±S.D., Student’s t test, *<0.05 (D). CYP2C8 mRNA expression in human liver biopsies and NAFLD score correlation, as determined by histology. Data were normalized for the lowest CYP2C8 mRNA expression value (shown in red) (E).
- Fig. 2.
Inhibitory effect of OCA on HFD-induced NASH. Serum ALT (A), serum hydroxyproline (B), hepatic NAFLD score (C). Hepatic mRNA levels of mCol1a1 (D), mCcl2 (E), mIcam (F), mTnfa (G), mIl1b (H) and mIl6 (I). n ≥ 6 mice/group. Data are means ± S.D., one-way ANOVA < 0.05, Tukey’s test, *<0.05. Representative images of immunostaining for the macrophage marker MAC387 (J) and CD4 (K) in liver sections from chow (a), HFD (b) and HFD+OCA (c) groups.
- Fig. 3.
Regulatory effect of OCA on arachidonic acid metabolism. Heat map of mRNA profiling of selected genes involved in arachidonic acid metabolism. The relative expression values of each target gene were measured in the chow, HFD, and HFD + OCA mice, normalized for the expression of β-actin and then expressed as HFD:chow (HFD) or HFD + OCA:chow (HFD + OCA) ratio. Each column represents an individual sample. Blue and red colors indicate downregulation and upregulation, respectively (A). Relative hepatic mRNA levels of arachidonate partitioning genes (B–E). Serum levels of LTB4 (F) and 14,15-EET (G). Ratio between serum LTB4 and serum 14,15-EET (H). Urinary levels of 14,15-DHET (I). Data are means ± S.D., one-way ANOVA < 0.05, Tukey’s test, *<0.05. n ≥ 6 mice/group.
- Fig. 4.
Effect of FXR activation on arachidonate metabolism and FFA-induced monocyte migration. Representative images showing crystal violet staining of THP-1 cells onto a 3-µm pore polycarbonate membrane insert upon exposure to the medium of Huh7 cells treated with 50 μM FFA in the presence or absence of 2 μM OCA (a-d) (A). Relative migration score (B). Levels of LTB4 (C), 14,15-EET (D), LTB4/14,15-EET ratio (E), and 14,15-DHET (F) in the medium of Huh7 cells exposed to 50 μM FFA in the presence or absence of 2 μM OCA. mRNA levels of CYP2C8 in Huh7 cells with the different treatments (G). Data are means ± S.D., one-way ANOVA < 0.05, Tukey’s test, *<0.05. n = 4/group.
- Fig. 5.
Effect of NF-kB and CYP450 epoxygenase modulation on FFA-induced migration. Migration score of THP-1 cells in the medium of Huh7 cells treated with 50 μM FFA in combination with 10 μM NF-kB inhibitor benzoxathiole derivative (BOT) (A) or 20 μM rifampicin (B). The effect of 50 μM FFA on the migration of THP-1 cells in the medium of Huh7 cells transiently overexpressing Cyp2c29 (C). mRNA expression levels of NF-kB target genes in Huh7 cells exposed to 50 μM FFA and 20 μM rifampicin (D and E). The mRNA expression levels of NF-kB target genes in Huh7 cells transiently overexpressing cyp2c29 and exposed to 50 μM FFA (F and G). Data represent the mean ± S.D., one-way ANOVA < 0.05, Tukey’s test, *<0.05. n = 3/group.
- Fig. 6.
Effect of gemfibrozil on OCA-mediated anti-inflammatory action. Representative images showing crystal-violet staining of THP-1 cells onto a 3-µm pore polycarbonate membrane insert upon exposure to the medium of Huh7 cells treated with 50 μM FFA in the presence or absence of 2 μM of OCA and 100 μM of GM (a-f) (A). Relative migration score (B). Levels of LTB4 (C), 14,15-EET (D) in the medium of Huh7 cells exposed to 50 μM FFA in the presence or absence of 2 μM OCA and 100 μM GM). *<0.05.
- Fig. 7.
Model of the FXR-mediated repression of NF-κB signaling. Increased LTB4 levels and decreased and EET levels promote NF-κB signaling, which triggers hepatic inflammation (A). Transactivation of P450 epoxygenase expression and EET synthesis by FXR, which, in turn, inhibits the NF-κB signaling (B).
Tables
- Table 1
Selected differentially expressed genes in liver from chow mice and high-fat diet (HFD) mice
Numbers in parentheses indicate gene expression levels quantified as log2 of fold changes.
Pathways Differentially Regulated in HFD Liver Lipid metabolism Gpat2 (0.7), Abhd4 (0.6), Acacb (1.2), Acsl5 (0.6), Acsm3 (1.0), Bche (0.7), Echs1 (0.5), Hadh (0.9), Acaca (0.7) TGFb-induced EMT TGFb2 (0.8), Jun (1.7), Fos (1.48), Fosl1 (0.8), Mmp2 (1.0), Edn1 (1.4), Vim (0.8), Ocln (−0.8) Fatty acid metabolism Cd74 (1.5), Elovl5 (1.6), Aacs (2.0), Acaa1b (1.3), Acacb (1.2), Acsf3 (0.7), Acsl5 (0.6), Acsm3 (1.0), Ch25h (1.3), Elovl2 (0.9), Echs1 (0.5), Fads1 (0.8), Fads2 (1.3), Fasn (1.5), Gpam (0.9), Hao2 (3.0), Hadh (0.9), Hsd17b4 (0.5), Myo5a (1.0), Elovl6 (0.9), Acaca (0.7), Scd1 (1.6), Scd2 (1.0), Scd3 (1.5) Arachidonic acid metabolism Cbr3 (1.6), Pla2g6 (0.9), Cyp2c29 (−1.2), Cyp2c37 (−0.8), Cyp2c39 (−0.7), Cyp2c44 (−1.0), Cyp2c50 (−0.7), Cyp2c54 (−1.2), Cyp2c55 (−1.3), Cyp2c70 (−1.6), Cyp4f14 (−0.7)
Data Supplement
- Supplemental Figures -
Supplemental Figure 1 - NASH assessment in mice fed a HFD
Supplemental Figure 2 - Arachidonic acid metabolism in mice fed a HFD
Supplemental Figure 3 - Effect of OCA on HFD-induced NASH
Supplemental Figure 4 - Effect of OCA on the expression of FXR target genes in Huh7 cells
Supplemental Figure 5 - Impact of FXR activation on FFA-induced monocyte migration and EET metabolism-related gene expression levels in primary cultured hepatocytes from mouse (A and B) and human (C and D)
Supplemental Figure 6 - Effect of the co-incubation with OCA and the FXR antagonist z-Guggulsterone (Gu) on FXR activation and FFA-induced monocyte migration
Supplemental Figure 7 - Effect of OCA, Gemfibrozil (GM) or Rifampicin (Rifa) on THP-1 cell migration induced by serum or exogenous LTB4
- Supplemental Figures -
