This study was approved by the Institutional Review Board of The First Affiliated Hospital, College of Medicine, Zhejiang University, China. All authors had access to the study data and reviewed and approved the final manuscript. The informed consent was obtained for experimentation with human subjects. The animals’ care and use involving experiments were in accordance with institution guidelines for the care and use of laboratory animals.
Patients and patient samples
Specimens (n=31) were acquired from normal laryngeal mucosa far from the negative margin during open partial or total laryngectomy for tumor excision or microsurgery for benign laryngeal lesions. Operations were performed in the Department of Otolaryngology of The First Affiliated Hospital, College of Medicine, Zhejiang University between October 2018 and May 2019. Specimens were frozen immediately in liquid nitrogen following surgical removal and stored until further analysis. Among the 31 patients, 28 were males, and 3 were females, with a mean age of 58.1 (range, 32–81) years. Exclusion criteria were as follows: 1) preoperative radiotherapy, chemotherapy, or immunotherapy, 2) treatment with PPIs, and 3) underlying metabolic or autoimmune diseases.
All patients voluntarily received 24 h combined multichannel intraluminal impedance and pH monitoring (MMS Ohmega Ambulatory Impedance and pH Recorder, the Netherlands preoperatively (after 4 h of fasting) and completed the self-administered reflux symptom index (RSI; Chinese version, 2015), which contains nine items. The maximum RSI was 45, with an RSI >13 considered suggestive of LPR. All patients underwent laryngoscopic examination and evaluation for the reflux finding score (RFS). The RFS is based on eight components (maximum score, 26), and an RFS >7 was considered suggestive of LPR. Abnormal gastroesophageal reflux was defined as a DeMeester score of ≥14.7 and syndrome association probability of ≥95% (gastroesophageal acid reflux) or syndrome association probability ≥95% (abnormal non-acid gastroesophageal reflux).
Seven-week-old male C57BL/6 mice (20–25 g) were purchased from Hubei Provincial Center for Disease Control and Prevention. Mice were housed in a specific-pathogen-free room with a controlled temperature (20 ± 2°C). The LPR animal model was established as described previously [19,20]. All mice were maintained under a 12 h light/dark cycle with free access to standard rodent chow and water. After 1 week of acclimation, the mice were randomly divided into three groups: control (treated with PBS), HCl (pH=3) treatment, and acidified pepsin (pH=3) treatment. HCl and acidified pepsin were administered by esophageal acid perfusion. Briefly, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (Sigma, P3761). Subsequently, 0.15 mL HCl (1 M, pH=3) with or without 2.5 mg/g pepsin (SINOPHARM, 7647-01-0) was perfused (8 drops/min) into the mid- and lower esophagus using a feeding tube. Drug perfusion was performed twice daily for 45 consecutive days. Control mice were perfused with PBS instead of HCl. After treatment, throat specimens were obtained and washed with 0.9% saline solution. The specimens were stored at −80°C until further analysis.
Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)
Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After sectioning (5 μM thick), the tissues were deparaffinized and rehydrated. Antigen retrieval was performed by incubating the sections at high pressure. After blocking with 3% hydrogen peroxide, all sections were incubated with normal non-immune serum for 10 min at room temperature. Subsequently, slides were incubated overnight with antibodies against gastric H+/K+-ATPase α subunit (D031-3, Clone 1H9; MBL International Corp., Woburn, MA, USA; 1:285 dilution), gastric H+/K+-ATPase β subunit (D032-3, Clone 1B6; MBL International Corp.; 1:285 dilution), polyclonal pepsin (PAA632Hu01, Cloud-Clone Corp.; 1:100 dilution), and Ki67 (Abcam, ab15580, 1:200 dilution), followed by incubation with the appropriate biotin-labeled secondary antibody. After adding the chromogen diaminobenzidine, sections were stained with H&E and dehydrated. After mounting in neutral balsam, the slides were observed under a microscope. Regions of interest were analyzed using Image-Pro Plus 6.0 software. The sections were evaluated by two experienced pathologists (YHT and YWW), who were blinded to the study treatments. Cell staining was scored as negative (0–10%), weakly positive (10–25%), moderately positive (26–50%), or strongly positive (50–100%).
For H&E staining, 5-μM-thick sections were deparaffinized, rehydrated, and stained with hematoxylin (Sigma, H9627) for 5 min. After plating in HCl alcohol for 10 s, the sections were stained with 0.5% eosin for 1 min, incubated with ammonium hydroxide for 10 s, washed under running water, and mounted in neutral balsam. Experienced pathologists evaluated the histology.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Apoptosis in the throat tissues of mice was assessed using the In Situ Cell Death Detection Kit-POD (Roche, Shanghai, China). Briefly, 5-μM-thick sections were deparaffinized, rehydrated, and incubated with proteinase for 25 min. After a 20-min incubation in cell-penetrating solution, sections were incubated in TUNEL reaction mixture at 37°C for 2 h in the dark. Subsequently, the sections were stained with 4,’6-diamidino-2-phenylindole (DAPI), mounted in mounting medium, and observed under a fluorescence microscope.
Isolation oflaryngeal epithelial cells
Primary laryngeal epithelial cells were isolated from C57BL/6 mice. Briefly, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (Sigma, P3761). Laryngeal mucosal specimens were obtained under a dissecting microscope and washed with Hanks’ solution
on ice. After cutting into 1 mm3 blocks, the specimens were treated with 10 mg/mL dispase II (Sigma-Aldrich, St. Louis, MO, USA) for 48 h at 4°C. The cell pellet was enzymatically dissociated in 0.05% Trypsin/EDTA at 37°C for 2–3 min and resuspended in 2 mL RPMI-1640 (SH30809.01; HyClone, USA) medium. The medium was replaced every 3 days, and the cells were expanded upon reaching 90% confluence.
Generation of H+/K+-ATPase-α-knockout (KO) cells and treatment
To establish an H+/K+-ATPase-α-subunit-KO cell line using CRISPR, we cloned a guide RNA (gRNA) targeting the H+/K+-ATPase α subunit (designed and synthesized by RIO Biotech) into the pUC57-T7-gRNA plasmid. The gRNA was amplified (forward, CACCGTATCAGACCAGCGCCACCA; reverse, AAACTGGTGGCGCTGGTCTGATAC), and the target and control gRNAs were cloned into the BsaI sites of the pUC57-T7-gRNA plasmid. Plasmids were transfected into laryngeal epithelial cells using Lipofectamine 2000 reagent (Invitrogen). After selecting for the transfected (puromycin-resistant) cells and confirming the knockout efficiency by western blotting, cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone, Waltham, MA, USA) at 37°C in a 5% CO2 humidified atmosphere. KO cells were treated with acidic medium (pH=3 or 5) or normal medium (pH=7) with or without pepsin for 1 h, followed by a 24 h incubation in fresh RPMI-1640. Pepsin was inactivated using 10 M NaOH (pH=8) at 37°C for 30 min, and the pH of the medium was adjusted to 7 using 1 M HCl. To induce autophagy and inhibit proton pumps, we also treated cells with 20 μM chloroquine (CQ) and 10 μg/mL pantoprazole, respectively.
Cells were seeded in 96-well plates (5×105/well) and treated with acidic medium containing pepsin and CQ/pantoprazole for 24 h. Subsequently, 20 µL cell counting solution (Beyotime Biological Technology Co. Ltd, C0037) was added, and the cells were incubated in the dark for an additional 4 h. The optical absorbance at 450 nm was measured using the Spectra Plus microplate reader (Multiskan MK3, Thermo).
For cell cycle analysis, we fixed cells in 700 µL ice-cold 80% ethanol at 4°C for at least 4 h. After incubating with 10 μL RNase (1 mg/mL) and 10 μL propidium iodide (APOAF, Sigma) in the dark, the cells were analyzed by flow cytometry. For apoptosis measurement, we incubated the cells in 500 µL Annexin V Binding Buffer. After adding 5 μL Annexin V–FITC and 5 μL propidium iodide, the cells were incubated for 10 min at room temperature in the dark. The percentage of apoptotic cells was determined by flow cytometry. To measure mitochondrial membrane potential (MMP), we incubated the cells in 1000 μL JC-1 solution (Beyotime Biological Technology) for 20 min at 37°C. After centrifugation, the cells were resuspended in 200 μL 10× incubation buffer and analyzed by flow cytometry. All flow cytometry data were analyzed using ModFit LT software (Becton Dickinson, Mountain View, CA, USA).
Total protein was extracted using RIPA lysis buffer. Equal amounts of protein (30 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (IPVH00010; Millipore Co., Boston, MA, USA). Subsequently, the membranes were incubated with primary antibodies against caspase-3 (ab214430, Abcam; 1:1000), LC3 (ab48394, Abcam; 1:1000), p62 (ab109012, Abcam; 1:1000), H+/K+-ATPase α subunit (D031-3, MBL International Corp.; 1:1000), and H+/K+-ATPase β subunit (D032-3, MBL International Corp.; 1:1000) at 4°C overnight. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab181602, Abcam) served as a loading control. After incubating with the appropriate secondary antibodies for 1 h, the signal was developed using an enhanced chemiluminescence assay kit (Beyotime Biological Technology) and visualized using the ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).
Immunofluorescence (IF) assay
LC3 and H+/K+-ATPase (α subunit) expression was assessed by IF. Briefly, cells were grown on glass slides for 24 h, fixed in 3% paraformaldehyde for 30 min at 4°C, and neutralized with 50 mM NH4Cl. After permeabilizing with 0.1% Triton X-100 for 15 min and washing with PBS, cells were incubated with antibodies against H+/K+-ATPase (α subunit) and LC3 at room temperature for 1 h, followed by incubation with the appropriate secondary antibody for 1 h and then with DAPI for 5 min. Stained cells were visualized under the LSM5 EXCITER laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
GFP-RFP-LC3 expression was also assessed by IF. Cells were treated with pepsin, CQ, and pantoprazole, followed by a 24 h incubation with adenoviruses carrying GFP-RFP-LC3. After fixing with 4% paraformaldehyde, LC3 expression was assessed by confocal laser scanning microscopy (LSM 800; Carl Zeiss). For analysis of mitophagy, cells were seeded on glass slides and, after the appropriate treatments, incubated with 100 nmol/L mitophagy dye for 30 min at 37°C. Fluorescence was measured by confocal laser scanning microscopy.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the TRIzol reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. After reverse transcription using the first-strand cDNA synthesis kit (Transgene, China), qRT-PCR was performed using SYBR green (Roche). The primer sequences used for PCR were as follows: mouse H+/K+-ATPase α subunit, 5′-GGAGATGGAGATTAACGACCACC-3′ (forward) and 5′-ACGGGCAAACTTCACATACTC-3′ (reverse); H+/K+-ATPase β subunit, 5′-TTCCGGCACTACTGTTGGAAC-3′ (forward) and 5′-TGACCACATAGAAACCTGCGTA-3′ (reverse); GAPDH, 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′- GGGGTCGTTGATGGCAACA-3′ (reverse). Target gene expression levels were normalized to GAPDH mRNA levels, and relative expression was calculated using the 2–∆∆Ct method.
Transmission electron microscopy
Cells grown on glass slides were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and gradually dehydrated in ethanol and acetone. Subsequently, the cells were embedded in epoxy resin and stained with uranyl acetate and lead citrate. Autophagy was assessed by transmission electron microscopy (HITACHI, HT7700-SS, Japan).
All experiments were independently performed at least three times. Data are expressed as means ± standard error. Statistical analyses were performed using SPSS 25.0 software (IBM Corp., Armonk, NY, USA) or GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). IHC and IF images were analyzed using Image-Pro Plus 6.0 software. Statistical significance was determined using one-way analysis of variance (ANOVA) or Student’s t-test. P-values <0.05 were considered statistically significant.