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.

Pepsin promotes IL-8 signaling-induced epithelial-mesenchymal transition in laryngeal carcinoma.

BACKGROUND: Laryngopharyngeal reflux (LPR), with its increasing morbidity, is attracting considerable attention. In recent years, the causal role between LPR and laryngeal carcinoma has been debated. The main harmful component of LPR is pepsin, which has been shown to induce mucosal inflammation by damaging the mucous membrane. Thus, pepsin is linked to an increased risk of laryngeal carcinoma, although the potential mechanism remains largely unknown. METHODS: The human laryngeal carcinoma cell lines Hep-2 and Tu212 were exposed to different pepsin concentrations and the morphology, proliferation, migration, secretion of inflammatory cytokines, and epithelial-mesenchymal transition (EMT) of the cells were assessed. To evaluate whether interleukin-8 (IL-8) had a causal relationship with pepsin and EMT, an IL-8 inhibitor was used to suppress IL-8 secretion during pepsin exposure and the expression of EMT markers, cell proliferation, and migration were analyzed. RESULTS: Pepsin promoted proliferation, colony formation, migration, and IL-8 secretion of Hep-2 and Tu212 cells in vitro. Furthermore, increased pepsin concentrations changed the morphology of Hep-2 and Tu212 cells; levels of the epithelial marker E-cadherin were reduced and those of mesenchymal markers vimentin and ß-catenin and the transcription factors snail and slug were elevated. A similar effect was observed in laryngeal carcinoma tissues using immunohistochemistry. IL-8 level was reduced and EMT was restored when pepsin was inhibited by pepstatin. EMT was weakened after exposure to the IL-8 inhibitor, with significant reduction in pepsin-induced cell proliferation and migration. CONCLUSIONS: Pepsin may induce EMT in laryngeal carcinoma through the IL-8 signaling pathway, which indicates that it has potential role in enhancing cell proliferation and metastasis of laryngeal carcinoma.

A Retrospective Study to Compare Five Induction Chemotherapy Regimens Prior to Radiotherapy in the Reduction of Regional Lymph Node Size in Patients with Nasopharyngeal Carcinoma.

BACKGROUND Combined chemotherapy and radiation therapy are used to treat nasopharyngeal carcinoma (NPC). Previous studies have shown that induction chemotherapy, given before radiotherapy, is beneficial in patients with local lymph node metastases. The aim of this study was to evaluate regional lymph node size in patients with NPC and the efficacy of five induction chemotherapy regimens given before radiotherapy. MATERIAL AND METHODS Between December 2007 and June 2011, 190 patients were included in this study, who had regionally advanced NPC (Stages II-IV). Five induction chemotherapy regimens were given prior to radiation: 98 patients (51.6%) received the TPF regimen (docetaxel, cisplatin, and fluorouracil); 56 patients (29.5%) received PF regimen (cisplatin and fluorouracil); 26 patients (13.7%) received the TP regimen (cisplatin and docetaxel); seven patients (3.7%) received combined nimotuzumab with TPF; three patients (1.6%) received a combination of the novel modified recombinant human endostatin (Endostar) with PF. The length and width of the regional lymph nodes were measured using neck B-mode (high-resolution grey scale) ultrasonography before chemotherapy and on the second day following completion of chemotherapy. Gastrointestinal tract and bone marrow suppression were also monitored during and after chemotherapy. RESULTS The TPF chemotherapy induction regimen resulted in an improved early response of lymph node size reduction, compared with the PF and TP chemotherapy induction regimens. The combined use of nimotuzumab with the TPF regimen improved efficacy by 15%. The combined use of Endostar improved the efficacy of the PF regimen by 56% (P<0.05). CONCLUSIONS In a retrospective study in patients with NPC, different induction chemotherapy regimens had different effects on lymph node size before radiation therapy.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1.

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence ß-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Optimized Generation of Primary Human Epithelial Cells from Larynx and Hypopharynx: A Site-Specific Epithelial Model for Reflux Research.

Laryngopharyngeal reflux (LPR) induces a differential damage effect on several anatomic sites within the larynx and hypopharynx; therefore, an in vitro model is needed for each anatomic site. This study aimed to establish a primary culture method for human laryngeal and hypopharyngeal epithelial cells derived from multiple anatomic sites. Surgical mucosa specimens were treated with a two-step enzymatic strategy to establish a primary culture. Of the 46 samples, primary cultivation was achieved successfully with 36 samples, and the positive ratio was 78.3%. In addition, flow cytometry revealed that these primary cells were epithelial cells with a purity of 94.9%. The proliferative ability was confirmed by positive staining for Ki-67. Laryngeal and hypopharyngeal epithelial cells from multiple sites exhibited similar epithelial morphology and positive cytokeratin expression. These cells can be cultured to passage 4. In summary, we successfully established the in vitro epithelial model of larynx and hypopharynx subsites, which may potentially be used as a platform for reflux research, especially for site-specific damage effect.

Exosomal miR-9 inhibits angiogenesis by targeting MDK and regulating PDK/AKT pathway in nasopharyngeal carcinoma.

BACKGROUND: Exosomes are small vesicles containing a wide range of functional proteins, mRNA and miRNA. Exosomal miRNAs from cancer cells play crucial roles in mediating cell-cell communication and tumor-microenvironment cross talk, specifically in enabling metastasis and promoting angiogenesis. We focused on miR-9 that was identified as a tumor suppressor previously in nasopharyngeal carcinoma (NPC) tumorigenesis. METHODS: Differential centrifugation, transmission electron microscopy and nanoparticle tracking analysis were used to isolate and identify exosomes. Quantitative PCR and western blotting analysis were used to detect miR-9, pri-miR-9, CD63, TSG101, MDK, P70S6K P-Ser424 and PDK1 P-Ser241 expression. Laser confocal microscopy was used to trace exosomal miR-9 secreted by NPC cells into HUVECs. The effect of exosomal miR-9 on cell migration and tube formation of HUVECs in vivo and vitro was assessed by using migration assay, tube formation assay and matrigel plug assay, respectively. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of exosomal miR-9 to the 3'untranslated region (3'-UTR) of MDK, while Phosphorylation Array was performed to identify AKT Pathway in HUVECs treated with exosomal miR-9. Furthermore, Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to detected miR-9, CD31 and MDK expression in human NPC tumor samples. RESULTS: NPC cells transfected with miR-9-overexpressing lentivirus, released miR-9 in exosomes. Exosomal miR-9 directly suppressed its target gene - MDK in endothelial cells. Mechanistic analyses revealed that exosomal miR-9 from NPC cells inhibited endothelial tube formation and migration by targeting MDK and regulating PDK/AKT signaling pathway. Additionally, the level of MDK was upregulated in NPC tumor samples and was positively correlated with microvessel density. Notably, the level of exosomal miR-9 was positively correlated with overall survival, and MDK overexpression was positively associated with poor prognosis in NPC patients, suggesting the clinical relevance and prognostic value of exosomal miR-9 and MDK. CONCLUSIONS: Taken together, our data identify an extracellular anti-angiogenic role for tumor-derived, exosome-associated miR-9 in NPC tumorigenesis and prompt further investigation into exosome-based therapies for cancer treatment.

Association between Laryngeal Pepsin Levels and the Presence of Vocal Fold Polyps.

Objective To determine whether pepsin, the main component of refluxed gastric contents, is significantly associated with vocal fold polyps and to evaluate the diagnostic value of pepsin in vocal fold polyps' tissues. Study Design Cross-sectional study. Setting Nanfang Hospital of Southern Medical University. Subjects and Methods The study included 32 patients with vocal fold polyps and 16 healthy controls between 2011 and 2012. Reflux symptom index and reflux finding score assessments, 24-hour combined multichannel intraluminal impedance and pH monitoring, and biopsy of the vocal fold polyp tissues or posterior laryngeal mucosa (healthy controls) for immunohistochemical pepsin staining were performed. Results The expression of pepsin was significantly higher in patients with vocal fold polyps than in controls (28/32, 75% vs 5/16, 31.25%; P < .001). The pepsin levels were significantly positively correlated with upright position pharyngeal acid reflux and esophageal reflux parameters adjusted by age. Based on pepsin staining data, the sensitivity and negative predictive values of 24-hour pH monitoring, the reflux symptom index, and the reflux finding score were 70% to 84.62%, whereas their specificity and positive predictive values were relatively low (20%-31.58%). Conclusion Pepsin reflux may be a risk factor for vocal fold polyps formation. In addition, pepsin immunohistochemical analysis of polyp biopsy samples appears to be a more sensitive and effective test for diagnosing laryngopharyngeal reflux than the reflux symptom index, the reflux finding score, and 24-hour pH monitoring in a clinical setting.