Galangin Modulates Neuroinflammation Pathways

Complete Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) was replaced with serum-free DMEM medium to starve the BV-2 cells for 4 h. Cells were pretreated with various concentrations of galangin for 1 h, followed by treatment with LPS (1 μg/mL) for different times. After the last behavioral test, the SNs of the rats were rapidly dissected out, frozen, and stored in a deep freezer at −80 °C until the assays were performed. The BV-2 cells and the rat SNs were lysed with lysis buffer for 30 min (Beyotime Inst. Biotech, Beijing, China). After centrifugation at 12,000× g at 4 °C, the supernatant protein was quantified with a bicinchoninic acid protein assay kit (Beyotime Inst. Biotech, Beijing, China). The detailed process is referenced in our previous work [24 (link)]. The source of the commercially available antibodies used for the western blot were OX-42 (1:1000), TH (1:1000), COX-2 (1:1000), iNOS (1:2000) (Abcam, Cambridge, CA, USA), phosphor-NF-κB p65 (1:1000), NF-κB p65 (1:1000), phosphor-AKT (1:2000), AKT (1:2000), phosphor-p38 (1:2000), p38 (1:1000), phosphor-ERK1/2 (1:2000), ERK1/2 (1:2000), phosphor-JNK1/2 (1:1000), JNK1/2 (1:2000), (Cell Signaling Technology, Danvers, MA, USA), and β-tubulin (1:2000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).