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

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.