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. 2025 Jan 24;20(1):e0315083. doi: 10.1371/journal.pone.0315083

Weak acid and pepsin reflux induce laryngopharyngeal mucosal barrier injury: A rabbit-model-based study

Chenjie Hou 1,2,3,#, Liqun Zhou 4,#, Yujin Zheng 5,#, Ting Chen 1,2,3,*, Renyou Hu 6, Jingyi Zheng 7, Chaofeng Liu 8, Yaqi Liu 1,2,3
Editor: Miquel Vall-llosera Camps9
PMCID: PMC11759997  PMID: 39854415

Abstract

Objective

Using rabbit models, this study simulated the laryngopharynx’s response to the synergistic effects of various acidic reflux environments and pepsin to investigate the response mechanism underlying weak acid reflux and pepsin in the mucosal barrier injury of laryngopharyngeal reflux.

Methods

The rabbits were divided into six groups, and the original larynx was recorded for each group. During the study period, rabbits were sprayed with different doses of acid and pepsin solutions and monitored for hypopharyngeal mucosal transient impedance before and after modeling. After the experiment, laryngeal mucosal tissues were collected, observed using hematoxylin and eosin staining, and assessed for E-cadherin expression. The width of the intercellular space and lanthanum staining penetrating the intercellular space were also observed using electron microscopy.

Results

Eight weeks post-modeling, evidence of laryngopharyngeal mucosa inflammatory responses was observed in each group. Downregulation of E-cadherin expression significantly positively correlated with acid strength (p < 0.05). The pepsin and acid intervention groups showed a significantly widened space between mucosal epithelial cells in the posterior ring area (p < 0.05). Meanwhile, in the experimental group, a large amount of stained lanthanum penetrated the intercellular spaces; however, no significant difference was observed in the mucosal impedance (MI).

Conclusion

This study demonstrated that acid, weak acid, and pepsin could damage the laryngeal mucosal barrier; pepsin was an independent factor associated with tissue damage; the downregulation of hypopharyngeal cadherin was associated with acid-intensity exposure. Transient LP-MI cannot be applied directly.

1. Introduction

Laryngopharyngeal reflux disease (LPRD) is an inflammatory disease condition resulting from gastroduodenal reflux, leading to morphological changes in the upper airways and digestive tract [1]. LPRD has been linked to the occurrence of various severe laryngeal diseases [2]; however, the pathophysiology of the injury in this disease remains unclear. Weak acids are believed to play significant roles in LPRD [3, 4]. We have previously reported the potential involvement of non-acidic reflux in laryngopharyngeal reflux [5]. In addition, pepsin plays an important role in the pathogenesis of LPRD [6]. Compared with that of the esophagus, the mucosa of the throat lacks a resistance mechanism to gastric acid and, hence, is more susceptible to reflux injury. Acid and pepsin connect perturbation decomposition of the complex barrier function of throat mucosa epithelial permeability and damage.

However, existing research mainly focuses on the damage caused by strong acids to the laryngeal mucosa. Current treatment methods for LPRD primarily target laryngeal reflux induced by strong acids, while the effectiveness against weak acid and non-acid (pepsin) reflux is relatively poor [7, 8]. This difference is related to the distinct mechanisms of injury that weak acids and pepsin exert on the larynx compared to the direct damage caused by strong acids. Therefore, this study aims to investigate the effects of weak acids and pepsin on laryngeal injury. E-cadherin is essential to maintaining epithelial integrity by mediating homogenous adhesion at junctions where epithelial cells attach. In a study involving 18 patients with LPRD, Gill et al. [9] found that E-cadherin expression in the laryngeal mucosal epithelium was significantly reduced, causing the destruction of the related intercellular barrier and the loss of mucosal continuity.

Notably, genetic diversity significantly contributes to the variations of calcium mucin expression and its underlying protection mechanisms. Therefore, diagnosing LPRD solely by pH monitoring may be unreliable, which necessitates novel diagnostic indicators that better reflect mucosal injury.

Mucosal impedance (MI) is determined by the intrinsic conductivity of the mucosa. In In vitro animal experiments [10], mucosal impedance can reflect the status and integrity of the mucosa, offering a new diagnostic index for reflux pharyngitis. This study simulated the laryngopharynx’s response to the synergistic effects of various acidic reflux environments and pepsin to investigate the response mechanism underlying weak acid reflux and pepsin in the mucosal barrier injury of laryngopharyngeal reflux.

2. Materials and methods

The study was approved by the Animal Ethics Committee of Fujian Medical University (No.: FJMU IACUC).

2.1. Animal

Thirty healthy male New Zealand albino rabbits with corporal mass between 2.5 and 3.5 kg were used in this study. To induce laryngitis, the rabbits received intramuscular anesthesia with diazepam (3 mg/kg; Xudong Haipu Pharma Co., Ltd., Shanghai, China), ketamine (40 mg/kg; Gutian Pharma Co., Ltd., Fujian, China), and Su-Mian-Xin (4 mg/kg; Shengda Pharma Co., Jilin, China). A special catheter (Fig 1) was inserted through the nasopharynx until it reached the hypopharynx. The external tip was sutured to the nasal lateral cartilage.

Fig 1. Schematic diagram of the catheter.

Fig 1

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The rabbits were randomly assigned into six groups. During the 8-week study, each group was treated with pH 3, 5, 7, and normal saline solution as the matrix (Table 1), with or without pepsin at 1 mL/kg for 3 min of slow spraying, twice per day, through the special catheter.

Table 1. Groups of experimental animals by intervention.

Acid (pH = 3) Weak acid (pH = 5) Non-acid
With pepsin Experiment Group 1: Acid + Pepsin (n = 4) Experiment Group 2: Weak acid + Pepsin (n = 5) Experiment Group 3: Normal saline + Pepsin (n = 5)
Without pepsin Experiment Group 4: Acid (n = 5) Experiment Group 5: Weak acid (n = 5) Control group: Normal saline (n = 6)
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All rabbits underwent endoscopic observation with a 3-mm endoscope (70° for nose, STORZ, Germany) and LP-MI detection with a transient Impedance-pH Reflux Monitoring Systems (Chongqing Jinshan Science & Technology (Group) Co., Ltd.) immediately and 8 weeks after intubation. At the end of the intubation period (8 weeks), the rabbits were euthanized, and the larynx and pharynx were removed for morphological and histological analysis.

3. Experimental method

3.1 Morphological evaluation: Laryngeal morphology assessment under laryngoscope

Following intramuscular anesthesia, a laryngoscopy was conducted. Since assessment of normal values for rabbits is lacking, we referred to the methods of Hu et al. and Lou et al. [11, 12], using a standard reflux finding score (RFS) system described by Belafsky et al. [13] to analyze the images. The RFS was calculated by two independent observers.

3.2 Instantaneous impedance monitoring of hypopharyngeal mucosa

After being anesthetized and fixed, the hypopharynx of all rabbits was monitored using a transient MI measurement system (Chongqing Kingsoft Technology Co., Ltd., China) (Fig 2) before and after catheter placement and removal. Data from experimental animals in a calm breathing state were recorded for 5 min.

Fig 2. Schematic diagram of impedance measurement.

Fig 2

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3.3 Histopathology

After 8 weeks, the rabbits were sacrificed using air embolism. The mucosal tissue on the inner side of the arytenoid area (non-catheterized side) was pruned into small tissue blocks of approximately 1 × 1 × 1 mm3 on an ice tray and examined using electron microscopy to calculate after lanthanum nitrate staining, dilated intercellular spaces (DIS), hematoxylin and eosin staining, and immunohistochemical examination.

3.4. Statistical analysis

Stata MP 13 statistical software package was employed for analyses. Normally distributed data are shown as mean ± standard deviation, whereas abnormally distributed data as median (interquartile range). Parametric differences were compared using the t-test, while non-parametric differences using the Kruskal—Wallis test. The correlation was compared using Spearman correlation analysis. p value < 0.05 was considered statistically significant.

4. Results

4.1. General information

No significant difference was identified in body weight between each group before and 4 and 8 weeks after modeling (p > 0.05).

4.2. Morphological evaluation under laryngoscope

The vocal cords of New Zealand rabbits are composed of non-keratinized laminated squamous epithelium. Before modeling, no significant differences were observed in the total RFS scores of each group (p > 0.05).

The total RFS scores were higher at 8 weeks post-modeling than before modeling. The mean score of the pepsin exposure group was significantly higher than that of the non-pepsin exposure group (Fig 3a). According to pH groups, the average score was positively correlated with acid exposure, and the difference between groups was significant (Fig 3b).

Fig 3. Results of RFS.

Fig 3

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(A)Grouped by pepsin. (B) Grouped by pH.

Significant differences were observed among the scores of false vocal fold sulcus, vocal fold edema, and diffuse laryngeal edema, regardless of whether in the pepsin or acid exposure group, and in the scores of laryngeal ventricular disappearance and posterior combined hyperplasia among the groups with different pH values.

4.3. Hypopharyngeal transient MI monitoring analysis

The implanted probe missed the arytenoid area. The mean MI values before and 8 weeks after modeling showed no significant differences (1,903.03 ± 132.23 and 2,050.83 ± 175.93, respectively) (p = 0.50). Moreover, no significant difference was found among the MIs of the groups (p = 0.52). However, the mucus in the larynx significantly reduced the impedance results.

5. Transmission electron microscope evaluation

5.1 Lanthanum dyeing observation

Transmission electron microscopy revealed that the space between the mucosal epithelial cells in the posterior ring area was significantly widened in all experimental groups, and a large amount of stained lanthanum passed through the intercellular space. The acid + pepsin group showed necrosis and lanthanum staining. In the saline control group, osmic acid staining did not, or very rarely, pass through the intercellular space; no cell necrosis was identified (Fig 4).

Fig 4. Transmission electron microscope lanthanum staining diagram.

Fig 4

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(A)Negative: lanthanum staining did not penetrate the barrier (control group). (B)Lanthanum staining penetrated the cellular barrier (experimental group). (C)Cell necrosis, lanthanum penetrated the cell (only in acid + pepsin experimental group).

5.2 Intercellular space measurement

The mean intercellular space of the pepsin exposure group was significantly higher than that of the non-pepsin exposure group (DIS) (Fig 5a). In the pH groups, the intercellular space was positively correlated with acid exposure, and the difference between the groups was significant (Figs 5b and 6).

Fig 5. Comparison of the DIS results.

Fig 5

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(A)Grouped by pepsin. (B) Grouped by pH.

Fig 6. Observation of transmission electron microscopy (conventional staining).

Fig 6

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(A) Apoptotic vacuoles with lysosomes. (B) Cell edema c. Expansion of intercellular space d. Cells are closely connected.

6. Inflammatory observation

Under a light microscope, unlike the normal saline control group, the experimental groups showed partial necrosis and shedding of the epithelial layer of the posterior ring area, infiltration of lymphocytes and plasma cells in the lamina propria, rupture of the muscular layer, hyperplasia and hypertrophy of the submucosal glands, vasodilatation, and congestion (Fig 7).

Fig 7. Degree of inflammation (white blood cell count diagram).

Fig 7

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(A)Grouped by pepsin. (B) Grouped by pH.

The inflammatory response was positively correlated with the exposure intensity, whether in the pepsin or acid group, and the differences between the groups were significant (p < 0.05).

7. E-cadherin expression

Spectrophotometry after immunohistochemical staining showed that the downregulation of E-cadherin was positively correlated with the acid exposure intensity (Fig 8). There was no significant difference when assessing pepsin exposure (Fig 9a). Data from each group were tested using the Shapiro—Wilk test; the p-values were > 0.05, indicating a normal distribution. Significant differences were observed among the three groups (Fig 9b).

Fig 8. Intensity grading of E-cadherin immunohistochemical staining using spectrophotometry.

Fig 8

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(A) Level 0 (B) Level 1 (C) Level 2 (D) Level 3.

Fig 9. Results of E-CAD staining compared by spectrophotometry.

Fig 9

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(A)Grouped by pepsin. (B)Grouped by pH.

8. Discussion

The study of weak acid, specifically non-acid, on laryngopharyngeal damage has attracted attention. In a prospective study of 99 patients, Palareti [14] found that glottic edema was positively and significantly associated with the number of non-acid LPR and non-acid esophageal reflux events. Laryngeal compartment disappearance and posterior junction hyperplasia significantly correlated with non-acidic and total reflux exposure times. A significant correlation was also found between granuloma/granulation score and the number of acidic LPR events.

In a study of 349 patients, Li et al. [15] found that non-acid reflux events play a considerable role in LPRD; non-acid is a potential risk factor for laryngeal inflammation, consistent with the results of this study. A considerable number of patients do not respond well to antacid therapy, which may be related to the poor effect of drugs on weak acid and non-acid reflux.

Existing studies suggest that pepsin is a primary component of laryngopharyngeal reflux injury, which induces mucosal inflammation by damaging the mucosa and is a specific and sensitive biomarker of LPR. Pepsin can be found in the laryngeal mucus or laryngeal mucosal cells of patients with reflux and is involved in the pathogenesis of vocal cord polyps, vocal cord leukoplakia, and laryngeal cancer [16].

Inactive but stable pepsin (pH 7 in non-acidic reflux) can be actively absorbed by laryngeal epithelial cells in a receptor-mediated manner via acid-independent endocytosis [17]. Pepsin can also be retained and reactivated in some intracellular vesicles [18], leading to intracellular damage and changes in pro-inflammatory cytokine gene expression. Johnston et al. [19] confirmed that endorsed pepsin causes mitochondrial damage and induces the expression of some stress and toxin-related genes. This suggests the involvement of pepsin in the signs and symptoms of weak acid and non-acid reflux. Samuels et al. [20] further investigated cell damage caused by pepsin and found that the gene expression profile of pro-inflammatory cytokines induced by endocytosed non-acidic pepsin was similar to that of severe gastroesophageal reflux disease (GERD).

Our study found that pepsin exposure independently resulted in significant laryngeal changes and severe inflammatory cell infiltration. In this study, the endocytosis of pepsin, independent of gastric acid, explains the observation of cell barrier destruction and morphological changes under laryngoscopy, even in the non-acid group (water + pepsin group). At the same time, our study established the animal model of weak acid and non-acid reflux for the first time and realized the effect of pepsin on the throat in the animal model without acid.

The downregulation of E-cadherin has been observed in the laryngeal mucosa of patients with LPRD [21], which causes laryngopharyngeal mucosa injury and discomfort. In our study, E-cadherin downregulation was also observed, and the degree of downregulation was negatively correlated with pH. E-cadherin, also known as epithelial cadherin or CD324, is a calcium-dependent cell adhesion molecule. Decreased E-cadherin expression is a marker of epithelial-mesenchymal transition and associated with an increased risk of cancer metastasis. The expression level of E-cadherin is correlated with the degree of tumor differentiation of head and neck squamous carcinoma and negatively correlated with the metastasis of head and neck carcinoma and patient prognosis [22]. A higher incidence of laryngeal and hypopharyngeal cancers has been reported in patients with laryngopharyngeal reflux; therefore, its relation to the decrease or loss of E-cadherin expression caused by reflux warrants further studies.

Samuels et al. [23] have demonstrated that exposure to pepsin only for a short duration could activate cancer-related signaling pathways in laryngeal cells. Li et al. [24] found that acidizing pepsin promoted metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis by reducing the activity of mitochondrial respiratory complex I and enhanced the growth and migration ability of vocal cord white spot epithelial cells. Active treatment of LPRD aims not only to improve the quality of life and relieve discomfort but also prevent and reduce the occurrence of hypopharyngeal and laryngeal cancer.

Simultaneously, the acid + pepsin group had the most serious mucosal damage, suggesting the expansion of the intercellular space, mucosal barrier damage, and apoptosis, consistent with previous findings.

Furthermore, obvious staining of lanthanum through the mucosal barrier in the weak acid and non-acid + pepsin experimental groups was also observed in this study, indicating that acid and non-acid reflux can induce expansion of the intercellular space and mucosal damage. The synergistic effect of pepsin cannot be ignored.

The esophageal mucosa of patients with GERD has ultrastructural changes, such as DIS [16]. The widening of the intercellular space is an early morphological marker of tissue damage in patients with GERD. Caviglia et al. [25] compared the intercellular spaces in the esophageal epithelia of patients with non-erosive esophagitis with those of normal individuals and concluded that a widened intercellular space is characteristic of patients with non-erosive esophagitis and can be used as an objective indicator of changes in the barrier structure.

Ravelli et al. [26] identified acid exposure as a major factor leading to space dilatation in the esophageal epithelium, and that the use of proton pump inhibitors reduces DIS, suggesting DIS as a feature of GERD. What’s more, laryngopharyngeal reflux has some similarities to GERD etiology.

Li et al. [27] used transmission electron microscopy to show that with the onset of erosive esophagitis, mucosal thickness increased, desmosomes significantly decreased, and the space between epithelial cells widened. However, this measurement is difficult to apply in clinical practice since it depends on a mucosal biopsy under an electron microscope. The breakdown of the mucosal barrier is stable during the disease state and is not susceptible to interference from other factors [10]. When no gas or fluid passes through, the lumen of the esophagus collapses, and the esophageal mucosa contacts the metal ring of the catheter, resulting in MI, which is determined by the inherent electrical conductivity of the esophageal mucosa. Therefore, MI may be a novel, reliable, highly sensitive, and specific diagnostic index for reflux pharyngitis and can reflect the integrity of the esophageal mucosa. A lower MI value indicates more severe damage to the esophageal mucosal integrity, which is negatively correlated with DIS. However, LPRD affects pharyngeal mucosal epithelial space enlargement.

In LPRD, this study found that DIS was proportional to the acid intensity. In addition, the stability of the laryngeal instantaneous MI was poor, and no significant difference between the two groups before and after modeling was observed. Possible reasons are as follows: laryngeal and esophageal anatomies differ, the detection mode of laryngeal MI cannot be directly applied to the detection mode of esophageal MI, and its placement and detection method requires further studies and optimization. However, several factors strongly affect MI detection. For example, we found that with more secretions in the larynx, MI could be significantly reduced. Breathing and swallowing can also affect MI measurements. Therefore, in future studies, the timing of MI testing should be optimized. For example, patients could gargle to reduce the secretion of the pharyngeal cavity and avoid swallowing to improve the test accuracy.

9. Conclusion

This study found that acid, weak acid, and pepsin could damage the laryngeal mucosal barrier and identified pepsin as an independent factor that may cause damage, as manifested by the downregulation of hypopharyngeal cadherin and enlargement of DIS. Downregulation of hypopharyngeal cadherin is related to acid-intensity exposure. However, transient throat MI cannot be directly applied unless the influencing factors are removed.

Supporting information

S1 Dataset

(XLSX)

pone.0315083.s001.xlsx (13.4KB, xlsx)

Acknowledgments

We would like to express our gratitude to Editage (www.editage.cn) for English language editing services. We also thank Dr. Linying Zhou, Dr. Minxia Wu, and Dr. Xi Lin from the Electron Microscopy Lab at the Public Technology Service Center, Fujian Medical University, for their generous technical assistance with electron microscopy.

Data Availability

The laboratory protocols is deposited in protocols.io (DOI: dx.doi.org/10.17504/protocols.io.eq2lywy8rvx9/v1). All relevant data are within the manuscript and its Supporting information files.

Funding Statement

All three of the funders — Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (Grant no. 2022ZQNZD001); Health youth research project of Fujian Province (Grant no. 2022QNH006) and Leading Project Science and Technology Innovation Joint Fund of Fujian Province (Grant no. 2023Y9351) — had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Mahmoud Kandeel

25 Jun 2024

PONE-D-24-20203weak acid and pepsin reflux  induced laryngopharyngeal mucosal barrier injury:a study  based on rabbit modelPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors,

I have carefully reviewed your manuscript titled "Response of the Laryngopharynx to Acidic Reflux and Pepsin in Rabbit Models" and find it to be a promising and insightful study presenting novel findings on the effects of pepsin on laryngopharyngeal reflux. However, I believe the manuscript would benefit significantly from the following considerations:

1- Enhancement of English Punctuation and Style:

It would be advantageous to improve the punctuation and overall writing style to enhance readability and ensure the manuscript meets the highest standards of academic writing.

Grammar and Style:

A thorough proofreading to address minor grammatical errors and improve the overall readability of the manuscript.

2- E-Cadherin Expression:

- The expression of E-cadherin alone may not be a definitive indicator of cancer progression. Downregulation of this marker could also result from the wound healing process, considering the tissue damage duration.

The images provided for grading and scoring E-cadherin expression are of low resolution and should be improved. Furthermore, the images depicting high expression levels show a concerning degree of background staining.

- The significance of E-cadherin expression depends on both the intensity and localization of the staining within the tissue, particularly at cell-cell contacts. The image showing the lowest expression still indicates presence at cell-cell contacts, suggesting functional E-cadherin. Therefore, the assertion that this expression is downregulated and indicative of cancer progression is not entirely substantiated by the current images. I recommend including more images and additional markers to provide a clearer and more comprehensive analysis.

3- Consider evaluating additional markers related to cancer development or gene mutations, as they could provide more conclusive evidence.

4- Inflammatory Markers:

The inclusion of inflammatory markers and corresponding tissue images would serve as strong evidence of an inflammatory response. Also, addition of images of the tissue showing inflammation, tissue damage or any other findings would be beneficial.

5- Effect of Catheter and Standardization:

The potential impact of the catheter on the results should be addressed, and it is crucial to standardize the methodology to ensure that procedural variations do not influence the outcomes. Otherwise, providing explanation of the measures taken to reduce variability in results would provide more clarity.

6- Treatment Dosage:

The manuscript mentions that the tissue was sprayed with the test solution, but it lacks details regarding the exact volume used and whether the dosage was standardized across subjects. Providing this information is essential to avoid variability in treatment dosage.

7- Expand on Pepsin's Role:

Elaborate on the potential mechanisms of pepsin-induced damage in non-acidic conditions and its clinical implications.

8- Improve Figure Legends:

Ensure that all figure legends are detailed and fully explain the data presented, enhancing the reader's understanding.

While your research is promising and offers valuable insights, addressing these points will significantly enhance the manuscript's clarity, robustness, and overall impact. I encourage you to incorporate these suggestions to provide a more comprehensive and rigorous presentation of your findings.

Best regards,

Reviewer #2: I would like to thank the authors for their effort in this study. There are many serious concerns regarding this manuscript as highlighted below:

Title

I do not see what is special about this topic, “Weak acid and pepsin reflux induced laryngopharyngeal mucosal barrier injury: a study based on rabbit model Study LPRD on rabbit model.”

1. Weak acid is generally believed to be gastric acid. Why are the authors avoiding writing the proper term? They should be specific! The weak acid could be anything.

2. It is established knowledge that LPRD causes reflux. What is so special about this study? The significance of the study is not properly documented.

3. The mention of “Study LPRD on rabbit model” is unnecessary.

Abstract

The method is not detailed! Sprayed with different concentrations of acid— which acid? Hydrochloric or sulfuric?

The tissues were observed with Hematoxylin and eosin. For what? What was the observer looking for? This is not informative at all!

The conclusion part is not understood and rather confuses the readers, especially the statement “This study found that acid, weak acid, and pepsin…”

Introduction

Why is mentioning weak acid instead of gastric acid important in this context?

The two statements here, “Compared with the mucosa of the esophagus, the mucosa of the throat lacks a resistance mechanism to gastric acid and is more susceptible to reflux injury. Acid and pepsin disrupt barrier function and epithelial permeability by perturbing junctional complexes,” lack references.

The statement “Gill et al. [6] studied 18 patients diagnosed with LPRD and found that the expression of E-cadherin in the laryngeal mucosal epithelium was significantly reduced, and pepsin appeared” is not understood with the ending words “pepsin appeared.”

The following sentence should have been broken into two for clarity.

The statement “Notably, as a result of genetic diversity, individual expression of calcium mucin protection mechanisms must be differences” is poorly written and not scientifically constructed.

The sentence “Therefore, the existence of throat mucosa damage is defined, and the diagnosis of LPRD is unreliable” is not clear. Who defined it, or what does the author mean here?

The next statement, “Therefore, we may need to look for objective diagnostic indicators that can reflect mucosal injury,” is poorly written. Also, the knowledge gap here is different from what was stated in the abstract: “In this study, rabbit models were used to simulate the response of the laryngopharynx under the synergistic effects of various acidic reflux environments and pepsin to reveal the response mechanism of weak acid reflux…”

Therefore, the authors sound inconsistent with their objectives!

Methodology

The methodology was poorly described. For instance, the statement “The animals were randomly assigned into six groups (five animals per group). During the 8-week study, the rabbits in each group were treated with pH 3, 5, 7, and normal saline solution as the matrix, adding or not adding pepsin at 1 mL/kg for 3 min of slow spraying, twice per day, through the special catheter” is unclear.

Which group received what? This was poorly described. No one understands what was done to the animals and how each group was treated.

Also, the phrase in this statement “each group were treated with pH 3, 5, 7” is ambiguous and confusing.

In the statement “After intramuscular anesthesia, the animals received laryngoscopy. Because there was no previous assessment of normal values for rabbits, we referenced the method reported by Zhang et al. and Lou et al.[8,9] using a standard RFS system described by Belafsky et al.[10] to analyze images,” what does this mean? The author did not provide a detailed explanation of what those references mean. Are they not referring to rabbits? Are they pointing to abnormal values?

Also, the authors just jumped to “What is instantaneous impedance?” They failed to provide some important background information in the introductory section. They just jumped to the methodology without detailed information. Overall, the authors sound non-communicating and unscientific in their writing form.

The statement “After 8 weeks, the animals were sacrificed by air embolism…” Why air embolism? What does this suggest? There was no detailed explanation of how this impacts the results.

Also, what does lanthanum nitrate do? What is DIS? What do the immunostainers reveal, and what are those immunomarkers under the section “2.3 HISTOPATHOLOGY”? The information provided is below expectations without any details.

And so on….

Overall, the manuscript lacks clarity, proper documentation, and scientific rigor in its writing. The authors need to provide detailed explanations, proper references, and clear descriptions of their methodology and findings to make the study comprehensible and valuable.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Arwa Flemban

Reviewer #2: Yes: Kamoru Adedokun

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Attachment

Submitted filename: REVIEWERS RESPONSE.pdf

pone.0315083.s002.pdf (100.6KB, pdf)
PLoS One. 2025 Jan 24;20(1):e0315083. doi: 10.1371/journal.pone.0315083.r002

Author response to Decision Letter 0

22 Aug 2024

Response to Reviewers

Reviewer #1

Dear Dr. Arwa Flemban,

Thank you very much for your detailed and constructive feedback on our manuscript entitled " Weak acid and pepsin reflux induce laryngopharyngeal mucosal barrier injury: a rabbit-model-based study". We appreciate the time and effort you have dedicated to evaluating our work, here is my response.

1- Enhancement of English Punctuation and Style:

The manuscript has been revised by the professional editing service: Editage (www.edita ge.cn).

2- E-Cadherin Expression:

Regarding E-Cadherin expression, we have updated the images with higher resolution and included references to E-Cadherin and cancer-related progression in the discussion section.

3- Consider evaluating additional markers related to cancer development or gene mutations, as they could provide more conclusive evidence.

We have also supplemented the discussion with relevant literature evidence on the link between inflammation and carcinogenesis. Due to the limited availability of rabbit-related antibodies, we are planning to search for more suitable markers for our future research.

4- Inflammatory Markers:

Added discussion on the inflammatory response section.

5- Effect of Catheter and Standardization:

To ensure the reliability of our experimental results, we used a blank control group (normal saline group) to account for any influence of the catheter. Additionally, we selected non-catheterized tissues to minimize any potential effects.

6- Treatment Dosage:

In our study, we applied different concentrations of solvents to treat 1 mL/kg of rabbit throat for 3 minutes using slow spraying.

7- Expand on Pepsin's Role:

We have discussed the mechanism of pepsin on cell damage and its potential impact on the development of laryngeal carcinoma.

8- Improve Figure Legends:

we have made modifications to part of the legend.

Best regards,

Reviewer #2

Dear Dr. Kamoru Adedokun,

Thank you for your insightful comments and suggestions on our manuscript titled " Weak acid and pepsin reflux induce laryngopharyngeal mucosal barrier injury: a rabbit-model-based study". We appreciate the time and effort you have invested in providing feedback. Below is my response.

1. Title

This study focuses on examining the effect of weak acid and pepsin on the laryngeal injury. Existing research has focused more on damage to the throat mucosa caused by strong acids. PPI treatment for laryngeal reflux effects the reflux caused by strong acid, while the treatment effect of weak acid and acid-free (pepsin) reflux is poor, which is related to the difference between the mechanism of damage caused by weak acid and pepsin to the throat and the mechanism of direct damage caused by strong acid. Therefore, this study focused on the effects of weak acid and pepsin on laryngeal injury. Common animal models include pigs, dogs, rabbits, and rats. The vocal cord structure of rabbits is the same as that of humans, and rabbits are silent animals, which can avoid the influence of vocal cord movement injury on the results. Therefore, we selected rabbits for modeling.

2. Abstract

Since the main component of stomach acid is hydrochloric acid, to simulate stomach acid, hydrochloric acid was used in this study. Hematoxylin and eosin were used to observe the tissue inflammatory response. The expression of some sentences was modified.

3. Introduction

Revised the wording in this section and added additional references.

4. Methodology

Since there is no specific rabbit laryngoscopy scoring standard, we also used the human laryngoscopy scoring standard previously reported (Belafsky et al.). Air embolization is necessary for rabbit tissue sampling and does not affect the experimental results. Lanthanum nitrate staining is one of the special staining methods for electron microscopy. When the mucosal barrier is intact, lanthanum nitrate does not penetrate the intercellular space; however, if the mucosal barrier is damaged, lanthanum staining can penetrate the intercellular space and inside the cell when the cell dies. DIS is the expansion of the cell gap; that is, when the mucosal barrier is normally intact, the cell connection is tight; however when broken, the cell gap can be observed under the electron microscope. Finally, the expression of E-Cadherin in cells was observed using immunohistochemical staining of arytenoid mucosa. Some background information regarding instantaneous impedance was added to the revised. And new references have been added.

Best regards,

Attachment

Submitted filename: Response to Reviewers.docx

pone.0315083.s003.docx (15.1KB, docx)

Decision Letter 1

Miquel Vall-llosera Camps

11 Sep 2024

PONE-D-24-20203R1Weak acid and pepsin reflux induce laryngopharyngeal mucosal barrier injury: a rabbit-model-based studyPLOS ONE

Dear Dr. Chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 25 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Miquel Vall-llosera Camps

Senior Staff Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Author,

I am writing to provide feedback on your manuscript, "[Manuscript Title]". I have carefully reviewed the original manuscript and the amendments you have provided.

I am pleased to note that many of the original comments have been addressed satisfactorily in the revised manuscript. The improvements you have made have significantly enhanced the clarity and overall quality of the work.

However, I would like to express some lingering concerns regarding the images of the E-cadherin staining. Based on my experience, E-cadherin, as an active molecule, is typically localized at the cell-cell adhesion points. While the images provided demonstrate two distinct intensity levels of the marker, neither image clearly shows staining at the cell membrane. The staining appears more like background noise.

I would recommend repeating the E-cadherin staining experiment with a different antibody or considering an alternative marker to assess EMT status or cellular epithelial integrity. Here are some suggestions for potential markers:

Occludin: A tight junction protein that is often downregulated during EMT.

ZO-1: Another tight junction protein that can be used to assess epithelial integrity.

Vimentin: An intermediate filament protein that is upregulated during EMT.

I believe that addressing these concerns will further strengthen the manuscript and provide a more compelling representation of your findings.

Thank you for your time and consideration.

Sincerely,

Reviewer #2: Thank you for explaining the concept. However, I find it concerning that your response to critiques, as well as those from other investigators I have come across in similar roles, often shows more clarity and strength than the original document. I notice that your revised explanation demonstrates greater energy and efficiency.

Could you incorporate this idea into your response to Reviewer 2, particularly regarding what your study contributes to existing knowledge on weak acids and why the treatment is important? You briefly touched on this in your response, but I believe this key information should have been highlighted in the abstract or introduction to provide a stronger foundation for the study.

It's essential for investigators to present their best insights upfront to engage and motivate readers.

Secondly, I would encourage you to pay close attention to capitalization in your title. It’s important to handle these details yourself, rather than relying on the journal office to perfect what should be done correctly from the start, especially after acceptance.

Good luck!

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Arwa Flemban

Reviewer #2: Yes: Kamoru A. Adedokun

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2025 Jan 24;20(1):e0315083. doi: 10.1371/journal.pone.0315083.r004

Author response to Decision Letter 1

10 Oct 2024

Response to Reviewers

Subject: Response to Reviewer #1’s Comments on Manuscript "Weak Acid and Pepsin Reflux Induce Laryngopharyngeal Mucosal Barrier Injury: A Rabbit-Model-Based Study"

Dear Dr. Flemban,

Thank you very much for your thoughtful suggestions and constructive feedback on our manuscript, "Weak Acid and Pepsin Reflux Induce Laryngopharyngeal Mucosal Barrier Injury: A Rabbit-Model-Based Study." We truly appreciate the time and effort you have dedicated to reviewing our work.

In response to your insightful comments regarding the E-cadherin staining images, we have chosen to upload new images that we believe more accurately reflect our experimental results.

We greatly value your recommendations on utilizing markers such as Occludin, ZO-1, and Vimentin in our future studies. E-cadherin, as you pointed out, is a specific adhesion molecule for epithelial cells, and its downregulation indeed signifies a loss of epithelial characteristics, thereby providing a clear indication of EMT occurrence. We also acknowledge that Occludin, ZO-1, and Vimentin can offer complementary insights that would enhance our understanding of the EMT process. Unfortunately, due to time constraints, we were unable to conduct additional experiments at this stage. We are committed to considering your suggestions in our ongoing research.

Thank you once again for your invaluable input and guidance.

Warm regards,

Ting Chen

Subject: Response to Reviewer #2’s Comments on Manuscript "Weak Acid and Pepsin Reflux Induce Laryngopharyngeal Mucosal Barrier Injury: A Rabbit-Model-Based Study"

Dear Dr. Adedokun,

Thank you very much for your valuable feedback and constructive suggestions regarding our manuscript, "Weak Acid and Pepsin Reflux Induce Laryngopharyngeal Mucosal Barrier Injury: A Rabbit-Model-Based Study."

We appreciate your observation about the clarity and strength of our response compared to the original document. In light of your feedback, we have revised the introduction to more clearly highlight the contribution of our study to the existing knowledge on weak acids and their implications for treatment. We have incorporated additional information and relevant references to provide a stronger foundation and underscore the importance of our findings.

Regarding the title capitalization, we have ensured that it adheres to the proper formatting guidelines. We understand the importance of these details and have taken care to address them appropriately in the revised manuscript.

Thank you once again for your insightful comments, which have significantly contributed to improving our manuscript.

Sincerely,

Ting Chen

Attachment

Submitted filename: Response to Reviewers.docx

pone.0315083.s004.docx (16.2KB, docx)

Decision Letter 2

Miquel Vall-llosera Camps

21 Nov 2024

Weak acid and pepsin reflux induce laryngopharyngeal mucosal barrier injury: a rabbit-model-based study

PONE-D-24-20203R2

Dear Dr. Chen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Miquel Vall-llosera Camps

Senior Staff Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I have no other comments. I think the reviewers have satisfied with the critiques. Best of luck.

#########################################

#########################################

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Kamoru Adedokun

**********

Acceptance letter

Miquel Vall-llosera Camps

25 Nov 2024

PONE-D-24-20203R2

PLOS ONE

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PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Dataset

    (XLSX)

    pone.0315083.s001.xlsx (13.4KB, xlsx)
    Attachment

    Submitted filename: REVIEWERS RESPONSE.pdf

    pone.0315083.s002.pdf (100.6KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pone.0315083.s003.docx (15.1KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pone.0315083.s004.docx (16.2KB, docx)

    Data Availability Statement

    The laboratory protocols is deposited in protocols.io (DOI: dx.doi.org/10.17504/protocols.io.eq2lywy8rvx9/v1). All relevant data are within the manuscript and its Supporting information files.

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