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

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.