Bax siRNA Protects against TNFα-Induced Hair Cell Loss

Abstract

Objective: 1) Understand the role of Bain tumor necrosis factor alpha (TNFα)-induced auditory hair cell (HC) death in rat organ of Corti (OC) explants in vitro 2. Determine the effectiveness of short interfering RNA that targets Bax mRNA (Bax siRNA) against TNFα-induced HC loss.

Method: OC explants dissected from 3-day-old rats were cultured in control media, TNFα, TNFα+BasiRNA, or Bax siRNA for 0, 48, and 96 hours in vitro. Real-time PCR, ELISA, HC viability studies, immunofluorescence, and confocal microscopy were performed. ANOVA with post hoc testing was used with P value set <.05.

Results: Explants exposed to TNFα-only demonstrated significant declines in the number of viable HCs while displaying increased Bax mRNA and protein expression, when compared to control explants. In contrast, the levels of viable HCs, Bax mRNA, and protein expression in explants treated with TNFα+Bax siRNA or Bax siRNA alone approached levels similar to OC explants cultured in control media. Immunolocalization studies demonstrated increased expression of Bax protein in the auditory HCs of the basal turn in TNFα-only treated explants, while OC explants cultured in control media, TNFα+Bax siRNA, or Bax siRNA alone did not display noticeable Bax expression.

Conclusion: TNFα initiates the programmed cell death of auditory HCs in vitro through up regulation of pro-apoptotic Bax gene and protein expression. Bax siRNA blocks TNFα-induced apoptosis of HCs as demonstrated by decreased Bax mRNA and protein expression, which results in increased numbers of viable HCs in the treated OC explants.