Collagen 1 col 1

Paeonol was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against to ANP, BNP, α-MHC, collagen I (Col I), connective tissue growth factor (CTGF), fibronectin-1, AKT, p-AKT, ERK1/2, p-ERK1/2, JNK, p-JNK and β-actin were purchased from Cell Signaling Technology (Boston, MA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6, TNF-α and ANP were purchased from R&D Systems Inc. (Minneapolis, MN).

The RIPA lysis solution (Beyotime, Beijing, China) was utilized to extract tissue and cell proteins, whereas the concentration of the protein was found through the bicinchoninic acid (BCA, Ricky, Shanghai, China). After separation by SDS-PAGE electrophoresis (Aowei, Shanghai, China), transfer to PVDF membrane, then block with 5% skim milk, add primary antibody HIF-1α, VEGF, collagen-I (Col-I), and GAPDH (1 : 1000, Cell Signaling Technology, MA, USA), incubate overnight at 4°C, and incubate the secondary antibody (CST) at 37°C for 1 h. Use the ECL Plus (Fluorescence, Beijing, China) to develop color and the gel imaging system (Image J) to scan and analyze the protein bands.