P iκbα

RPMI-1640, fetal bovine serum (FBS), trypsin, 12-O-tetradecanoylphorbol-13-acetate (TPA), penicillin G, and streptomycin were purchased from Fisher Scientific (Waltham, MA, USA). Cleaved caspase-3, p-IκBα, p-p38, p-NF-κBp65, p-ERK, p-JNK, ERK, JNK, p38, IκBα, NF-κBp65, Bax and β-actin antibodies were obtained from Santa Cruz Biotechnology (Paso Robles, CA, USA). Anti-rabbit/mouse IgG secondary antibodies were obtained from Abcam (Cam-bridge, MA, USA). N-acetylcysteine (NAC) was obtained from Tocris (Minneapolis, MN, USA). Bay 11-7082, and SP600125 were purchased from Med Chem Expresss (Monmouth Junction, NJ, USA). Kaempferol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and had a purity of over 98%.

Kidney homogenate and podocytes were collected and lysed in cell lysis buffer (Beyotime, Haimen, China) with protease inhibitor cocktail and phosphatase inhibitor (both from Sigma-Aldrich) for protein extraction. Equal amount of protein lysates (30 μg) were separated by 10% serum dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electrotransferred onto nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% non-fat dry milk in PBS for 1 h, the membranes were probed with the primary antibodies against TLR4, phosphorylated-p65 (p-p65), p65, p-IκBα, IκBα, Cleaved Caspase-3, Bcl-2 and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, followed by incubated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. Peroxidase-labeled protein bands were detected by enhanced chemiluminescence reagents (Millipore) and the protein intensity was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Carboxymethyl Pachymaran was a gift from Hunan Butian Pharmaceutical Co., Ltd. (Huaihua, China)country; Fluorouracil Injection was provided by Shanghai XudongHaipu Pharmaceutical Co., Ltd. (Shanghai, China); alanine aminotransferase (ALT) assay kitand aspartate aminotransferase (AST) assay kit were purchased from Beijing Leadman Biochemistry Co., Ltd. (Beijing, China); Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione and catalase (GSH-Px), and reactive oxygen species (ROS) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); ELISA kits specific for interleukin -1β (IL-1β), interleukin-6 (IL-6) and interferon-γ (IFN-γ) were purchased from the Huamei Institute of Biotechnology (Wuhan, China); β-actin, NF-κB, p-NF-κB, IκB-α, p-IκB-α, p-p38, p-JNK, GCL, Bax, Bcl-2, Keap1, Nrf2, p38, and HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies were purchased from Cowin Biotech (Beijing, China).