Quantifying Liver Fibrosis Markers

The Western blotting procedure is similar to our previous reports [17 (link), 23 (link)]. Briefly, the liver tissue from the rat model was lysed in RIPA buffer containing a proteinase inhibitor cocktail (Biocolor BioScience & Technology, Shanghai, China). The protein concentration was determined using bicinchoninic acid (Bioss, Beijing, China). Protein was loaded at 30 μg/gel each lane, separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with rabbit primary antibodies anti-MMP-1 (1:1000, sc-241561) or TIMP-1 (1:1000, sc-5538) (Santa Cruz Biotech, USA) and anti-GAPDH (rabbit monoclonal antibody, 1:1000, Ab181602, Abcam, UK) at 4 °C overnight, then washed extensively with 0.1% Tween-20 in PBS and incubated with a secondary antibody conjugated to horseradish peroxidase (1:5000; sc-2004, Santa Cruz, USA) at room temperature for 3 h. The blot was visualized using the ECL system (Amersham, UK).
The serum levels of Smad7, collagen I, collagen III, laminin and hyaluronic acid were detected using ELISA according to the manufacturer’s instruction. The ELISA kits for Smad7 (# CSB-E09225r) and hyaluronic acid (# CSB-E08120r) were purchased from CUSABIO Technology LLC (Wuhan, China), and the kits for collagenase I (# CX20064), collagenase III (# kt210320) and laminin (# KT20202) were from MSKBIO (Wuhan, China).