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

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.