Figure 6.

Puerarin promoted neurite outgrowth via PR signaling. (a) siRNA-mediated silencing of PR in PC12 cells. PC12 cells were transiently silenced by PR siRNAs and negative control siRNAs. The expression of PR was determined by Western blot analysis with anti-PR antibody. Representative blots were shown. (b) Visualization of neurite outgrowth. After siRNA transfection, PC12 cells were treated with puerarin and NGF for 72 h. Neurite outgrowth was determined by a commercial neurite staining kit and analyzed under a fluorescence microscope from Carl Zeiss (Jena, Germany). The representative images were shown. Scale bar, 50 μm. (c) Quantification of neurite outgrowth. Neurites from three nonoverlapping fields for each slice were recorded and analyzed. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (d) Luciferase assay for promoter activity. PC12 cells were transiently transfected with 2xPRE-TK-Luc and pRL-TK and cultured for 24 h. The cells were subsequently treated with puerarin and progesterone for 24 h. The promoter activity was assayed with the Dual-Luciferase Reporter System under a Clariostar microplate reader from BMG Labtech (Ortenberg, Germany). Results were expressed as luciferase activity and normalized to Renilla activity for three independent experiments. ^∗∗p < 0.01, ^∗∗∗p < 0.001. (e) Molecular docking of puerarin and progesterone. Puerarin and progesterone were docked into PR structure (PDB: 3D90) by AutoDock Vina. (f) Potential mechanism. Puerarin may interact with PR and activate the transcriptional factor activity of PR to induce neuroprotective genes and neurotrophic factors.