Objective To investigate the molecular mechanism by which receptor activator of nuclear factor-κB ligand (RANKL) regulates autophagy
Objective To investigate the molecular mechanism by which receptor activator of nuclear factor-κB ligand (RANKL) regulates autophagy and osteoclast differentiation through the ROS-HIF-1α signaling axis. Methods Mouse monocytic/macrophage RAW264.7 cells were randomly divided into control group, RANKL group, and 3 combined intervention groups, that is, RANKL+CQ group (intervention with autophagy inhibitor, CQ), RANKL+FG-4592 group (intervention with HIF-1α stabilizer, FG-4592), and RANKL+NAC group (intervention with ROS scavenger, NAC). The number of mature multinucleated osteoclasts was quantitatively detected with tartrate-resistant acid phosphatase (TRAP) staining; the protein levels of HIF-1α and LC3 were detected by Western blotting and the mRNA levels of osteoclast differentiation-related genes (TRAP, Cath K and MMP-9) were analyzed by qRT-PCR; autophagy flux and activity were observed by adenovirus mRFP-GFP-LC3 labeling system combined with confocal laser microscopy; and the production of intracellular reactive oxygen species (ROS) was measured by DCFH-DA probe. Results Compared with the control group, the RANKL group obtained significantly increased number of TRAP-positive multinucleated osteoclasts (P
