Inhibition of protein disulfide isomerase mitigates steroid-induced osteonecrosis of the femoral head by suppressing osteoclast activity through the reduction of cellular oxidative stress

Abstract

Osteonecrosis of the femoral head (ONFH) is a devastating and irreversible hip disease usually associated with increased oxidative stress due to the clinical application of high-dose or long-term glucocorticoids (GCs). Previous publications have demonstrated protein disulfide isomerase (PDI) plays a critical role in regulating cellular production of reactive oxygen species (ROS). We therefore ask whether interfering PDI could affect GCs-stimulated osteoclastogenesis. To test the hypothesis, we conducted bioinformatics and network analysis based on potential gene targets of steroid-induced osteonecrosis of the femoral head (SIONFH) in light of multiple databases and concomitantly verified the associated biological effect via the in vitro model of dexamethasone (DEX)-stimulated osteoclastogenesis. The results revealed 70 potential gene targets for SIONFH intervention, including the P4HB gene that encodes PDI. Further analysis based on network topology-based analysis techniques (NTA), protein-protein interaction (PPI) networks, and mouse cell atlas database identified the importance of PDI in regulating the cellular redox state of osteoclast during ONFH. Western blotting (WB) validations also indicated that PDI may be a positive regulator in the process of DEX-stimulated osteoclastogenesis. Hence, various PDI inhibitors were subjected to molecular docking with PDI and their performances were analyzed, including 3-Methyltoxoflavin (3 M) which inhibits PDI expression, and ribostamycin sulfate (RS) which represses PDI chaperone activity. The binding energies of DEX, 3 M, and RS to PDI were -5.3547, -4.2324, and -5.9917 kcal/mol, respectively. The Protein-Ligand Interaction Profiler (PLIP) analysis demonstrated that both hydrogen bonds and hydrophobic interactions were the key contributions to the DEX-PDI and 3M-PDI complexes, while only hydrogen bonds were identified as the predominant driving forces in the RS-PDI complex. Subsequent experiments showed that both 3 M and RS reduced osteoclast differentiation and bone resorption activity by stifling the expression of osteoclastic markers. This reduction was primarily due to the PDI inhibitors boosting the antioxidant system, thereby reducing the production of intracellular ROS. In conclusion, our results supported PDI's involvement in SIONFH progression by regulating ROS in osteoclasts and highlighted PDI inhibitors may serve as potential options for SIONFH treatment.

Keywords: Bioinformatics; Osteoclast; Protein disulfide isomerase (PDI); Reactive oxygen species (ROS); Steroid-induced osteonecrosis of the femoral head (SIONFH).