Betulinic acid

Spironolactone was purchased from Sigma, USA (CAS50-01-7). DMEM, FBS, streptomycin, penicillin, and 0.25% pancreatic enzyme were all obtained from Gibco, NY, USA. Recombinant rat IL-1β was purchased from R&D Systems (Abingdon, UK), and collagenase II, dimethyl sulfoxide, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid assay kit and radioimmunoprecipitation assay (RIPA) buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Asiatic acid and betulinic acid were purchased from Selleck Chemicals.

Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai,China) provided GLA (CAS No. 79498-31-0, Batch No. B20698, 98% purity). The chemical structure of GLA is illustrated in Fig. 1A. To obtain the original solution (50 mM), we dissolved the GLA in dimethyl sulfoxide and kept it at − 80 °C. Pancreatic enzyme, Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12), radio-immunoprecipitation assay (RIPA) buffer, 4, 6-diamidino-2-phenylindole (DAPI) solution and toluidine blue staining solution were purchased from Beijing Solaybao Technology (Beijing, China). Betulinic acid (Batch No. S360301, CAS No. 472-15-1, 99.88% purity) was purchased from selleck (Shanghai, China). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Boster Biological Technology (Wuhan, China). Primary antibodies against Primary antibodies against (GAPDH), COL2A1, MMP13, SOX9, iNOS, COX-2 were obtained from Abcam (Shanghai, China). Primary antibodies against extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), NF-κB p65, phosphorylated-ERK (p-ERK;Thr202/Tyr204),phosphorylated-JNK (p-JNK;Thr183/Tyr185), phosphorylated-NF-κB p65 (p-P65;Ser536) were from Cell Signaling Technology (Danvers, MA, USA).