As primary antibodies, a rabbit anti-nuclear factor-κ B (NF-κB) (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), a rabbit anti-TLR4 (diluted 1:1000, Cell Signaling Technology), a rabbit anti-SMAD2 (diluted 1:1000, Cell Signaling Technology), a rabbit anti-p-SMAD2 (diluted 1:1000, Cell Signaling Technology), a rabbit anti-SMAD3 (diluted 1:1000, Sangon Biotech), a rabbit anti-p-SMAD3 (diluted 1:1000, Sangon Biotech), and a rabbit anti-SMAD4 (diluted 1:1000, Wanleibio, Shenyang, China) were used. Protein extracts were stored at –80°C until use. Equal amounts of denatured protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were transferred to polyvinylidene difluoride membranes (Solarbio Life Sciences, Beijing, China). Nonspecific binding was blocked with BSA (Dalian Meilun Biotechnology) for 1 hour at room temperature. The membranes were then incubated overnight at 4°C with antibodies against NF-κB, SMAD2, p-SMAD2, SMAD3, p-SMAD3, SMAD4, and β-actin at the dilutions specified by the manufacturers. The membranes were then incubated with anti-rabbit IgG (DyLight 680 Conjugate; Cell Signaling Technology) forx 2 hours at room temperature. The protein bands were imaged with a LI-COR automatic chemiluminescence image analysis system (LI-COR, Nebraska USA). Quantification of Western blotting signals was achieved with Odyssey Fc Imaging System (LI-COR, Nebraska USA).