Diosgenin Promotes Bacterial Clearance in Bladder Epithelial Cells by Regulating Rab27b Expression: A Study Based on Virtual Screening and Experimental Validation.

Introduction: The annual rise in antimicrobial resistance (AMR) rates highlights the critical significance of exploring non-antibiotic therapies for infectious diseases. Urinary tract infection (UTI) is one of the most common bacterial infectious diseases, and uropathogenic Escherichia coli (UPEC), its primary pathogenic bacterium, invades bladder epithelial cells (BECs) to survive and proliferate intracellularly, thereby evading the host's immune clearance. Rab27b mediates the expulsion of intracellular bacteria, and upregulating Rab27b expression can effectively reduce the number of intracellular bacteria. Methods: In this study, virtual screening and molecular dynamics simulation were used to screen for compounds capable of stably binding to Rab27b. Surface plasmon resonance (SPR) was subsequently performed to validate the direct binding affinity of diosgenin (DSG) to Rab27b. The CCK-8 assay was adopted to determine the experimental concentrations of the compounds for cellular experiments. Western blot (WB), immunofluorescence (IF), colony counting, siRNA transfection and other assays were used to clarify the effects of the screened compounds on Rab27b expression and the number of intracellular bacteria in BECs. Additionally, IF, hematoxylin-eosin (HE) staining, scanning electron microscopy (SEM), and colony counting were employed to investigate the impacts of DSG on Rab27b expression, epithelial integrity, the number of intracellular bacterial CFU, and inflammatory responses in the bladder of mice with UPEC-induced UTI models. Results: Virtual screening and experimental validation demonstrated that DSG and ST5330591 (ST) could stably bind to Rab27b. DSG upregulated Rab27b protein expression without affecting its mRNA level, suggesting a post-transcriptional mechanism, which contributes to accelerated intracellular bacterial clearance. After Rab27b downregulation via siRNA transfection, DSG lost its ability to clear intracellular bacteria. Furthermore, DSG reduced the number of intracellular Colony-Forming Unit (CFU) in BECs of mice with urinary tract infection, alleviated bladder inflammatory responses, and mitigated the efflux and damage of the bladder epithelial layer. Conclusion: This study demonstrated that DSG directly binds to Rab27b and upregulates its protein expression via a post-transcriptional mechanism (without altering mRNA levels), thereby promoting the clearance of intracellular UPEC in BECs and exerting therapeutic effects on UTI.