Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1ß signaling pathway.

BACKGROUND: Laryngopharyngeal reflux (LPR) is one of the most common disorders in otorhinolaryngology, affecting up to 10% of outpatients visiting otolaryngology departments. In addition, 50% of hoarseness cases are related to LPR. Pepsin reflux-induced aseptic inflammation is a major trigger of LPR; however, the underlying mechanisms are unclear. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has become an important bridge between stimulation and sterile inflammation and is activated by intracellular reactive oxygen species (ROS) in response to danger signals, leading to an inflammatory cascade. In this study, we aimed to determine whether pepsin causes LPR-associated inflammatory injury via mediating inflammasome activation and explore the potential mechanism. METHODS: We evaluated NLRP3 inflammasome expression and ROS in the laryngeal mucosa using immunofluorescence and immunohistochemistry. Laryngeal epithelial cells were exposed to pepsin and analyzed using flow cytometry, western blotting, and real-time quantitative PCR to determine ROS, NLRP3, and pro-inflammatorycytokine levels. RESULTS: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1ß levels in laryngeal tissues. Intracellular ROS levels were elevated by increased pepsin concentrations, which were attenuated by apocynin (APO)-a ROS inhibitor-in vitro. Furthermore, pepsin significantly induced the mRNA and protein expression of thioredoxin-interacting protein, NLRP3, caspase-1, and IL-1ß in a dose-dependent manner. APO and the NLRP3 inhibitor, MCC950, inhibited NLRP3 inflammasome formation and suppressed laryngeal epithelial cell damage. CONCLUSION: Our findings verified that pepsin could regulate the NLRP3/IL-1ß signaling pathway through ROS activation and further induce inflammatory injury in LPR. Targeting the ROS/NLRP3 inflammasome signaling pathway may help treat patients with LPR disease.

Association of pepsin and DNA damage in laryngopharyngeal reflux-related vocal fold polyps.

PURPOSE: This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR. METHODS: Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously. Hematoxylin-eosin staining was performed for pathologic analysis. Immunohistochemistry and ELISA were used to detect pepsin in tissue and saliva of VFP patients. 8-OHdG and p-H2AX expression was detected to measure oxidative DNA damage in tissue. DNA damage was investigated in human immortalized laryngeal epithelial cells exposed to pepsin. RESULTS: The pepsin concentration in saliva was significantly higher (t = 2.38, P = .024) in the pepsin positive group. There was no significant difference in pepsin expression at different sites and pathological subtypes of VFPs. The levels of 8-OHdG and p-H2AX were significantly higher in the pepsin positive group and positively correlated with the tissue expression of pepsin. The concentration of pepsin in saliva also showed a significant correlation with 8-OHdG levels. Expression of 8-OHdG and p-H2AX, and tail moment of the comet assay were elevated in human immortalized laryngeal epithelial cells following treatment with pepsin. CONCLUSION: Patients with VFPs have higher levels of oxidative DNA damage in the presence of pepsin reflux. Pepsin may induce DNA damage in laryngeal epithelial cells and participate in the pathogenesis of VFPs.