Am J Otolaryngol 41 (2020) 102681

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Am J Otolaryngol
journal homepage: www.elsevier.com/locate/amjoto

Association of pepsin and DNA damage in laryngopharyngeal reflux-related T

vocal fold polyps
Yuan-Feng Dai1, Jia-Jie Tan1, Chao-Qun Deng, Xiong Liu, Ze-Hong Lv, Xiang-Ping Li
⁎

Department of Otolaryngology – Head and Neck Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

ARTICLE INFO ABSTRACT

Keywords: Purpose: This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold
Vocal fold polyp polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR.
Laryngopharyngeal reflux Methods: Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was
Pepsin performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously.
Oxidative DNA damage
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.

1. Introduction
Hoarseness is the most common clinical symptom of voice disease,
which damages patients' communication ability and reduces their
quality of life [1]. Vocal fold polyp (VFP) is one of the most common
voice diseases associated with hoarseness. [2]. In recent years, lar-
yngopharyngeal reflux (LPR) has received more attention in academic
circles as a risk factor for VFPs [3]. A recent study suggested that vocal
cord mucosa may be more vulnerable in the presence of LPR, which
reduces some of the protective mechanisms of mucosa (carbonic an-
hydrase III, mucin, and E-cadherin) [4–7].
Reflux of gastric contents causing a series of throat, voice and even
respiratory diseases are referred to as LPR [8]. Approximately 10% of
clinic patients in otolaryngology have LPR-related symptoms, and up to
55% of patients with hoarseness have reflux in their throat [9]. Beltsis
et al. [10] reported that 72.2% of VFP patients had significantly higher
level of laryngeal reflux (LPR); we further [11] discovered that 75% of
VFP patients had mild to moderate pepsin expression (a biomarker of
LPR [12]). Cumulative studies suggest LPR as a risk factor for VFP, and
pepsin to be the key mediator. However, the underlying pathogenesis
and pathophysiological mechanisms remain unknown.
It has been gradually recognized that VFPs may result from time-
dependent accumulation of damaged cells and biomacromolecules ag-
gravated by persistent stimulation of LPR [13], in which chronic in-
flammation plays a part. Oxidative DNA damage is closely associated
with a variety of chronic inflammatory diseases (such as cardiovascular
disease [14], chronic obstructive pulmonary disease [15], etc.), and
plays prerequisite roles in gastroesophageal reflux disease pathogenesis
[16–18]. However, the role of oxidative damage in the pathogenesis of
VFPs—in the presence of LPR—remains unclear.
Therefore, in this study, the detection of pepsin levels in polyp tis-
sues was first performed to evaluate LPR. The levels of p-H2AX and 8-

Abbreviations: LPR, laryngopharyngeal reflux; VFPs, vocal fold polyps

⁎
Corresponding author at: Department of Otolaryngology – Head and Neck Surgery, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue
North, Guangzhou 510515, China.
E-mail address: li321162@qq.com (X.-P. Li).
1
Contributed equally to this work.

https://doi.org/10.1016/j.amjoto.2020.102681
Received 21 May 2020
Available online 13 August 2020
0196-0709/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y.-F. Dai, et al. Am J Otolaryngol 41 (2020) 102681

Table 1 Table 2
Pepsin IHC in the tissue of VFP patients.⁎ Clinical features/ pathological classification of the subjects.
Pepsin IHC levels Clinical features/pathological classification Number of cases

− + ++ +++ Total Unilateral/bilateral

Unilateral polyps 14(47%)
5(16.67%) 17(56.67%) 8(26.67%) 0(0%) 30(100%) Bilateral polyps 16(53%)
Anatomic site
⁎
IHC, Immunohistochemistry. The anterior commissure 7(23%)
The edge of the vocal cord 23(77%)
Pathological changes
OHdG in polyp tissues were also detected to assess the level of DNA
Edematous polyps 18(60%)
oxidative damage, further analyzing the relationship between DNA Vascular polyps 8(26.7%)
damage and LPR-related VFPs. In addition, cytological experiments Fibrous polyps 4(13.3%)
were conducted using immortalized laryngeal epithelial cells to in-
vestigate if pepsin causes DNA damage in laryngeal epithelial cells,
further exploring the potential role of LPR in the pathophysiology of cultured in serum-free keratinocyte growth media KGM™-2 (LONZA,
VFPs. Basel, Switzerland).

2. Materials and methods 2.3. Histological analysis

2.1. Subjects
In this study, 30 patients (15 males and 15 females, average age of
35.13 years [95% confidence interval, 30.46–39.80 years]) with VFPs
were recruited from the Department of Otolaryngology of Nanfang
Hospital, China between 2017 and 2018. Patients with acute or chronic
infection of the upper respiratory tract, history of pharyngeal surgery,
malignant tumor or diabetes were excluded [11]. This study was ap-
proved by the Ethics Committee of Nanfang Hospital (NO. NFEC-
201607-K2–01). Informed consent was signed by each participant prior
to the start of any procedure.
2.2. Cell culture
The human immortalized ventricle Bmi1 cell line (hV-Bmi1) and the
human immortalized subglottic Bmi1 cell line (hSG-Bmi1) induced with
Bmi-1 were established as described previously [19]. They were
Specimens were paraffin-embedded and made into continuous sec-
tions for histological analysis. Hematoxylin-Eosin (HE) staining was
evaluated and subjects were divided into four groups according to
histological features: Edematous polyps, vascular polyps, and fibrous
polyps.
2.4. Immunohistochemistry (IHC)
After being dewaxed and rehydrated, tissue sections were incubated
in 0.05 M (pH 6.0) citrate buffer at high temperature and high pressure
(150 Kpa, 100 °C) for 5 min to retrieve the antigen. Thereafter, sections
were stained with primary antibodies against pepsin (1:400, USCN
LIFE, Texas, USA), 8-OHdG (1:200, BIOSS, Beijing, China) and p-H2AX
(1:500, Cell Signaling Technology, Beverly, MA, USA) at 4 °C for 16 h.
Sequential sections from the same tissue blocks were stained at least
twice by the same procedure. Sections were evaluated by two in-
vestigators blinded to this study and scored as follows: The proportion

Fig. 1. Immunohistochemical analysis of pepsin (400×). (A) positive control: (gastric mucosa) intracytoplasmic brown granules in chief cells. (B) Negative control
(phosphate-buffered saline (PBS) replacing primary antibody): no brown intracytoplasmic granules in most cells. (C) Moderately positive sample in pepsin-positive
group: light brown colour in the cytoplasm of laryngeal squamous epithelial cells interstitially. (D) Weakly positive sample in the pepsin-negative group: yellowish
brown in the cytoplasm of a few epithelial cells. (E) Negative sample in the pepsin-negative group: no brown staining. (F) Salivary pepsin levels: pepsin-positive
group had a higher level of pepsin in saliva than did the pepsin-negative group. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

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Fig. 2. Clinical features and pathological classifications of vocal fold polyps (VFPs): (A) Unilateral polyp. (B) Bilateral polyp. (C) Polyp in anterior commissure. (D)
polyp at the edge of vocal fold. (E) Edematous type: lamina propria with edema, inflammation cell infusion (Hematein-Eosin (HE) staining, ×400). (F) Vascular type:
vessel proliferation, dilatation thrombosis, lamina propria with red cells and a few inflammation cells (HE staining, ×400). (G) Fibrous type: lamina propria with
fibrous and a few inflammation cells (HE staining, ×400). (H) Tissue expression of pepsin in unilateral polyp and bilateral polyp groups. (I) Tissue expression of
pepsin in polyps that occurred in different positions (anterior commissure, or the edge of vocal cord). (J) Tissue expression of pepsin in polyps with different
pathological changes (edematous, vascular or fibrous polyps). (K) Salivary pepsin levels in unilateral or bilateral polyps. (L) Salivary pepsin levels of polyps occurred
in different positions (anterior commissure, or the edge of vocal cord). (M) Salivary pepsin levels in polyps with different pathological changes (edematous, vascular
or fibrous polyps). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

score of positive cells: < 1% stained cells, 0; 2–25%, 1; 26–50%, 2;
51–75%, 3; > 75%, 4; intensity score of staining: no staining, 0; di-
luted, 1; moderate, 2; strong, 3 points. The gross score was calculated
by multiplying the proportion score and intensity score, graduated as
negative (0–1), weak (1–4), medium (5–8) and strong positive (9–12)
[20].
2.5. Enzyme-linked immunosorbent assay (ELISA)
Pepsin concentration in saliva was detected by a rigorous proce-
dure. Sputum (≥3.0 mL) in the hypopharynx was collected with a deep
cough from all participants and stored in liquid nitrogen until detection.
The assay was performed by an investigator blinded to the study. All
specimens were rewarmed and attenuated with deionized water prior to

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Table 3 2.7. Comet assay

Pepsin expression in different types of VFPs.⁎
Pepsin IHC t P The comet assay was performed to determine DNA damage induced
by pepsin. In brief, both hV-Bmi1 and hSG-Bmi1 cells were treated with
− + ++ pepsin continuously for 6 h each day for 5 d, isolated by enzymatic
digestion, and mixed with (low-melting) agarose on agarose-covered
Unilateral/bilateral
Unilateral polyps (n = 14) 3 10 1 −0.93 0.36 frosted slides at 4 °C. After 1 h, slides were incubated in lysis buffer
Bilateral polyps (n = 16) 5 7 4 (10 mM Tris, pH 10, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10%
Anatomic site DMSO) for 1 h, and separation of fragmented DNA was performed by
The anterior commissure (n = 7) 3 3 1 −0.86 0.40 gel electrophoresis (25 V; 300 mA, 20 min) in alkaline conditions.
The edge of vocal cord (n = 23) 5 14 4
Following neutralization (400 mM Tris buffer, pH 7.5), the tail moment
Pathological changes
Edematous polyps (n = 18) 5 11 2 0.873 0.43 was calculated for 50 cells from each of the two duplicate slides using
Vascular polyps (n = 8) 1 5 2 TriTek CometScore™ software. DNA damage was quantitated by mea-
Fibrous polyps (n = 4) 2 1 1 suring the tail moment, product of the tail length and fraction of total
DNA [21].
⁎
IHC, Immunohistochemistry.

detection. Thereafter, specimens were centrifuged at 2500g for 20 min
at 4 °C (Eppendorf, AG, Germany). The supernatants were assayed using
an ELISA kit (USCN LIFE, Houston, TX, USA) and a pepsin standard
curve fitted by a dose-response curve was used to calculate the con-
centration of pepsin in units of ng/mL.
2.6. Immunocytochemical analysis
Both hV-Bmi1 and hSG-Bmi1 cells were treated with different con-
centrations of pepsin (0 mg/mL, 0.1 mg/mL, or 1 mg/mL) at a pH 7 for
6 h/d for 5 d. Thereafter, cells were fixed in 10% formaldehyde and
cytospun onto microscopic slides. Fixed cells were hydrated with
varying concentrations of alcohol and phosphate-buffered saline (PBS),
incubated with the primary antibodies against 8-OHdG (1:200, BIOSS,
Beijing, China) overnight at 4 °C, and detected using HRP-conjugated
secondary antibody. Cells were stained with hematoxylin and imaged
using an inverted microscope (Olympus, Beijing, China).
2.8. Western blot analysis
The hV-Bmi1 and hSG-Bmi1 cells were collected after pepsin
treatment using RIPA buffer. Lysates were separated on 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
subsequently transferred to polyvinylidene difluoride (PVDF) mem-
branes (Millipore, Burlington, MA, USA). Membranes were incubated
with primary p-H2AX antibody (1:1000, Cell Signaling Technology)
overnight at 4 °C, and analyzed by western blotting.
2.9. Statistical analysis
Percentages are used to describe qualitative variables, whereas
mean values (maximum and minimum) are used to describe continuous
variables. Mann-Whitney U test was used for discontinuous variables
and Student's t-test was used for continuous variables. Spearman's
correlation coefficient was used to analyze the correlation between the
pepsin positive group and negative group. All statistical analyses were
performed using SPSS version 19.0 (SPSS, Inc., IBM, Armonk, NY, USA).
A P value < .05 was considered statistically significant. A sufficient
number of cases should be enrolled to ensure the statistical power

Fig. 3. Immunohistochemical analysis of 8-OHdG (400×). (A) Positive control (laryngeal carcinoma): sepia nucleus in most cells. (B) Negative control (PBS replacing
primary antibody). (C) Strong positive sample in 8-OHdG positive group: sepia nucleus in parts of cells. (D) Moderately positive sample in 8-OHdG positive group:
light brown nuclei in parts of cells. (E) Weakly positive sample in 8-OHdG-negative group: yellowish brown nuclei in a few epithelial cells. (F) Negative sample in 8-
OHdG-negative group: no brown staining observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

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Fig. 4. Immunohistochemical analysis of p-H2AX (400×). (A) Positive control (laryngeal carcinoma): sepia nucleus in most cells. (B) Negative control (PBS replacing
primary antibody). (C) Strong positive sample in p-H2AX-positive group: sepia nucleus in cells. (D) Moderately positive sample in p-H2AX-positive group: light
brown nuclei in parts of cells. (E) Weakly positive sample in p-H2AX-negative group: yellowish brown nuclei in a few epithelial cells. (F) Negative sample in p-H2AX-
negative group: no brown staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 4 (3.94 ± 0.93 ng/mL) (Fig. 1F).

Expression of 8-OHdG in pepsin positive group and pepsin negative group
tissue.
3.2. Clinical features and pathological classification of VFPs
8-OHdG IHC Z P

− + ++ +++ As shown in Table 2, the subjects were further grouped according to

clinical and pathological characteristics, such as unilateral polyps and
Pepsin positive group 1 6 10 5 −2.358 0.018⁎ bilateral polyps (Fig. 2A and B); the lesions located in the anterior
(n = 22)
commissure or in the edge of the vocal cord (Fig. 2C and D); edematous
Pepsin negative group 4 2 1 1
(n = 8) polyps (manifested as edema in the mesenchyme, with moderate in-
flammatory cell infiltration), vascular polyps (manifested as angiogen-
IHC, Immunohistochemistry; P < 0.05. esis, dilatation and occasional thrombosis, with invasion of some red
⁎

blood cells and a large number of inflammatory cells in the me-

Table 5 senchyme) and fibrous polyps (manifested as fibrinoid degeneration
Expression of P-H2AX in pepsin positive group and pepsin negative group tissue. and slight inflammatory cell infiltration in the mesenchyme) (Figs. 2E-
P-H2AX IHC Z P G).

− + ++ +++
3.3. Pepsin expression in different types of VFPs
Pepsin positive group 1 4 14 3 −2.873 0.004⁎
(n = 22)
Pepsin negative group 4 2 2 0
Tissue expression of pepsin in different clinical classifications of
(n = 8) VFPs is described in Table 3. There was no significant difference in
tissue expression of pepsin between unilateral and bilateral polyps
⁎
IHC, Immunohistochemistry; P < .05. (t = −0.93, P = .36, Fig. 2H). There was also no significant difference
in pepsin level in saliva between the unilateral group (5.97 ± 4.88 ng/
(1 − β) > 0.75. mL) and bilateral group (8.11 ± 7.91 ng/mL) (t = −0.87, P = .39,
Fig. 2K). By analyzing the different locations of polyps, we found that
3. Results there was no significant difference in tissue expression of pepsin be-
tween the anterior commissure group and the group at the edge of the
3.1. Pepsin IHC in patients with VFPs vocal cord (t = −0.86, P = .40, Fig. 2I). There was also no significant
difference in the pepsin level of saliva between the anterior commissure
The result of pepsin IHC in the tissue is shown in Table 1 and group (4.63 ± 3.40 ng/mL) and the group at the edge of vocal cord
Figs. 1A–E. According to the result, patients were divided into a pepsin (7.87 ± 7.25 ng/mL) (t = −1.13, P = .27, Fig. 2L).
positive group (10 men, 12 women; average age, 35.55 years [95% Further pathological analysis showed that there was no significant
confidence interval (CI), 30.48–40.61 years]) and a negative group (5 difference (P > .05, Fig. 2J) in tissue expression of pepsin among the
men, 3 women; average age, 34.00 years [95% CI, 20.68–47.32 years]). edematous, vascular, and fibrous groups. There was still no significant
Further analysis showed that pepsin concentration in saliva was difference in pepsin level of saliva between the edematous group
8.26 ± 1.56 ng/mL in the pepsin positive group, which was sig- (8.85 ± 7.77 ng/mL), the vascular group (4.75 ± 3.49 ng/mL), and
nificantly higher (t = 2.38, P = .024) than that in the negative group the fibrous group (4.02 ± 3.35 ng/mL) (P > .05, Fig. 2M).

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(caption on next page)

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Y.-F. Dai, et al.
Fig. 5. (A) Expression of 8-OHdG in tissue pepsin positive and negative groups. (B) Expression of p-H2AX in tissue pepsin positive and negative groups. (C)
Continuous sections from three different tissue showed pepsin, 8-OHdG, and p-H2AX expressed congruously (mostly expressed in basal layer of epithelium). (D)
Correlation analysis between concentrations of pepsin in saliva and 8-OHdG level in tissue. (E) Correlation analysis between concentrations of pepsin in saliva and p-
H2AX level in tissue.
3.4. Further analysis of pepsin expression and 8-OHdG/p-H2AX levels
As shown in Figs. 3 and 4, 8-OHdG and p-H2AX were localized in
the epithelial layer, which was similar to pepsin, but mainly in karyon.
Mann-Whitney rank-sum test showed significant differences in the IHC
score of 8-OHdG (P = .018, Table 4) and p-H2AX (P = .004, Table 5) in
tissue between the pepsin positive and negative groups. Tissue ex-
pression of 8-OHdG and p-H2AX were higher in the pepsin positive
group than the negative group (Fig. 5A and B). Moreover, pepsin ex-
pression positively correlated with the expression of 8-OHdG
(r = 0.527, P = .001) and p-H2AX (r = 0.460, P = .005, Fig. 5C). As
illustrated in Fig. 5D and E, pepsin concentration in saliva positively
correlated with 8-OHdG (r = 0.406, P = .013) in tissue but not with P-
H2AX (r = 0.293, P = .058).
3.5. Pepsin induced DNA damage in human immortalized laryngeal
epithelial cells
As DNA damage may be a risk factor for LPR related VFPs, we
sought to understand the regulation of DNA damage by pepsin in
human immortalized laryngeal epithelial cells. To investigate if pepsin
causes DNA damage, hV-Bmi1 and hSG-Bmi1 cells, two types of human
immortalized laryngeal epithelial cells, were incubated with pepsin.
Fig. 6 shows representative pictures. Pepsin was able to induce 8-OHdG
in both hV-Bmi1 and hSG-Bmi1 cells and increased concurrently with
increasing pepsin concentrations (Fig. 6A). Furthermore, we examined
P-H2AX by western blotting and found that P-H2AX expression in-
creased following exposure to different concentrations of pepsin in hV-
Bmi1 and hSG-Bmi1 cells (Fig. 6B). Comet assay showed that the tail
moment significantly increased from 0.14 ± 0.04 to 1.00 ± 0.21 and
from 0.14 ± 0.04 to 2.88 ± 0.29 when hV-Bmi1 cells were treated
with 0.1 mg/mL and 1 mg/mL pepsin, respectively (t-test, P < .0001).
Similarly, the tail moment increased from 0.08 ± 0.03 to 0.91 ± 0.20
and from 0.08 ± 0.03 to 2.40 ± 0.30 in hSG-Bmi1 cells (t-test,
P < .0001, Fig. 6C), indicating that pepsin may cause DNA damage.
4. Discussion
It is generally believed that LPR is a risk factor for VFP formation,
but the specific molecular biological mechanisms have not been fully
elucidated. This study explores the direct effects of different reflux
substances such as pepsin, bile acid and gastric acid on the laryngeal
mucosa and effectively and accurately clarifies the role of LPR to vocal
cord polyp. Here, pepsin which the main invasive component of LPR
[22] was detected in tissue of patients with VFPs. The positive rate of
pepsin is 73.33%, confirming that laryngeal refluxes were widely pre-
sent in VFPs. Further analysis of pepsin ELISA in saliva showed that
patients with pepsin positive expression in tissue also had a higher level
of pepsin in saliva. Normally, gastric gas may carry a small amount of
pepsin through the esophagus to the surface of the airway, but this part
of pepsin can eventually be expelled with airway secretions. However,
patients with VFPs usually show excessive movement of the exhalation
muscle owing to abnormal pronunciation, which increases the regur-
gitation of gastric content and pepsin level [23]. This pepsin cannot be
completely excreted from the airway, and adheres to the mucosal epi-
thelium of the pharynx and larynx (manifested as a higher level of
pepsin in saliva). It is taken up by receptor mediated endocytosis and
found in late endosomes and the trans-reticular Golgi. When reflux
reoccurred, pH in the airway mucosal surface could fall below 6, and
pepsin adsorbed on the surface of mucosal epithelial cells or stored in
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Am J Otolaryngol 41 (2020) 102681
vesicles may be activated, causing mitochondrial dysfunction and cell
damage [24].
Patients with VFPs present varying clinical symptoms and signs, and
different sizes and locations of polyps can affect pronunciation quality
differently [25]. By analyzing the pathological changes of the epithelial
layer, basal layer, and lamina propria layer in tissue samples of patients
with VFPs, Martins et al. [26] found that morphological changes of
VFPs mainly occurred in lamina propria layer, including edema, an-
giogenesis, and inflammation. The most common changes were inter-
stitial edema, vascular proliferation with congestion, and congestion
[26]. Previous studies confirmed that LPR is associated with the oc-
currence of VFPs [3], but there is no report about the association be-
tween LPR and different clinical or pathological classifications. In this
study, we analyzed the association between pepsin expression and
different positions of VFPs (unilateral or bilateral, anterior commissure
or edge of the vocal cord) and different pathological types (edematous,
vascular, and fibrous). We found that there is no significant difference
in the pepsin expression between groups of different locations and pa-
thologic types of VFPs, suggesting that pepsin does not cause specific
locations of polyps, nor does it cause specific pathological classifica-
tions.
Current literature illustrates that the main etiology of VFPs is micro
trauma of the vocal cord, which resulted from vibrations, and magni-
fied by external invasive factors (such as LPR). Expansion of vocal cord
impingement and repeated injury-repair will initiate the inflammatory
response and generate Reinke's space edema, vascular dilation, or
congestion, etc. [27–29]. DNA damage, well known as a critical factor
of apoptosis, aging and tumorigenesis, has attracted the attention of
scientists again in recent years, owing to its role in chronic inflamma-
tion [30–32]. 8-OHdG and p-H2AX are regarded as biomarkers of DNA
damage. As a typical product of guanine oxidative damage in DNA, 8-
OHdG is utilized to quantify oxidative damage of nucleic acids [33]. P-
H2AX, the phosphorylated form of histone H2AX, is a specific indicator
in detection of early DNA double-stranded structure damage [34]. This
study showed that expression of 8-OHdG and p-H2AX increased in
pepsin positive group tissue and 8-OHdG expression in tissue positively
correlated with pepsin concentration in patient's saliva, suggesting that
DNA damage, especially oxidative damage, was aggravated in the lar-
yngeal epithelium of VFP patients with LPR. Furthermore, IHC analysis
revealed that 8-OHdG and p-H2AX levels were raised as the pepsin level
increased. In cells with pepsin, 8-OHdG and p-H2AX expression was
enhanced, whereas in cells without pepsin, the expression of 8-OHdG
and p-H2AX was weak, which further suggested that the presence of
pepsin in laryngeal epithelial mucosa was closely related to the oc-
currence of DNA damage. Thus, we believe that pepsin reflux to the
laryngeal epithelial mucosa may promote DNA oxidative damage and
double-stranded breaks.
DNA oxidative damage is closely related to several diseases (such as
cardiovascular disease [35], chronic obstructive bronchitis [36], etc.).
The damage to the human body is multi-linked, wide-ranging, and plays
a decisive role in the pathogenesis of several human diseases. Studies
have confirmed that gastroesophageal reflux stimulation can cause
DNA damage to esophageal mucosal epithelium. Li [37] proved that in
Barrett's esophagus acid reflux increases intracellular calcium, activates
NOX5-S, and increases ROS production, which causes DNA damage.
Bhardwaj [38] certified that acidic bile salts induce ROS, such as hy-
drogen peroxide and superoxide in esophageal cells, which in turn,
rapidly induces DNA damage. Pepsin, gastric acid, and bile acid are the
main components of gastric reflux, and reflux to non-gastric tissue can
cause serious damage to the epithelial mucosa. It is clear that gastric
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Y.-F. Dai, et al.
Fig. 6. (A) Induction of 8-OHdG by pepsin in human immortalized laryngeal epithelial cells. Cells were treated with different concentrations of pepsin, and 8-OHdG
was determined by immunohistochemical staining; (B) Treatment with pepsin induces DNA damage in human immortalized laryngeal epithelial cells. Treated cells
were analyzed for p-H2AX following treatment with different concentrations of pepsin using western blotting; (C) DNA damage was analyzed following treatment
with different concentrations of pepsin using the alkaline comet assay. Representative comet images are shown.
acid and bile acid stimulation can cause DNA damage to esophageal
mucosal epithelium, but it is not clear if pepsin—the main invasive
component of LPR—can cause DNA damage to laryngeal mucosa.
Therefore, we detected DNA damage by stimulating human im-
mortalized laryngeal epithelial cells with different concentrations of
pepsin, and showed that the expression of 8-OHdG and p-H2AX, and the
tail moment of the comet assay significantly increased. It was proved
further that pepsin might cause DNA damage in laryngeal epithelial
cells. The existence of pepsin in LPR is tantamount to a continuous or
low-intensity stimulation of the pharynx and larynx mucosa. This long-
term and repeated chemical stimulation will damage laryngeal epi-
thelial cells and cause changes in DNA structure. DNA damage can in-
crease genetic instability and lead to changes in enzyme and protein
function [39], up-regulate pro-inflammatory cytokines at the tran-
scriptional level to induce increased secretion of cytokines, which am-
plifies the inflammatory cascade [40]. Therefore, we speculated that
pepsin reflux might aggravate DNA damage in laryngeal epithelial cells,
thereby promoting abnormal repair of laryngeal epithelial mucosal
damage, increasing the secretion of inflammatory cytokines, which
results in a chronic inflammatory microenvironment and promotes the
formation of inflammatory lesions such as VFPs.
We believe that these findings reveal a novel mechanism of pepsin-
induced cell injury and have an important clinical significance for pa-
tients with VFP, especially for those who continue to use maximum acid
inhibition therapy. Based on our preliminary evidence, testing for
pepsin in VFP patients may help determine if LPR is involved in the
pathogenesis of the disease, potentially aiding clinicians to consider
anti-reflux treatment strategies. Pepsin, which stimulates laryngeal
epithelial cells and aggravates DNA damage in laryngeal epithelial
mucosa, may play a fundamental role in the occurrence of LPR-related
vocal cord polyps. The pathway of DNA damage caused by pepsin, as
well as the types and efficacy of commercial drugs used to inhibit DNA
damage can be explore in future studies. The outcomes of these studies
will be beneficial to patients with LPR disease, of whom account for 5%
of the outpatient department of ENT and 55% of patients with hoar-
seness.
This present study further confirmed the relationship between
pepsin and the development of VFPs. To the best of our knowledge, this
is the first report on the role of DNA oxidative damage in LPR pro-
moting VFPs. However, there were several limitations with this study.
First, we recruited only a small number of subjects; however, a larger
sample size is required to reinforce our conclusions. In addition, as this
study is a cytological and histological experiment, further demonstra-
tions on an animal model of LPR is necessary. Second, although we
preliminarily showed that DNA damage of laryngeal mucosa was ag-
gravated in tandem with pepsin stimulation, potential mechanisms,
such as the involvement of ROS, MAPKs, and NLRP3. need to be further
studied.
In conclusion, we found that pepsin had a high positive rate in tissue
and saliva of patients with VFPs, confirming the widespread presence of
laryngopharyngeal reflux in patients with VFPs. Pepsin reflux has no
correlation with the location and pathological classification of VFPs,
but it can aggravate injury and DNA damage of laryngeal epithelial
mucosa. We hypothesize that pepsin may further promote the infiltra-
tion of inflammatory cells and expand the inflammatory micro-
environment in laryngeal epithelial mucosa by promoting oxidative
DNA damage, accelerating formation and development of VFPs. Our
study enriched the theoretical basis of pepsin in promoting in-
flammatory diseases such as VFPs. Further research will be carried out
on the constructed primary epithelial cells [41] and animal models of
9
Am J Otolaryngol 41 (2020) 102681
laryngopharyngeal reflux disease.
Funding
This work was supported by the National Natural Science
Foundation of China (grant number 81400452).
Declaration of competing interest
There are no potential conflicts of interest regarding the study,
authorship, or publication of this article.
Acknowledgments
The authors acknowledge the collaboration of participants in this
study.
CRediT author statement
Yuan-Feng Dai: Conceptualization, Writing - original draft, formal
analysis; Jia-Jie Tan: Writing – original draft, Formal analysis; Chao-
Qun Deng: Formal analysis, Writing – review & editing; Xiong Liu:
Data curation, Writing – review & editing; Ze-Hong Lv: Data curation,
Writing – review & editing; Xiang-Ping Li: Conceptualization, Writing -
original draft. All authors are accountable for all aspects of the work
and approved the final manuscript.
References
[1] Wallis L, Jackson-Menaldi C, Holland W, et al. Vocal fold nodule vs. vocal fold
polyp: answer from surgical pathologist and voice pathologist point of view. J Voice
2004;18:125–9.
[2] Fang R, Jiang JJ, Smith BL, et al. Expression of hypoxia inducible factor-1α and
vascular endothelia growth factor in vocal polyps. Laryngoscope 2013;123:2184–8.
[3] Lechien JR, Saussez S, Nacci A, et al. Association between laryngopharyngeal reflux
and benign vocal folds lesions: a systematic review. Laryngoscope
2019;129:E329–41.
[4] Lechien JR, Saussez S, Harmegnies B, et al. Laryngopharyngeal reflux and voice
disorders: a multifactorial model of etiology and pathophysiology. J Voice
2017;31:733–52.
[5] Samuels TL, Handler E, Syring ML, et al. Mucin gene expression in human laryngeal
epithelia: effect of laryngopharyngeal reflux. Ann Otol Rhinol Laryngol
2008;117:688–95.
[6] Gill GA, Johnston N, Buda A, et al. Laryngeal epithelial defenses against lar-
yngopharyngeal reflux: investigations of E-cadherin, carbonic anhydrase isoenzyme
III, and pepsin. Ann Otol Rhinol Laryngol 2005;114:913–21.
[7] Mayr D, Durst F, Berghaus A. E-cadherin but not beta-catenin expression is de-
creased in laryngeal biopsies from patients with laryngopharyngeal reflux. Eur Arch
Otorhinolaryngol 2008;265(8):937–42.
[8] Lechien JR, Saussez S, Schindler A, et al. Clinical outcomes of laryngopharyngeal
reflux treatment: a systematic review and meta-analysis. Laryngoscope
2019;129:1174–87.
[9] Koufman JA, Amin MR, Panetti M. Prevalence of reflux in 113 consecutive patients
with laryngeal and voice disorders. Otolaryngol Head Neck Surg 2000;123:385–8.
[10] Beltsis A, Katsinelos P, Kountouras J, et al. Double probe pH-monitoring findings in
patients with benign lesions of the true vocal folds: comparison with typical GERD
and the effect of smoking. Eur Arch Otorhinolaryngol 2011;268:1169–74.
[11] Wang L, Tan J-J, Wu T, et al. Association between laryngeal pepsin levels and the
presence of vocal fold polyps. Otolaryngol Head Neck Surg 2017;156:144–51.
[12] Samuels TL, Johnston N. Pepsin as a marker of extraesophageal reflux. Ann Otol
Rhinol Laryngol 2010;119:203–8.
[13] Chung JH, Tae K, Lee YS, et al. The significance of laryngopharyngeal reflux in
benign vocal mucosal lesions. Otolaryngol Head Neck Surg 2009;141(3):369–73.
[14] Kroese LJ, Scheffer PG. 8-Hydroxy-2′-deoxyguanosine and cardiovascular disease: a
systematic review. Curr Atheroscler Rep 2014;16:452.
[15] Yang S, Wu H, Zhao J, et al. Feasibility of 8-OHdG formation and hOGG1 induction
in PBMCs for assessing oxidative DNA damage in the lung of COPD patients.
Respirology 2014;19:1183–90.
[16] Li D, Cao W. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-
induced DNA damage in Barrett’s cells and Barrett’s esophageal adenocarcinoma
cells. Am J Physiol Gastrointest Liver Physiol 2014;306:G863–72.
[17] Bhardwaj V, Horvat A, Korolkova O, et al. Prevention of DNA damage in Barrett’s
Y.-F. Dai, et al.
esophageal cells exposed to acidic bile salts. Carcinogenesis 2016;37:1161–9.
[18] Bhardwaj V, Gokulan RC, Horvat A, et al. Activation of NADPH oxidases leads to
DNA damage in esophageal cells. Sci Rep 2017;7:9956.
[19] Tan J-J, Wang L, Mo T-T, et al. Establishment of immortalized laryngeal epithelial
cells transfected with Bmi1. Cell Transplant 2020;29:1–10.
[20] Tan J-J, Wang L, Mo T-T, et al. Pepsin promotes IL-8 signaling-induced epithe-
lial–mesenchymal transition in laryngeal carcinoma. Cancer Cell Int 2019;19:64.
[21] Bajpayee M, Kumar A, Dhawan A. The comet assay: assessment of in vitro and in
vivo DNA damage. Methods Mol Biol 2013;1044:325–45.
[22] Johnston N, Dettmar PW, Bishwokarma B, et al. Activity/stability of human pepsin:
implications for reflux attributed laryngeal disease. Laryngoscope
2007;117(6):1036–9.
[23] Schreiber S, Garten D, Sudhoff H. Pathophysiological mechanisms of extra-
esophageal reflux in otolaryngeal disorders. Eur Arch Otorhinolaryngol
2009;266:17–24.
[24] Johnston N, Wells CW, Samuels TL, et al. Pepsin in nonacidic refluxate can damage
hypopharyngeal epithelial cells. Ann Otol Rhinol Laryngol 2009;118:677–85.
[25] Cho J-H, Choi Y-S, Joo Y-H, et al. Clinical significance of contralateral reactive
lesion in vocal fold polyp and cyst. J Voice 2018;32:109–15.
[26] Martins RH, Defaveri J, Domingues MA, et al. Vocal polyps: clinical, morphological,
and immunohistochemical aspects. J Voice 2011;25:98–106.
[27] Eckley CA, Swensson J, Duprat AC, et al. Incidence of structural vocal fold ab-
normalities associated with vocal fold polyps. Braz J Otorhinolaryngol
2008;74:508–11.
[28] Volić SV, Klapan I, Seiwerth S, et al. Extracellular matrix of Reinke’s space in some
pathological conditions. Acta Otolaryngol 2004;124:505–8.
[29] Yumoto E. Aerodynamics, voice quality, and laryngeal image analysis of normal
and pathologic voices. Curr Opin Otolaryngol Head Neck Surg 2004;12:166–73.
[30] Kuilman T, Michaloglou C, Vredeveld LCW, et al. Oncogene-induced senescence
10
Am J Otolaryngol 41 (2020) 102681
relayed by an interleukin-dependent inflammatory network. Cell
2008;133:1019–31.
[31] Freund A, Orjalo AV, Desprez P-Y, et al. Inflammatory networks during cellular
senescence: causes and consequences. Trends Mol Med 2010;16:238–46.
[32] Rodier F, Coppé J-P, Patil CK, et al. Persistent DNA damage signalling triggers se-
nescence-associated inflammatory cytokine secretion. Nat Cell Biol 2009;11:973–9.
[33] Valavanidis A, Vlachogianni T, Fiotakis C. 8-Hydroxy-2′ -deoxyguanosine (8-
OHdG): a critical biomarker of oxidative stress and carcinogenesis. J Environ Sci
Health C Environ Carcinog Ecotoxicol Rev 2009;27:120–39.
[34] Takahashi A, Ohnishi T. Does gammaH2AX foci formation depend on the presence
of DNA double strand breaks? Cancer Lett 2005;229:171–9.
[35] Kroese LJ, Scheffer PG. 8-Hydroxy-2′-deoxyguanosine and cardiovascular disease: a
systematic review. Curr Atheroscler Rep 2014;16(11):452.
[36] Yang S, Wu H, Zhao J, et al. Feasibility of 8-OHdG formation and hOGG1 induction
in PBMCs for assessing oxidative DNA damage in the lung of COPD patients.
Respirology 2014;19(8):1183–90.
[37] Li D, Cao W. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-
induced DNA damage in Barrett’s cells and Barrett’s esophageal adenocarcinoma
cell. Am J Physiol Gastrointest Liver Physiol 2014;306(10):G863–72.
[38] Bhardwaj V, Horvat A, Korolkova O, et al. Prevention of DNA damage in Barrett’s
esophageal cells exposed to acidic bile salts. Carcinogenesis 2016;37(12):1161–9.
[39] Horn V, Triantafyllopoulou A. DNA damage signaling and polyploid macrophages
in chronic inflammation. Curr Opin Immunol 2018;50:55–63.
[40] Karakasilioti I, Kamileri I, Chatzinikolaou G, et al. DNA damage triggers a chronic
autoinflammatory response, leading to fat depletion in NER progeria. Cell Metab
2013;18(3):403–15.
[41] Mo T-T, Tan J-J, Wang M-G, et al. Optimized generation of primary human epi-
thelial cells from larynx and hypopharynx: a site-specific epithelial model for reflux
research. Cell Transplant 2019;28:630–7.