Pparα

Fucoxanthin with a purity of 70% was purchased by Shandong Jiejing Group Corporation (Rizhao, China). AST, ALT, TC, TG, SOD, CAT, GSH-Px, MDA, and Oil Red O staining solution kits were all obtained from the Jiancheng Bioengineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits and a bicinchoninic acid protein assay kit were obtained from BOSTER (Wuhan, China). AMPK, phosphorylated AMPK, PPARα, SREBP-1c, ACC, phosphorylated ACC, FAS, and CPT-1 antibodies were on sale at Affinity Biosciences, Inc. (Cincinnati, OH, USA). HO-1, Keap-1, GCLM, Nrf2, NQO1, GAPDH, MyD88, TLR4, phosphorylated IκBα, and phosphorylated NF-κBp65 antibodies were obtained from Wuhan Sanying Biotechnology Co. (Wuhan, China).

Total protein was extracted and quantified from liver tissue homogenates for western blotting. Primary antibodies included PPARα (Affinity, Changzhou, China), PGC-1α (Proteintech, Wuhan, China), GAPDH (Proteintech, Wuhan, China), and Blots were analysed using chemiluminescence with imaging system (Bio-Rad, CA, USA).

Total cellular proteins were extracted using the RIPA lysis buffer, and a BCA assay kit (Beyotime) was used to quantify the protein concentrations. The same amounts of proteins (20 μg) were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% low-fat milk for 1 h, and then incubated with primary antibodies against SERCA1 (1:500; Affinity Biosciences), SERCA2 (1:5000; Abcam), SERCA3 (1:5000; Proteintech), IP3R (1:1000; Affinity Biosciences), PLCβ1 (1:1000; Proteintech), P2Y2 (1:1000; Affinity Biosciences), P2Y4 (1:1000; Affinity Biosciences) P2Y12 (1:2000; Abcam), Col1a1 (1:1000; Abcam), Runx2 (1:1000; Affinity Biosciences), Osx (1:1000; Bioss), β-catenin (1:1000; Proteintech), OPG (1:1000; Abcam), RANKL (1:1000; Proteintech), DKK1 (1:2000; Proteintech), NFATc1 (1:500; Santa Cruz), Calcr (1:1000; Proteintech), Ctsk (1:1000; Affinity Biosciences), TRAP (1:2000; Abcam), PPARα (1:1000; Affinity Biosciences), p-PPARα (1:1000; Affinity Biosciences) or β-actin (1:4000; Proteintech) overnight at 4  °C. The blots were incubated with HRP-conjugated goat-anti-rabbit secondary antibody (1:5000; Abcam) or HRP-conjugated goat-anti-mouse secondary antibody (1:5000; Abcam), and then visualized using an enhanced chemiluminescence system (Image Quant 350, GE Healthcare).