Lauric acid attenuates 14-day DDC-induced cholangiopathy in association with reduced NF-κB-CCL2 signaling and restored LRH-1-regulated gene expression

Abstract

BACKGROUND

Chronic inflammatory cholangiopathies involve bile duct injury, biochemical cholestasis, hepatocellular injury, immune cell recruitment, oxidative stress, and fibrogenic remodeling. We previously demonstrated that 20% dietary lauric acid (LA) protected against early 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholangiopathy (proof of concept) by activating liver receptor homolog-1 (LRH-1).

AIM

To determine whether concurrent low-dose 5% dietary LA supplementation attenuates 14-day DDC-induced cholangiopathy and to characterize the associated inflammatory and metabolic responses.

METHODS

Eight-week-old male C57BL/6J mice were fed for 14 days with either Chow, 0.1% DDC, 5% LA, or a 0.1% DDC diet supplemented with 5% LA.

RESULTS

DDC feeding induced severe hepatocellular and cholestatic liver injury, macrophage infiltration, ductular reaction, oxidative stress, hepatocyte senescence, and fibrogenic remodeling. LA supplementation significantly reduced DDC-induced elevations of serum aminotransferases, bilirubin, and total bile acids, and decreased hepatic F4/80-positive macrophage infiltration and Picrosirius red collagen staining. LA supplementation restored hepatic expression of LRH-1-dependent genes involved in bile acid homeostasis, which DDC suppressed. Furthermore, DDC suppressed ileal farnesoid X receptor-fibroblast growth factor 15 signaling, which LA normalized, and LA attenuated DDC-induced hepatic oxidative and nitrosative stress and improved the expression of mitochondrial fatty acid metabolism genes. LA was associated with suppressed hepatic fibrosis markers and promoted anti-inflammatory macrophage genetic signatures. Notably, DDC activated nuclear factor-kappa B binding to the C-C motif chemokine ligand 2 promoter and repressed LRH-1 transcription, which were reversed by LA supplementation.

CONCLUSION

Dietary LA attenuated chronic DDC cholangiopathy which was associated with reduced nuclear factor-kappa B-dependent inflammatory signaling and restoring LRH-1-mediated bile acid homeostasis. This positions LA as a potential therapeutic strategy for inflammatory cholangiopathy.

Key Words: Lauric acid; Liver receptor homolog-1; Cholestasis; Hepatocyte; Macrophage; Nuclear factor-kappa B; 3,5-diethoxycarbonyl-1,4-dihydrocollidine

Core Tip: Lauric acid (LA), a dietary medium-chain fatty acid, was studied in a 14-day 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced mouse model. Concurrent 5% LA supplementation attenuated biochemical cholestasis, hepatocellular injury, ductular reaction, collagen deposition, oxidative stress, senescence, and cell-death markers. These effects were associated with reduced nuclear factor-kappa B - C-C motif chemokine ligand 2 signaling, restored liver receptor homolog-1-regulated gene expression, and increased hepatic dilauroylphosphatidylcholine abundance. This study demonstrates a decrease in the development of liver injury but was not designed to demonstrate reversal of established liver injury. Further investigation will be needed to determine if liver receptor homolog-1 or dilauroylphosphatidylcholine is required for LA protection in this model.

INTRODUCTION

Inflammatory cholangiopathies, including primary sclerosing cholangitis, primary biliary cholangitis, and biliary atresia, are characterized by progressive bile duct injury, cholestasis, inflammation, and fibrosis that ultimately lead to cirrhosis and liver failure[1-4]. Despite advances in understanding disease pathogenesis, effective medical therapies that end disease progression remain limited. Cholangiopathy progression is driven by complex interactions among cholangiocytes, hepatocytes, and hepatic immune cells, particularly macrophages, resulting in persistent inflammatory signaling, oxidative stress, bile acid dysregulation, and tissue remodeling[5]. Accumulation of toxic bile acids is a major contributor to hepatocellular cholestatic liver injury[6]. Excessive bile acid retention promotes hepatocyte oxidative stress, mitochondrial dysfunction, inflammatory cytokine production, and cellular senescence[7-9]. In parallel, injured cholangiocytes secrete chemokines and cytokines that recruit inflammatory monocytes and activate hepatic macrophages, amplifying liver injury and fibrogenic responses. Among these mediators, C-C motif chemokine ligand 2 (CCL2) has emerged as a critical regulator of monocyte recruitment and inflammatory macrophage activation in cholestatic liver diseases. However, the upstream mechanisms that coordinate inflammatory signaling with metabolic dysfunction during cholangiopathy remain incompletely defined[10].

Nuclear factor-kappa B (NF-κB) is a central regulator of inflammation and disease progression. Canonical activation by tumor necrosis factor, interleukin-1, and Toll-like receptor signaling converges on the inhibitor of NF-κB kinase complex, leading to inhibitor of NF-κB degradation, p65/p50 nuclear translocation, and transcription of cytokines, chemokines, adhesion molecules, survival genes, and oxidative-stress and tissue-remodeling programs. NF-κB activity can also be shaped by epigenetic regulators, such as WDR54, which was reported to interact with RBBP5 and increase H3K4me3 enrichment at the p65 promoter in hepatocellular carcinoma[11].

Nuclear receptors play essential roles in maintaining hepatic and intestinal bile acid homeostasis. Liver receptor homolog-1 (LRH-1, NR5A2) regulates bile acid metabolism, cholesterol transport, mitochondrial function, and anti-inflammatory signaling, while farnesoid X receptor (FXR) controls enterohepatic bile acid feedback pathways through induction of intestinal fibroblast growth factor 15/19 (FGF15/19) signaling[5,12]. We previously demonstrated that activation of LRH-1 protects against parenteral nutrition-associated cholestasis and that inflammatory NF-κB signaling directly suppresses LRH-1 transcription through promoter binding, thereby disrupting hepatoprotective metabolic pathways[13]. More recently, in a proof-of-principle study in a 3-day 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) acute cholangiopathy model, supplementation with high-dose (20%) dietary lauric acid (LA), a medium-chain fatty acid and precursor of the endogenous LRH-1 agonist dilauroylphosphatidylcholine (DLPC), was protective by restoring LRH-1/FXR signaling. This protection was associated with suppression of oxidative stress, reducing hepatocyte senescence, and increased expression of macrophage-associated reparative markers. Although these findings established proof-of-concept that high-dose LA can modulate very early cholangiopathic injury, it is essential to determine the effects of a more physiologic dose of LA on a model of chronic inflammatory cholangiopathy. Furthermore, the relationship between inflammatory NF-κB signaling, CCL2-mediated immune activation, and repression of LRH-1 pathways during chronic cholestatic injury has not been defined[14,15].

The DDC mouse model recapitulates several histopathological features relevant to human cholangiopathies, including bile duct injury, cholestasis, inflammatory macrophage infiltration, ductular reaction, and periportal fibrogenic remodeling[16-18]. Therefore, in the present study, we utilized a chronic 14-day DDC model to investigate whether concurrent low-dose dietary LA supplementation attenuates the development of DDC-induced injury and to characterize associated inflammatory and metabolic responses. We hypothesized that LA supplementation would be associated with reduced NF-κB-CCL2 signaling and restored LRH-1-regulated bile acid homeostasis. Because LA was initiated at the start of DDC exposure, the study evaluated protection during injury development rather than reversal of established cholestasis or fibrosis. The findings are limited to this DDC model and do not establish efficacy in other cholangiopathy models or human disease. The 14-day experimental period characterizes a relatively short-term evaluation of DDC-induced cholangiopathy. Although early periportal collagen deposition and fibrogenic gene expression were assessed, the study did not assess long-term fibrosis progression or fibrosis regression.

MATERIALS AND METHODS

Animal studies

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus. Experimental procedures were performed in accordance with ARRIVE guidelines and institutional regulations for the ethical use of laboratory animals.

Eight-week-old male C57BL/6J mice were obtained from Jackson Laboratory and maintained in a pathogen-free animal facility under controlled environmental conditions (12-hour light/12-hour dark cycle). Animals were allowed unrestricted access to food and water and were acclimated for one week before initiation of the study. Male mice were selected to minimize potential variability associated with estrogen-dependent regulation of bile acid metabolism, nuclear receptor signaling, and biliary physiology that could influence cholestatic responses.

Following acclimation, mice were randomly allocated into the following treatment groups: Control (Chow): Mice received a standard laboratory chow diet. DDC: Mice were fed a diet containing 0.1% (w/w) DDC (Sigma-Aldrich, Cat. No. 137030, St Louis, MO, United States) for 14 days to induce cholestatic liver injury. LA: Mice received a diet supplemented with 5% (w/w) LA for 14 days. DDC/LA: Mice were fed a diet containing 0.1% (w/w) DDC and 5% (w/w) LA for 14 days. Experimental diets were custom manufactured by Inotiv according to study specifications. Animals were monitored daily throughout the experimental period.

Tissue and blood collection

At study completion, mice were anesthetized with pentobarbital administered intraperitoneally. Whole blood was collected from the retro-orbital sinus, and serum was isolated for biochemical analyses. Liver and terminal ileum tissues were excised, rapidly frozen, and stored at -80 °C for subsequent molecular studies or processed for histological and immunofluorescence analyses. Bone marrow-derived macrophages were harvested for gene expression and functional assays.

Serum markers of hepatocellular injury [alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] and biochemical evidence of cholestasis (alkaline phosphatase, total bilirubin, and total serum bile acids) were measured in the University of Colorado Hospital Clinical Chemistry Laboratory using coded samples, with the exception of total serum bile acids. Total serum bile acid concentrations were quantified using a colorimetric Total Bile Acid Assay Kit (Cat. No. DZ042A-KY1, Diazyme Laboratories, Poway, CA, United States) according to the manufacturer’s protocol.

Histological and immunohistochemical analyses

At the time of tissue collection, livers were excised, fixed in neutral-buffered formalin, processed for paraffin embedding, and sectioned at a thickness of 4 μm. For evaluation of liver morphology and injury, sections were stained with hematoxylin and eosin using standard histological procedures. For immunohistochemical analyses, paraffin-embedded liver sections were stained for macrophage and cholangiocyte markers using antibodies against F4/80 (Cell Signaling Technology, Cat. No. 70076, MA, United States, clone D2S9R; 1:200 dilution) and cytokeratin 19 (Proteintech, Cat. No. 14965-1-AP; San Diego, CA, United States, polyclonal; 1:1000 dilution). Heat-induced antigen retrieval was performed in sodium citrate buffer (10 mmol/L, pH 6.0) containing 0.1% Tween-20 using a pressure-based decloaking system, followed by cooling to room temperature. Immunostaining was carried out on a Discovery Ultra automated staining platform (Roche). Tissue sections were incubated with primary antibodies at 37 °C according to optimized staining conditions. Detection of bound antibodies was achieved using an HRP-conjugated secondary detection system (OmniMAP anti-rabbit HRP; Ventana/Roche, AZ, United States) followed by visualization with 3,3’-diaminobenzidine chromogen. Sections were subsequently counterstained with hematoxylin, differentiated, dehydrated through graded ethanol solutions, cleared in xylene, and mounted with permanent mounting medium. Liver sections were stained with Picro-sirius red solution according to the protocol described in our previous study (Ghosh et al[19]). All tissue processing, staining, and slide preparation were performed by the University of Colorado Cancer Center Research Histology Core Facility. Histological images were acquired using bright-field microscopy and analyzed in a blinded manner.

RNA isolation and quantitative real-time polymerase chain reaction

Total RNA was isolated from liver tissue, primary hepatocytes, intrahepatic mononuclear cells, bone marrow-derived macrophages, and cultured cell lines using TRIzol reagent (Invitrogen; Cat. No. 15596026, Waltham, MA, United States) or the RNeasy Mini Kit (Cat. No. 74104 QIAGEN, Hilden, Germany) according to the manufacturers’ protocols. RNA concentration and purity were determined spectrophotometrically before reverse transcription. Complementary DNA was synthesized using the iScript complementary DNA Synthesis Kit (Cat. No. 1708891; Bio-Rad, Hercules, CA, United States). Quantitative real-time polymerase chain reaction was performed using TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, United States), MicroAmp™ Optical 96-Well Reaction Plate with Barcode (Cat. No. 4306737, Thermo Fisher Scientific, Waltham, MA, United States), and MicroAmp™ Optical Adhesive Film (Cat. No. 4311971 Thermo Fisher Scientific, Waltham, MA, United States). Relative gene expression was calculated using the comparative Ct (2^-ΔΔCt) method after normalization to the appropriate housekeeping gene. The probe information used in this study is provided in Supplementary Table 1.

Nitrite determination

Nitric oxide production was assessed indirectly by measuring nitrite levels in isolated hepatocytes using a Nitrite Assay Kit (Cat. No. MAK367; MilliporeSigma, St. Louis; MO, United States). Assays were performed according to the manufacturer’s instructions, and absorbance was recorded at 540 nm using a microplate reader.

Malondialdehyde quantification

Malondialdehyde, a marker of lipid peroxidation and oxidative stress, was measured in primary hepatocyte lysates using a commercially available assay kit (Cat. No. E-BC-K025-M, Elabscience, Wuhan, Hubei, China). Following the manufacturer’s protocol, absorbance was determined at 532 nm with a microplate reader.

Lipid peroxide measurement

Lipid peroxidation in primary hepatocytes was quantified using a commercial Lipid Peroxide Assay Kit (Cat. No. E-BC-K176-M, Elabscience, Wuhan, Hubei, China) according to the manufacturer’s protocol. Absorbance was measured at 586 nm using a microplate spectrophotometer, and values were normalized as recommended by the assay guidelines.

Isolation of intrahepatic mononuclear cells and hepatocytes

Intrahepatic mononuclear cells were isolated from mouse liver as previously described with minor modifications[18]. Briefly, freshly excised liver tissue was finely minced and digested in Hanks’ Balanced Salt Solution containing Liberase R (Cat. No. 05989132001, Roche, Basel, Switzerland) and DNase I (Cat. No. 1010415900, Sigma-Aldrich, St Louis, MO, United States) at 37 °C for 30 minutes. The digested suspension was sequentially passed through 100-μm and 70-μm cell strainers to remove debris. Hepatocytes were depleted by low-speed centrifugation (25 × g, 3 minutes), and the remaining non-parenchymal cell fraction was collected by centrifugation at 300 × g for 5 minutes. Cells were subsequently enriched using a 40% Histodenz (Cat. No. D2158; MilliporeSigma, St Louis, MO, United States) density gradient and centrifuged at 1500 × g for 20 minutes. Mononuclear cells recovered from the interphase were collected and used for downstream analyses. Hepatocytes were washed several times and used for culture.

Nicotinamide adenine dinucleotide, reduced form measurement

Nicotinamide adenine dinucleotide, reduced form (NADH) in primary hepatocytes was quantified using a commercial NADH Assay Kit (AAT Bioquest, Cat. No. 15273, Pleasanton, CA, United States) according to the manufacturer’s protocol. Absorbance was measured at 460 nm using a microplate spectrophotometer, and values were normalized as recommended by the assay guidelines.

Protein extraction and western blot analysis

Total cellular protein was extracted from isolated liver or hepatocytes using Mammalian Protein Extraction Reagent or Tissue Protein Extraction Reagent or nuclear or cytoplasmic extraction kit NE-PER (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocols. Protein concentrations were determined prior to electrophoresis, and equal amounts of protein were loaded onto 4%-20% gradient sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA, United States) for separation.

Following electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, United States). Membranes were blocked and subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence detection reagents and captured using a digital imaging system. Antibodies used for immunoblotting and their corresponding sources are listed in Supplementary Table 2.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) analyses were conducted using isolated primary hepatocytes and liver tissue homogenates with the EZ-Magna ChIP™ G ChIP Kit (MilliporeSigma, Burlington, MA, United States) according to the manufacturer’s instructions. Briefly, chromatin was cross-linked, sheared, and immunoprecipitated with antibodies against NF-κB. Enriched DNA fragments were purified and analyzed by quantitative polymerase chain reaction using promoter-specific primers targeting established transcription factor binding regions within the Ccl2 and Nr5a2 genes. Results were expressed relative to input chromatin and appropriate control immunoprecipitations as previously described[19].

Senescence-associated β-galactosidase staining

Cellular senescence was evaluated by senescence-associated β-galactosidase staining using a Cellular Senescence Detection Kit (Cat. No. GXS0003; OZ Biosciences, Marseille, France). Primary hepatocytes isolated from experimental groups were cultured overnight before analysis. Staining was performed according to the manufacturer’s protocol, and senescent cells were identified by the presence of characteristic blue cytoplasmic staining under bright-field microscopy.

DLPC analysis by liquid chromatography-tandem mass spectrometry

For lipid extraction, liver tissue was suspended in ice-cold 100% methanol at a concentration of 15 mg/mL. Samples were subjected to glass bead beating for 5 minutes, followed by vigorous vortexing for 30 minutes at 4 °C. The lysates were then centrifuged at 18213 × g for 10 minutes, and the resulting supernatants were collected for analysis. Supernatants were analyzed using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (MS/MS) (Vanquish and Exploris, Thermo Fisher Scientific, Waltham, MA, United States). Samples were injected at a volume of 10 μL, and non-polar lipid metabolites were separated on a Waters Acquity BEH C18 column (1.7 μm, 2.1 mm × 100 mm) using a 5-minute gradient in MS/MS mode, as previously described[20]. An analytical DLPC standard (1,2-dilauroyl-sn-glycero-3-phosphocholine; MedChemExpress, Monmouth Junction, NJ, United States, CAS 18194-25-7) was prepared at 500 nM in methanol and analyzed alongside the extracted samples to confirm DLPC identity based on retention time and MS/MS fragmentation pattern. Raw data files were converted to mzXML format using RawConverter, and peaks were integrated using EI-Maven software (Princeton University, Princeton, NJ, United States). Quality control was performed using technical replicate samples analyzed at the beginning, middle, and end of the instrument run[20].

CCL2 enzyme-linked immunosorbent assay

Serum concentrations of CCL2 (monocyte chemoattractant protein-1) were quantified using a commercially available mouse CCL2 enzyme-linked immunosorbent assay kit (Cat. No. 6721, Chondrex, Inc., Woodinville, WA, United States) following the manufacturer’s instructions. Absorbance was measured using a microplate reader, and CCL2 concentrations were calculated from a standard curve generated with recombinant standards supplied with the kit.

Statistical analysis

Quantitative gene expression data were generated from technical triplicates for each biological sample. For animal studies, values from individual mice were averaged and presented as mean ± SE of the mean (SEM). The number of biological replicates per experimental group ranged from 6 animals to 9 animals and is indicated in the corresponding figure legends. For in vitro studies, measurements were obtained from at least three independent experiments, each performed with technical replicates.

Statistical analyses were conducted using GraphPad Prism. Comparisons among multiple groups were performed using one-way analysis of variance followed by Tukey’s post hoc test to correct for multiple comparisons. Differences between two groups were assessed using an unpaired two-tailed Student’s t-test. P < 0.05 was considered statistically significant. Some of the selected Chow/DDC experiment data were also used in our previous study (Ghosh et al[19]).

To minimize bias, all biological samples were assigned coded identifiers before processing, and data acquisition and analysis were performed in a blinded manner without knowledge of treatment allocation until completion of statistical analyses.

RESULTS

Concurrent dietary LA attenuates biochemical cholestasis, hepatocellular injury, ductular reaction, and macrophage infiltration during 14-day DDC exposure

In our previous proof-of-concept study, dietary supplementation with 20% LA protected against early DDC-induced cholangiopathy (3-day model)[5]. To determine the effect of a lower dose of LA on the progression of DDC cholangiopathy over 14 days, adult male mice were fed a 0.1% DDC diet for 14 days with or without 5% (5 g/100 g diet) dietary LA supplementation. We selected the 14-day DDC model to evaluate chronic inflammatory cholangiopathy and determine whether low-dose LA supplementation protects against DDC-induced liver injury. Because DDC feeding can reduce food intake and body weight, we analyzed initial and final body weights, liver weight, final liver weight ratio, and daily dietary intake (Table 1). DDC and DDC-LA mice showed reduced final body weights and liver weights compared with chow-fed mice, whereas LA alone did not cause weight loss. Liver weight ratios were similar among the groups. Dietary intake was lower in DDC, LA, and DDC-LA mice than in chow-fed mice but was comparable among treatment groups. Importantly, LA supplementation did not further reduce DDC diet intake compared with DDC mice, indicating that the protective effects of LA were not due to differences in food consumption or body weight (Table 1). As shown in Figure 1A, 14 days of DDC feeding resulted in marked hepatocellular injury (elevated AST, ALT) and cholestasis (elevated serum alkaline phosphatase, total bilirubin, and total bile acids) compared with Chow-fed and LA-supplemented controls, which did not differ from each other. LA diet supplementation in the DDC-treated mice significantly reduced serum AST, ALT, and alkaline phosphatase levels and markedly decreased circulating bile acids and bilirubin, indicating attenuation of hepatocellular injury and cholestasis. Liver histology demonstrated that DDC feeding induced hepatocyte swelling, bile pigment accumulation, periportal inflammation, and ductular reaction (Figure 1B). These pathological alterations were reduced in DDC mice receiving LA supplementation. Immunohistochemical staining revealed substantial infiltration of F4/80-positive macrophages in DDC livers, whereas DDC/LA mice had reduced hepatic macrophage infiltration (Figure 1C). Cytokeratin 19 staining demonstrated a pronounced ductular reaction in DDC mice, which was reduced by LA administration (Figure 1D). These biochemical and histologic findings demonstrate that dietary LA reduced liver injury, macrophage infiltration, and hepatocellular cholestasis in this chronic model of DDC-induced cholangiopathy.

Figure 1 Dietary lauric acid reduces cholestatic liver injury and macrophage infiltration in 14-day 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. A: Serum liver chemistries including aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and total bile acids were measured in Chow (n = 6), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (n = 6), lauric acid (LA) (n = 6), and DDC/LA (n = 6) mice after 14 days of treatment. DDC feeding significantly increased biochemical markers of hepatocellular injury and cholestasis, whereas 5% dietary LA supplementation attenuated these changes; B: Representative hematoxylin and eosin-stained liver sections (200 ×; scale bar: 100 μm) from Chow, DDC, LA, and DDC/LA mice. DDC feeding induced periportal inflammation, cholestatic injury, and ductular expansion, which were reduced by LA treatment. Photomicrographs are representative of 6 mice in each group; C: Representative immunohistochemical staining and quantification of F4/80-positive macrophages (100 ×; scale bar: 200 μm) in liver sections. DDC mice exhibited marked hepatic macrophage accumulation, whereas LA supplementation significantly reduced inflammatory cell infiltration. Photomicrographs are representative of 6 mice in each group; D: Representative immunohistochemical staining and quantification of cytokeratin 19-positive bile duct epithelia (100 ×; scale bar: 200 μm). DDC feeding induced a robust ductular reaction which was attenuated in DDC/LA mice. Photomicrographs are representative of 6 mice in each group. Data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance with Tukey’s multiple comparison test. ^aP < 0.05 vs all groups. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; CK-19: Cytokeratin 19; HE: Hematoxylin and eosin.

Figure 2 Dietary lauric acid restores hepatocyte bile acid homeostasis, suppresses oxidative stress and senescence, and improves expression of mitochondrial enzymes. A: Relative mRNA expression of bile acid synthesis and transporters and nuclear receptor signaling genes in isolated hepatocytes from Chow (n = 5), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (n = 5), lauric acid (LA) (n = 4), and DDC/LA (n = 4) mice. DDC feeding reduced expression of genes involved in bile acid transport (Abcb11/BSEP, Abcc2/MRP2 and Abcc4) and synthesis (Cyp7a1) and nuclear receptor signaling (Nr5a2, Nr0b2/SHP, Nr1h4/FXR), whereas LA restored expression in DDC mice of Abcb11/BSEP, Cyp7a1, Nr0b2/SHP, Abcc2, Fgfr4, and Nr5a2/LRH-1; B: Relative mRNA expression of Fgf15, Slc10a2/ASBT, and Nr1h4/FXR in terminal ileum of experimental groups. DDC feeding suppressed Fgf15 and Nr0b2 and increased Slc10a2/ASBT expression. LA supplementation upregulated intestinal Fgf15, Nr1h4, and Nr0b2 expression and suppressed Slc10a2 expression in DDC mice; C: Relative expression of oxidative stress-related genes and markers of oxidative stress in hepatocytes. DDC mice showed upregulated Nqo1, Hmox1, Txnip, Sod2, Krt18, and Hmgb1, whereas DDC was associated with suppressed Cyp2e1, Nfe2 l2/NRF2, and Gpx4. LA supplementation significantly reversed the effects of DDC, except for Krt18. DDC diet increased hepatic nitrites, lipid peroxides, and malondialdehyde, whereas LA supplementation significantly reversed these findings; representative immunoblot and quantitation of cleaved caspase 3 in hepatocytes. Growth factor receptor-bound protein 2 was used as a loading control (n = 3 per group); D: Relative expression of hepatocyte senescence-associated genes. DDC mice exhibited increased expression of Cdkn1a/p21, Cdkn1b and E2f1, which were markedly reduced by LA treatment. β-galactosidase staining, an indicator of senescence, of primary hepatocytes (overnight culture) isolated from the 4 groups of mice (n = 4 per group) showed senescence in DDC hepatocytes, which was prevented by LA. Representative images are shown; E: Relative expression of mitochondrial fatty acid oxidation and tricarboxylic acid cycle genes in hepatocytes. LA treatment increased expression of Aco2, Cpt1a, Cpt2 and Hadhb consistent with improved mitochondrial homeostasis. Measurement of nicotinamide adenine dinucleotide, reduced form concentration in liver tissue supernatant showed reduced nicotinamide adenine dinucleotide, reduced form in DDC mice and increased levels in LA supplemented mice, consistent with increased fatty acid oxidation. Data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance with Tukey’s multiple-comparison test. ^aP < 0.05 vs all groups; ^bP < 0.05 vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine. DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; FXR: Farnesoid X receptor.

Figure 3 Dietary lauric acid downregulates hepatic fibrosis, and alters macrophage-associated transcriptional markers in 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice. A: Representative Picrosirius red staining of liver sections from Chow, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), lauric acid (LA), and DDC/LA mice following 14 days of treatment. DDC feeding induced periportal collagen deposition that was prevented by dietary LA supplementation; B: Quantification of Picrosirius red-positive (100 ×) staining area in liver sections (n = 4 mice per group) mice; C: Relative hepatic mRNA expression of fibrosis-associated genes Adam1, Col1a1, Spp1, Mmp13, and Timp2. DDC increased the expression of profibrotic and matrix-remodeling genes, whereas dietary LA reduced their expression (except Timp2). Groups included 5 Chow, 5 DDC, 4 LA, and 4 DDC/LA mice; D: Relative mRNA expression of pro-inflammatory genes Ccl2, Itgam, Cxcl2, and Cd68 in isolated intrahepatic mononuclear cells. DDC feeding induced inflammatory immune activation, which was attenuated by dietary LA treatment. Mice included Chow (n = 5-6), DDC (n = 5), LA (n = 4), and DDC/LA (n = 4); E: Relative mRNA expression of anti-inflammatory macrophage markers Retnlg, Chil3, Arg1, and Clec10a1 in isolated intrahepatic mononuclear cells showing upregulation in DDC/LA mice compared to DDC mice. Data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance with Tukey’s multiple-comparison test. ^aP < 0.05 vs all groups; ^bP < 0.05 vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine. DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; IHMC: Intrahepatic mononuclear cells.

Figure 4 Dietary lauric acid suppresses nuclear factor-kappa B-dependent inflammatory signaling and restores liver receptor homolog-1 transcriptional activity. A: Serum C-C motif chemokine ligand 2 (CCL2) concentrations in Chow, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), lauric acid (LA), and DDC/LA mice following 14 days of treatment. DDC feeding significantly increased circulating CCL2 Levels, whereas dietary LA normalized CCL2 Levels in DDC mice (n = 3-4 mice per group); B: Representative immunoblots and quantitation of phospho-nuclear factor-kappa B (NF-κB) p65 and total NF-κB p65 in liver homogenates. DDC-induced cholangiopathy increased NF-κB phosphorylation, which was suppressed by LA treatment. Growth factor receptor-bound protein 2 was used as a loading control (n = 3); C: Chromatin immunoprecipitation analysis of NF-κB binding to the Ccl2 promoter in liver tissue. DDC feeding increased NF-κB occupancy at the promoter, whereas LA supplementation significantly reduced NF-κB promoter binding (n = 4 mice per group); D: Relative mRNA expression of NFKB1, CCL2, STAT1, and CDKN1B in Huh7 cells following transfection with control or NF-κB siRNA. NF-κB knockdown reduced expression of pro-inflammatory and senescence-associated genes; E: Relative mRNA expression of oxidative stress gene HMOX1 in Huh7 cells following NF-κB siRNA knockdown. Suppression of NF-κB signaling reduced HMOX1 expression; F: Chromatin immunoprecipitation analysis of NF-κB binding to the Nr5a2/Liver receptor homolog-1 (LRH-1) promoter in liver tissue. DDC increased NF-κB occupancy at the promoter, while LA supplementation reduced promoter binding. Relative mRNA expression of the LRH-1 target gene NR0B2/SHP in Huh7 cells following NF-κB siRNA knockdown. Silencing NF-κB increased NR0B2/SHP expression, consistent with NF-κB-mediated repression of LRH-1 signaling; G: Dilauroylphosphatidylcholine relative abundance, measured in liver tissue by liquid chromatography-mass spectrometry, was increased by LA supplementation in DDC-treated mice (n = 4-5 per group); H: Relative mRNA expression of NR0B2/SHP and CCL20 in Huh7 cells following transfection with control or NR5A2-siRNA in the presence or absence of LA; I: Proposed mechanisms of LA protection in DDC mouse model. DDC activates NF-κB/p65, increases p65 binding to the Ccl2 promoter, and promotes CCL2-mediated macrophage infiltration and inflammation. DDC also suppresses LRH-1 promoter activity/signaling, contributing to impaired Nr0b2, Abcb11, and Abcc2 expression, oxidative/nitrosative stress, hepatocyte senescence, cholestasis, ductular reaction, and fibrosis. LA reverses these DDC-induced effects by reducing NF-κB/p65-Ccl2 promoter activation, restoring LRH-1 signaling, inducing bile salt export pump and multidrug resistance-associated protein 2, promoting bile acid and bilirubin canalicular transport, respectively, and enhancing anti-inflammatory macrophage polarization. Together, LA reduces DDC-induced cholestatic injury and fibrogenic remodeling. Data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance with Tukey’s multiple-comparison test or Student’s t-test where appropriate. ^aP < 0.05 vs all groups. n = 4-6 mice per group or three independent cell culture experiments. ^eP < 0.05 vs siRNA-NR5A2. DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; NF-κB: Nuclear factor-kappa B; GRB2: Growth factor receptor-bound protein 2; DLPC: Dilauroylphosphatidylcholine; LA: Lauric acid; LRH-1: Liver receptor homolog-1; CCL2: C-C motif chemokine ligand 2; BSEP: Bile salt export pump; MRP2: Multidrug resistance-associated protein 2.

Table 1 Liver, body weight, and diet intake by mouse group, mean ± SEM.

Mouse group	Chow (n = 8)	DDC (n = 9)	Lauric acid (n = 6)	DDC/Lauric acid (n = 6)
Initial body weight (g)	23 ± 0.37	23.67 ± 0.55	24.00 ± 0.36	25.83 ± 0.95c
Final body weight (g)	25.13 ± 0.91b	17.99 ± 0.50	24.37 ± 0.49b	17.73 ± 0.68
Liver weight (g)	1.24 ± 0.07b	0.95 ± 0.01	1.15 ± 0.05b	0.88 ± 0.05
Liver weight/final body weight ratio	0.04 ± 0.001	0.05 ± 0.001	0.05 ± 0.002	0.05 ± 0.001
Diet intake (g/day/mouse)	4.47 ± 0.2a	2.94 ± 0.43	2.85 ± 0.05	2.65 ± 0.29

^aP < 0.05 vs all other groups.

^bP < 0.05 vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

^cP < 0.05 vs Chow.

DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

Dietary LA restores hepatocyte bile acid homeostasis and suppresses oxidative stress and senescence during chronic DDC-induced cholangiopathy

We first examined hepatocyte-specific gene expression for selected pathways that were altered in the short-term DDC model[5]. Quantitative real-time polymerase chain reaction analysis of freshly isolated hepatocytes revealed that chronic DDC exposure significantly suppressed expression of genes involved in bile acid synthesis and transport and nuclear receptor signaling, including Abcb11/BSEP, Cyp7a1, Nr0b2/SHP, Fgfr4, and Nr5a2/LRH-1 (Figure 2A). Dietary LA supplementation restored expression of these genes toward Chow control levels. In addition, expression of the compensatory hepatocyte bile acid efflux transporters Abcc2 and Abcc4 was increased in DDC/LA mice, suggesting enhanced hepatocellular adaptation to cholestatic stress.

We next examined ileal expression of genes involved in enterohepatic bile acid homeostasis. DDC markedly suppressed terminal ileal expression of Fgf15, while significantly increasing expression of the apical bile acid transporter Slc10a2/ASBT (Figure 2B). LA supplementation partially restored Fgf15 expression, reduced Slc10a2/ASBT, and significantly increased Nr1h4/FXR expression in the terminal ileum.

We next assessed hepatocyte oxidative stress gene expression, which is upregulated in hepatocellular cholestatic injury. DDC feeding markedly increased hepatocyte expression of the oxidative stress and cell death marker genes, Nqo1, Hmox1, Txnip, Sod2, Krt18, and Hmgb1, whereas it suppressed Cyp2e1, Nfe2 l2/NRF2, and Gpx4. LA supplementation was associated with significant reversal of these effects, with the exception of Krt18 (Figure 2C) in DDC mice. To assess oxidative injury, total nitrites, lipid peroxides, and malondialdehyde levels were measured in hepatocytes isolated from each group of mice. DDC mice displayed significantly elevated nitrite production and lipid peroxidation, whereas these markers were markedly reduced by LA treatment (Figure 2C). DDC also increased hepatocyte cleaved caspase-3 protein, providing molecular and biochemical evidence of cell injury and apoptotic signaling beyond serum aminotransferases (Figure 2C). LA treatment markedly lowered cleaved caspase-3 in DDC mice.

Hepatocyte senescence is increasingly recognized as a driver of the progression of chronic cholangiopathy[5,9]. Consistent with this concept, DDC mice exhibited robust induction of the senescence-associated genes Cdkn1a/p21, Cdkn1b and E2f1 (Figure 2D), which were completely normalized by dietary LA supplementation. Furthermore, β-galactosidase staining demonstrated increased numbers of senescent hepatocytes in DDC livers, which were substantially reduced following LA supplementation (Figure 2D).

Since mitochondrial dysfunction is associated with cholestatic liver injury[21], we next evaluated genes involved in fatty acid oxidation and mitochondrial metabolism. LA supplementation increased hepatic expression of Aco2, Cpt1a, Cpt2, and Hadhb compared with DDC mice (Figure 2E), presumably compensating for mitochondrial injury caused by the DDC cholangiopathy. In parallel with these transcriptional changes, hepatic levels of NADH were decreased in DDC mice and significantly increased following LA supplementation, consistent with increased fatty acid oxidation induced by LA (Figure 2E). Together, these findings demonstrate that dietary LA restores hepatocyte bile acid regulatory pathways, reduces oxidative stress, induces mitochondrial gene expression, and suppresses hepatocyte senescence.

Dietary LA attenuates fibrogenic responses, and alters macrophage-associated transcriptional signatures

Because persistent liver inflammation and cholangiocyte injury promote fibrogenesis during chronic cholangiopathy, we next examined whether dietary LA modulates hepatic fibrotic pathways in DDC-fed mice. Picrosirius red staining demonstrated increased periportal deposition of collagen in DDC livers compared with Chow controls. LA supplementation in DDC mice significantly reduced hepatic periportal collagen deposition/early fibrogenic remodeling (Figure 3A and B). Consistent with these findings, DDC mice had significantly upregulated hepatic expression of the fibrosis-associated genes Adam1, Col1a1, Spp1, Mmp13, and Timp2, all of which were reduced following LA treatment, except Timp2 (Figure 3C). Because the study ended after 14 days, these data demonstrate attenuation of early fibrogenic remodeling and collagen accumulation, not regression of established long-term fibrosis.

To determine the effect of LA treatment on immune-associated transcriptional responses in the DDC model, we next evaluated immune-associated gene expression in isolated intrahepatic mononuclear cells, which contain multiple immune-cell populations, including macrophages. DDC mice displayed increased expression of the pro-inflammatory genes Ccl2, Itgam, and Cxcl2, indicative of inflammatory macrophage activation and immune cell recruitment (Figure 3D). In contrast, LA treatment markedly suppressed expression of these pro-inflammatory genes. In addition, LA supplementation significantly increased the expression of Retnlg, Chil3, Arg1 and Clec10a1, which have been associated with reparative macrophage responses (Figure 3E). These findings indicate that LA not only suppressed inflammatory macrophage activation but also promoted a phenotypic shift toward a reparative macrophage program. Because isolated intrahepatic mononuclear cells contain multiple immune-cell populations and no flow-cytometric, spatial, or functional polarization analysis was performed, these results support a shift in macrophage-associated transcriptional signatures but do not definitively establish macrophage polarization or cell-specific function.

Dietary LA suppresses the NF-κB-CCL2 inflammatory axis and restores LRH-1 signaling

Our previous studies demonstrated that activation of NF-κB signaling is a critical driver of cholestatic liver injury and directly represses expression of the hepatoprotective nuclear receptor LRH-1 (Nr5a2) through promoter binding[13]. Because DDC-induced cholangiopathy is characterized by persistent inflammation and suppression of LRH-1 signaling, we investigated whether dietary LA modulates this NF-κB-dependent regulatory pathway. DDC-fed mice showed markedly elevated serum levels of the pro-inflammatory chemokine CCL2 compared with Chow controls (Figure 4A), which were significantly attenuated in DDC/LA (Figure 4A). DDC livers exhibited increased phosphorylation of NF-κB p65, indicating activation of the canonical NF-κB pathway, which was markedly suppressed in DDC/LA mouse liver (Figure 4B). CCL2 is a known NF-κB target gene. Previously, we identified CCL2 as a major mediator of hepatocyte injury and macrophage recruitment in DDC-induced cholangiopathy. Therefore, we next examined the effect of LA on the NF-κB binding at the Ccl2 promoter. ChIP analysis demonstrated robust NF-κB enrichment at the Ccl2 promoter in DDC mice, whereas LA supplementation markedly reduced NF-κB occupancy (Figure 4C). To further define the downstream consequences of NF-κB activation, Huh7 hepatocytes were transfected with NF-κB siRNA. NF-κB knockdown significantly reduced expression of CCL2 together with the senescence-associated and inflammatory genes STAT1, CDKN1B, and HMOX1 (Figure 4D and E), supporting a central role for NF-κB in driving hepatocyte inflammation, oxidative stress, and senescence.

NF-κB signaling also negatively regulated the protective LRH-1 pathway. ChIP analysis revealed markedly increased NF-κB binding to the Nr5a2/LRH-1 promoter in DDC livers compared with Chow controls (Figure 4F), consistent with our previous findings that NF-κB represses LRH-1 transcription[13]. LA supplementation significantly reduced NF-κB occupancy at the Nr5a2 promoter. Supporting this mechanism, NF-κB knockdown increased expression of the LRH-1 downstream target NR0B2/SHP (Figure 4F). In our previous studies, we reported that activation of LRH-1 by its agonist DLPC protects against parenteral nutrition-associated cholestasis and acute DDC-induced liver injury[5,13,18].

DLPC, a strong LRH-1 agonist, is a phospholipid containing LA at both the sn-1 and sn-2 positions and is typically present at very low or undetectable levels under physiological conditions. However, DLPC synthesis can be induced by dietary LA supplementation. To determine whether 5% LA supplementation increases hepatic DLPC production in DDC-fed mice, we quantified hepatic DLPC levels by liquid chromatography-mass spectrometry. Hepatic DLPC abundance was low in both Chow and DDC mice but was significantly increased following LA supplementation, with the highest levels observed in the DDC/LA group (Figure 4G). These findings demonstrate that dietary LA serves as a substrate for endogenous DLPC synthesis and may contribute to LRH-1 activation during DDC-induced cholestatic liver injury. To assess the functional contribution of LRH-1, Huh7 cells were transfected with control siRNA or NR5A2 siRNA. NR5A2 knockdown significantly reduced the relative mRNA expression of SHP, and induced CCL20, compared with control siRNA, which were not reversed by LA treatment. These findings support a role for LRH-1 in mediating the effects of LA on genes associated with bile acid homeostasis and inflammatory signaling (Figure 4H).

Collectively, these findings identify NF-κB as a central molecular switch linking inflammation and metabolic dysfunction during chronic cholangiopathy. DDC-induced NF-κB activation promotes inflammatory CCL2 signaling while simultaneously suppressing LRH-1-dependent hepatoprotective pathways. Dietary LA supplementation, likely through inducing DLPC synthesis, interrupts this pathogenic NF-κB program by reducing NF-κB activation and promoter occupancy, thereby suppressing the CCL2-STAT1 inflammatory axis and restoring LRH-1/SHP signaling (Figure 4I).

DISCUSSION

The pathogenesis of inflammatory cholangiopathies entails cholangiocyte injury, hepatocyte dysfunction, macrophage activation, cholestasis, and progressive periportal fibrosis[4,22,23]. In the current study, we demonstrate that low-dose dietary LA supplementation is associated with significant attenuation of hepatocellular injury, cholestasis, cell death pathway activation, and early stages of fibrosis responses in chronic DDC-induced cholangiopathy. Associated findings induced by LA supplementation include reduced oxidative stress and hepatocyte senescence, restoration of bile acid homeostasis, and changes in macrophage-associated inflammatory and reparative transcriptional markers. Mechanistically, we propose NF-κB as a central regulator that links inflammatory signaling to the repression of LRH-1-dependent hepatoprotective pathways[5,18].

Chronic inflammation and fibrogenesis in cholangiopathies are driven not only by biliary injury but also by persistent inflammatory responses that amplify tissue damage. Consistent with this concept, chronic DDC feeding induced substantial macrophage infiltration, ductular reaction, and expression of inflammatory mediators, including CCL2 and CXCL2. CCL2 is a potent chemokine involved in the recruitment of inflammatory monocytes and has been implicated in the pathogenesis of primary sclerosing cholangitis, biliary atresia, and experimental cholestatic liver injury. We previously demonstrated that cholangiocyte-derived CCL2 promotes hepatocyte injury and senescence in acute DDC-induced cholangiopathy[5]. The current study extends these observations by demonstrating that chronic DDC injury is associated with sustained activation of the NF-κB-CCL2 signaling axis, which dietary LA effectively suppresses.

NF-κB is widely recognized as a master regulator of inflammatory responses in chronic liver disease[13]. Canonical activation by tumor necrosis factor, interleukin-1, and Toll-like receptor signaling converges on the inhibitor of NF-κB kinase complex, leading to inhibitor of NF-κB degradation, p65/p50 nuclear translocation, and transcription of cytokines, chemokines, adhesion molecules, survival genes, and oxidative-stress and tissue-remodeling programs. NF-κB activity can also be shaped by epigenetic regulators[14]. However, our findings suggest that its role extends beyond activation of pro-inflammatory cytokines and chemokines. We previously reported that NF-κB directly binds to the Nr5a2 promoter and suppresses transcription of LRH-1, a nuclear receptor that regulates bile acid metabolism and anti-inflammatory signaling[13]. In the present study, DDC-induced NF-κB activation was associated with increased NF-κB occupancy at both the Ccl2 and Nr5a2 promoters. This dual activity establishes NF-κB as a molecular switch that simultaneously promotes inflammatory signaling while suppressing protective LRH-1 pathways. Importantly, LA supplementation reduced NF-κB activation and promoter occupancy, thereby reducing CCL2 expression and restoring LRH-1-SHP signaling. These findings identify a previously underappreciated NF-κB-CCL2-LRH-1 regulatory axis that coordinates inflammatory and metabolic responses during chronic cholangiopathy[10,24,25].

A major consequence of cholestatic injury is disruption of bile acid homeostasis. Excessive accumulation of hydrophobic bile acids contributes to oxidative stress, mitochondrial dysfunction, and hepatocellular injury. In our previous acute DDC study, LA restored LRH-1 and FXR signaling pathways and normalized bile acid metabolism. The current study demonstrates that these effects persist during chronic disease progression. LA restored hepatocyte expression of LRH-1 target genes, including Nr0b2/SHP, Abcb11/BSEP, and Fgfr4, and improved intestinal FXR-FGF15 signaling. Because LRH-1 and FXR cooperate to regulate bile acid synthesis, transport, and enterohepatic feedback mechanisms[5,13,26], restoring this signaling network is likely to substantially reduce cholestatic injury in LA-treated mice.

Oxidative stress and hepatocyte senescence are increasingly recognized as important contributors to disease progression in cholangiopathies. Senescent hepatocytes produce pro-inflammatory mediators that perpetuate tissue injury and fibrosis through the senescence-associated secretory phenotype. In the present study, chronic DDC feeding induced expression of oxidative stress markers, increased lipid peroxidation and nitrosative stress, and promoted hepatocyte senescence as evidenced by elevated Cdkn1a, E2f1, and β-galactosidase activity. These changes were markedly attenuated by LA treatment. Furthermore, NF-κB silencing reduced expression of STAT1, CDKN1B, and HMOX1, suggesting that NF-κB signaling directly contributes to oxidative stress and senescence programs in this model.

Macrophages play a critical role in the pathogenesis of cholestatic liver injury. Consistent with our previous findings in an acute DDC model, LA shifted hepatic immune responses away from a pro-inflammatory phenotype toward a reparative program. LA reduced expression of inflammatory markers in intrahepatic mononuclear cells while increasing expression of Chil3 and Retnlg, indicative of a reparative macrophage-associated transcriptional signature polarization. Therefore, these results demonstrate changes in macrophage-associated transcriptional signatures rather than definitive macrophage polarization or functional reprogramming. This phenotypic switch was accompanied by reduced fibrosis and collagen deposition. Because LRH-1 activation has previously been linked to STAT6-dependent macrophage reprogramming, restoration of LRH-1 signaling may provide an important mechanistic link between metabolic regulation and immune homeostasis in progressive cholangiopathies[18].

An important strength of this study is the demonstration that a relatively low dose of dietary LA remains effective during established disease. Unlike our previous acute study, which used 20% dietary LA for 3 days[5], the present work shows that 5% dietary LA administered for 14 days during DDC feeding retains substantial hepatoprotective activity. This finding enhances the translational relevance of LA as a potential nutritional or therapeutic intervention for chronic cholestatic liver diseases.

Several limitations should be acknowledged. The study was performed in a single murine model of cholangiopathy that is relatively short-term and did not directly evaluate long-term fibrosis progression or regression. Validation of these findings in additional murine models of cholestasis (e.g., Mdr2 knock out mice and bile duct ligation mice) would help establish the broader applicability of these findings, however that was out of the scope of the present study. Although our data strongly support a role for NF-κB-mediated regulation of LRH-1, additional genetic studies targeting NF-κB and LRH-1 specifically in hepatocytes and macrophages will be required to establish cell-specific mechanisms. Although hepatic DLPC abundance increased with LA, the present study was not designed to determine whether DLPC is the key mediator or whether LRH-1 activation is required, because DLPC blockade or depletion and rescue experiments were not performed. Furthermore, it remains to be determined whether LA supplementation or its metabolite, DLPC, exerts similar protective effects in human studies. Finally, it should be noted that the current study demonstrates LA, when given concurrently with DDC exposure, provides protection against the development of cholangiopathic injury rather than therapeutic reversal of established cholestasis or fibrosis. The latter will require delayed treatment protocols after the establishment of cholestatic injury.

CONCLUSION

In conclusion, this study suggests that dietary LA attenuates chronic murine DDC-induced cholangiopathy (including biochemical cholestasis, hepatocellular injury, oxidative stress, senescence, cell death-associated markers, immune cell accumulation, and early fibrogenic remodeling) by coordinating the suppression of NF-κB-dependent inflammatory signaling and the restoration of LRH-1-mediated metabolic homeostasis. Protection was associated with reduced NF-κB-CCL2 signaling, restored LRH-1-regulated gene expression, and increased hepatic DLPC abundance. These findings define a proposed coordinated response in the DDC model but do not establish therapeutic reversal of established disease or prove that LRH-1, DLPC, or the entire NF-κB-to-LRH-1 pathway is required for this effect. Validation of this protection in delayed-treatment protocols, additional cholangiopathy models, and cell-specific mechanistic studies are indicated in future studies. Nevertheless, the findings in the current study support further investigation of LA-based therapeutic strategies for chronic cholestatic liver diseases.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge E. Erin Smith, HTL(ASCP) CM, and Helen DuBois, HTL(ASCP)CM, of the University of Colorado Anschutz Medical Campus, Pathology Shared Resource, Research Histology Laboratory.