Modulation of Pepsin‐Mediated Inflammatory Responses in Vocal Cord Epithelial Cells by Amprenavir

ABSTRACT Objectives Pepsin is a key driver of epithelial injury and inflammation in laryngopharyngeal reflux (LPR). Prior work in esophageal cells implicates hypoxia‐inducible factor‐2α (HIF‐2α) in refluxate‐mediated cytokine secretion. We investigated whether pepsin increases HIF‐2α under neutral (pH 7) conditions in immortalized human true vocal cord (TVC) cells and whether amprenavir, a pepsin inhibitor, can reverse this response. Methods TVCs were treated with 0.1–1 mg/mL pepsin and/or 10 μM amprenavir at pH 7 for 2, 4, 6, and 24 h. HIF‐2α protein levels were evaluated by Western blot. Based on the most inflammatory condition, expression of proinflammatory genes was assessed with or without a HIF‐2α transcriptional inhibitor. Results The 6‐h 1 mg/mL pepsin treatment induced the highest HIF‐2α levels and was selected for further analysis. Pepsin significantly increased HIF‐2α expression ( p < 0.01), and amprenavir reduced it numerically ( p = 0.056). Pepsin significantly upregulated ICAM1 , IL1B , and IL8 ( p < 0.05), while amprenavir significantly reduced ICAM1 , IL8 , and TNF ( p < 0.05). Inhibition of HIF‐2α selectively reduced IL1B expression ( p < 0.05). Conclusion Pepsin exposure increased HIF‐2α and upregulated key inflammatory genes. While amprenavir significantly reduced cytokine expression, HIF‐2α inhibition alone had only a modest effect, suggesting additional pathways contribute to pepsin‐induced inflammation. Amprenavir's ability to attenuate this response supports its therapeutic potential in LPR. Level of Evidence N/A.