Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/ NLRP3/IL-1β 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-inflammatory cytokine levels. Results: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1 beta 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-1 beta 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-1 beta 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.