P ask1

Protein was extracted from homogenized renal tissues in lysis buffer containing a cocktail of proteinase inhibitors (Beyotime, Shanghai, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China). Proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Bedford, MA). After blocking with 5% nonfat milk, the membranes were incubated with indicated primary antibodies against Bax, Bcl-xl, thioredoxin 1 (TRX1), thioredoxin-interacting protein (TXNIP), phospho-P38 MAPK, P38 MAPK (all 1 : 1000 dilution; CST, Danvers, MA), phospho-apoptosis signal-regulating kinase 1 (p-ASK1), or ASK1 (both 1 : 500 dilution; Affinity Biosciences, Changzhou, China) at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000; Bio-Rad, California, USA) for 60 min. The protein bands were visualized using enhanced chemiluminescence (ECL; Bio-Rad, California, USA) reagents and captured with the ChemiDoc system. Intensity of the protein bands was semiquantified and normalized against GAPDH using ImageJ analysis software.

Byakangelicin (purity ≥ 98.5%) was obtained from Push Bio‐technology Co., Ltd. Silibinin (purity > 97%) was obtained from Meilunbio Co., Ltd. Olive oil and carbon tetrachloride were obtained from Shanghai Macklin Biochemical Co., Ltd. byakangelicin was dissolved in dimethylsulphoxide (DMSO) from Sigma‐Aldrich Corporation for cell experiment. Recombinant human PDGF‐BB was obtained from R&D system. Recombinant Human TGF‐β1 was obtained from PeproTtech. Primary antibodies including anti‐α‐smooth muscle actin (α‐SMA), anti‐collagen Ⅰ (COL‐1), anti‐GAPDH and anti‐p‐Stat3, Stat3, β‐tubulin, cyclin D1, P53, p‐ASK‐1, ASK‐1, IL‐1β, NF‐κB and Nrf‐2 were acquired from Affinity Biosciences; anti‐p‐Smad3, Smad3, p‐PDGFR, PDGFR, p‐ERK, ERK, p‐AKT, AKT, PARP, cleaved caspase‐3 and caspase‐3 were received from (Cell Signaling Technology); anti‐p‐JNK, JNK from Signalway Antibody LLC; and anti‐4‐HNE from Abcam. Reagents and antibodies are prepared according to the instructions.