Puerarin rescued Dex-induced Osteoblast Apoptosis via AIF-mediated Caspase-independent pathway

Abstract GIOP is the most common secondary osteoporosis, and the increased apoptosis of osteoblasts is thought to be the main cause of GIOP. Puerarin is the major isoflavone extracted from Pueraria lobata. Our previous study indicated that Puerarin protects osteoblast apoptosis partly through the caspase-dependent pathway. So, we concluded that there existed other anti-apoptotic mechanisms in Puerarin. Therefore, in the current study, we focused on the role of the caspase-independent pathway, specifically the apoptosis-inducing factor (AIF) pathway. MTS assay, flow cytometry, TUNEL staining, Western blotting, and immunofluorescence staining were used in this study. Caspase inhibitor Z-VAD-FMK and RNA silencing (siRNA) AIF were used to assist Puerarin in detecting the anti-apoptosis mechanisms. The results showed that Puerarin significantly alleviated Dex-induced osteoblast cell proliferation inhibition and cell cycle arrest at G0/G1 phase. Puerarin can restore the protein expression of cell cycle-related proteins (cyclin D1) and cyclin-dependent kinase (CDK4). Flow cytometry and TUNEL staining revealed that the pan-caspase inhibitor Z-VAD-FMK and siRNA AIF partially inhibited Dex-induced osteoblast apoptosis, while Puerarin significantly reduced Dex-induced osteoblast cell apoptosis. Immunofluorescence and Western blotting results showed that nucleus translocation of AIF from mitochondria in Dex-treated cells also inhibited by Puerarin. Our results suggest that Puerarin can restore Dex-induced cell cycle inhibition and apoptosis of osteoblasts through the AIF-regulated caspase-independent pathway.