Pharmaco-nutraceutical improvement of the response to obeticholic acid with omega-3 polyunsaturated fatty acids

Abstract

Obeticholic acid (OCA) is the second-line therapy for primary biliary cholangitis. While efficient in promoting bile acid (BA) detoxification and limiting liver fibrosis, its clinical use is restricted by severe dose-dependent side effects. We tested the hypothesis that adding n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids to OCA may improve the therapeutic effect of the low drug dosage. Several liver cell lines were exposed to vehicle, low or high OCA dose (1-20 μM) in the presence or absence of EPA/DHA for 24 h. To induce ER stress, apoptosis, and fibrosis, HepG2 cells were exposed to a 400 μM BA mixture or to 2 ng/ml transforming growth factor-β (TGF-β). For inflammation analyses, THP-1 cells were activated with 100 ng/ml lipopolysaccharides (LPS). The impact of OCA+EPA/DHA was assessed using transcriptomic (qRT-PCR), proteomic (ELISA, caspase-3), and metabolomic (LC-MS/MS) approaches. The addition of EPA/DHA reinforced the ability of low OCA dose to down-regulate the expression of genes involved in BA synthesis (CYP7A1 and CYP8B1) and uptake (NTCP) and to up-regulate the expression of MRP2 and 3 genes. EPA/DHA also enhanced the anti-inflammatory response of the drug by reducing the expression of the LPS-induced cytokines: tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-1β, and monocyte chemoattractant protein-1 in THP-1 macrophages. OCA+EPA/DHA decreased the expression of BIP, CHOP, and COL1A1 genes and the caspase-3 activity. EPA+DHA potentiate the response to low OCA doses on BA toxicity and provide additional benefits on ER stress, apoptosis, inflammation, and fibrosis. These observations support the idea that adding n-3 PUFAs to the drug may reduce the risk of dose-related side effects in patients treated with OCA.

Keywords: autoimmune liver diseases; bile acid detoxification; drug response improvement; inflammation and fibrosis resolution; n-3 polyunsaturated fatty acids.

Figure 1. N-3 PUFAs potentiate the effects of low OCA concentrations on the expression of genes governing bile acid synthesis and transport in hepatoma HepG2 cells.

Human hepatoma HepG2 cells were treated with vehicle (DMSO and ethanol), OCA (1 or 20 μM) in the absence or presence of EPA/DHA (40/40 μM) for 24 h. Total RNA was extracted. CYP7A1 (A), CYP27A1 (B), CYP8B1 (C), NTCP (D), MRP2 (E), MRP3 (F), and MRP4 (G) transcript levels were quantified by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells set at 1. Statistical significances as determined by a one-way ANOVA followed by Tukey’s multiple comparison post-hoc were as follows: vehicle vs. OCA-treated cells: *:P<0.05; **:P<0.01; ****:P<0.0001. OCA vs. OCA+EPA/DHA: §:P<0.05; §§:P<0.01; §§§:P<0.001; §§§§:P<0.0001. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MRP2, multidrug-resistance protein 2; NTCP, Na⁺-taurocholate cotransporting peptide; OCA, obeticholic acid.

Figure 2. EPA and DHA modulate the ability of OCA to regulate the transcription of genes involved in bile acid homeostasis in differentiated human HepaRG hepatocytes.

Differentiated human HepaRG hepatocytes were cultured in the presence of vehicle (DMSO and ethanol), EPA/DHA (50/50 µM), OCA (1 or 20 µM) or a combination of EPA/DHA (50/50 µM) and OCA (1 or 20 µM) for 24 h. Total RNA was extracted. CYP7A1 (A), CYP27A1 (B), CYP8B1 (C), NTCP (D), BSEP (E), MRP2 (F), MRP3 (G), and MRP4 (H) transcript levels were quantified by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells set at 1. Statistical significances as determined by a one-way ANOVA followed by Tukey’s multiple comparison post-hoc were as follows: vehicle vs. OCA-treated cells: *:P<0.05; **:P<0.01, ***:P<0.001, ****:P<0.0001. OCA vs. OCA+EPA/DHA: §:P<0.05. BSEP, bile salt export pump; DHA, docosahexaenoic acid ; EPA, eicosapentaenoic acid; MRP2, multidrug-resistance protein 2; NTCP, Na⁺-taurocholate cotransporting peptide; OCA, obeticholic acid.

Figure 3. The eicosapentaenoic and docosahexaenoic acids affect the bile acid-related transcriptomic signature of OCA in primary human hepatocytes in a gene-dependent manner.

Human hepatocytes in primary culture were treated with vehicle (DMSO), EPA/DHA (25/25 µM), OCA (1 or 20 µM) or a combination of EPA/DHA (25/25 µM) and OCA (1 or 20 µM) for 24 h. Total RNA was extracted. CYP7A1 (A), CYP27A1 (B), CYP8B1 (C), NTCP (D), BSEP (E), MRP2 (F), MRP3 (G), and MRP4 (H) transcript levels were quantified by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells set at 1. Statistical significances were as follows: vehicle vs. OCA-treated cells: *:P<0.05; **:P<0.01; ***:P<0.001; ****:P<0.0001. OCA vs. OCA+EPA/DHA: §:P<0.05. BSEP, bile salt export pump; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MRP2, multidrug-resistance protein 2; NTCP, Na⁺-taurocholate cotransporting peptide; OCA, obeticholic acid.

Figure 4. N-3 PUFAs modulate the bile acid-related transcriptomic signature of the obeticholic acid in murine hepatocytes in primary culture.

Hepatocytes were purified as described in the Materials and Methods section and subsequently cultured in the presence of vehicle (DMSO and Ethanol), OCA (1 or 20 μM) in the absence or presence of EPA/DHA (50/50 μM) for 6 h. Total RNA was extracted. Cyp7a1 (A), Cyp27a1 (B), Cyp2c70 (C), Cyp8b1 (D), Ntcp (E), Bsep (F), Mrp2 (G), Mrp3 (H), and Mrp4 (I) transcript levels were quantified by qRT-PCR. MRNA levels were expressed relatively to control cells set at 1. Statistical significances were as follows: vehicle vs. OCA-treated cells: *:P<0.05; **:P<0.01; ***:P<0.001; ****:P<0.0001. OCA vs. OCA+EPA/DHA: §:P<0.05; §§:P<0.01; §§§:P<0.001. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MRP2, multidrug-resistance protein 2; NTCP, Na⁺-taurocholate cotransporting peptide; OCA, obeticholic acid.

Figure 5. N-3 PUFAs reinforce the ability of OCA to limit bile acid secretion by HepG2 cells.

Human hepatoma HepG2 cells were treated with vehicle (DMSO and Ethanol), OCA (1 μM) in the absence or presence of EPA/DHA (50/50 μM) for 36 h. Cell media were collected and profiled for the presence of 20 bile acid species using LC-MS/MS as detailed in the Materials and Methods section. Statistical significances as determined by a one-way ANOVA were as follows: vehicle vs. OCA-treated cells: *:P<0.05; **:P<0.01; ***:P<0.001. OCA vs. OCA+EPA/DHA: §§:P<0.01; §§§:P<0.001. CDCA: chenodeoxycholic acid; GCDCA: glycochenodeoxycholic acid.

Figure 6. Eicosapentaenoic and docosahexaenoic acids protect liver cells against the bile acid-induced apoptosis (A) and ER stress (B and C), as well as against TGF-β-induced fibrosis (D) in the OCA-treated HepG2 (B)

(A) Cells were treated with vehicle (DMSO), EPA/DHA, OCA or a combination of OCA+EPA/DHA at the indicated doses for 24 h and subsequently exposed for 2 h to the vehicle (ethanol) or a toxic BA mixture (CA, CDCA, LCA, and DCA, 100 µM each). The caspase-3 activity was then quantified as detailed in the Materials and Methods section and expressed relatively to control cells set at 1. (B and C) Cells were exposed to vehicle or the same toxic BA mixture as above for 24 h in the absence or presence of EPA/DHA, OCA (1 or 20 µM) or the OCA+EPA/DHA combinations at the indicated doses. Total RNA was extracted and analyzed for transcript levels of the ER-stress BIP (B) and CHOP (C) markers by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells set at 1. (D) Cells were treated with vehicle, EPA/DHA, OCA, or a combination of OCA+EPA/DHA at the indicated doses in the presence of TGF-β for 24 h. Total RNA was extracted and analyzed for transcript levels of the fibrosis marker COL1A1 by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells set at 1. Statistical significances as determined by a one-way ANOVA followed by Tukey’s multiple comparison post-hoc were as follows: vehicle vs. untreated cells exposed to bile acids or TGF-β: ††:P<0.01; †††:P<0.001; ††††:P<0.0001. Vehicle cells vs. OCA-treated cells: ‡‡:P<0.01; ‡‡‡:P<0.001; ‡‡‡‡:P<0.0001. BA or TGF-β-exposed cells vs. BA or TGF-β+OCA±EPA/DHA-exposed cells: ***:P<0.001; ****:P<0.0001. OCA-treated cells vs. OCA+EPA/DHA-treated cells: §§:P<0.01; §§§:P<0.001; §§§§:P<0.0001.

Figure 7. Addition of n-3 PUFAs to OCA leads to anti-inflammatory events in macrophages

THP-1 monocytes were differentiated into macrophages with PMA for 72 h and then stimulated with 100 ng/μl LPS for 24 h in the presence or absence of EPA/DHA, OCA, or their combination as described in the Materials and Methods section. Total RNA was extracted. TNFα (A), IL-6 (B), IL-1β (C), and MCP-1 (D) transcript levels were quantified by qRT-PCR as detailed in the Materials and Methods section, and mRNA levels were expressed relatively to control cells (i.e. without LPS challenge) set at 1. (E) The level of IL-6 in culture media was measured by ELISA as described in the materials and methods section and is expressed relatively to control cells set at 1. Data represent the mean of three independent experiments in which each treatment was performed in triplicate. Each data point therefore corresponds to the mean of nine replicates ± SD. Statistical significances as determined by a one-way ANOVA followed by Tukey’s multiple comparison post-hoc were as follows: untreated differentiated THP-1 exposed to vehicle vs. untreated differentiated THP-1 exposed to LPS: ††:P<0.01; †††:P<0.001; ††††:P<0.0001; vehicle differentiated THP-1 vs. OCA-treated differentiated THP-1: ‡‡:P<0.01; ‡‡‡:P<0.001; ‡‡‡‡:P<0.0001; LPS-exposed differentiated THP-1 vs. LPS+OCA±EPA/DHA-exposed differentiated THP-1: *:P<0.05; **:P<0.01; ***:P<0.001; ****:P<0.0001; OCA-treated differentiated THP-1 vs. OCA+EPA/DHA-treated differentiated THP-1: §:P<0.0001; §§:P<0.0001; §§§§:P<0.0001.

Figure 8. OCA and n-3 PUFAs activate different nuclear and/or membrane receptors to additively reduce the successive events involved in the pathogenesis of inflammatory and autoimmune hepatobiliary diseases.

As illustrated by blue arrows, OCA controls the intracellular levels of pro-inflammatory, pro-apoptotic, and pro-fibrotic bile acids in hepatocytes through the selective activation of FXR. EPA and DHA modulate bile acid synthesis and metabolism, ER-stress, apoptosis, inflammation, and fibrosis in a direct manner through the activation of PPAR and GPCR receptors or after conversion into active SPM derivatives that also activate GPCRs. Activation of these complementary transcriptional pathways may be responsible for the additive effects observed during the present investigations. SPMs, specialized pro-resolving mediators.