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Shui-Hong Zhou
Zhejiang University School of Medicine First Affiliated Hospital
Corresponding Author
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Background: Although laryngopharyngeal reflux (LPR) has been implicated in various upper aerodigestive tract and laryngeal diseases, the underlying mechanisms remain elusive. In this study, we investigated the role of gastric acidified pepsin in laryngeal precancerosis.
Results: Acidified pepsin (pH=3) enhanced the growth and survival of mouse laryngeal epithelial cells in vitro and promoted laryngeal mucosal thickening and laryngeal epithelial cell growth in vivo. Furthermore, acidified pepsin promoted autophagy/mitophagy induction, accompanied by a significant decrease in mitochondrial membrane potential (MMP). Inhibition of autophagy by chloroquine abolished the ability of acidified pepsin to promote mitophagy and cell growth in laryngeal epithelial cells. Additionally, chloroquine promoted cell apoptosis and further reduced MMP in laryngeal epithelial cells treated with acidified pepsin. The expression levels of pepsin and H+/K+-ATPase α and β subunits in 31 human laryngeal mucosa specimens were 51.6%, 48.4%, and 48.4%, respectively. Importantly, the pepsin level was correlated with the H+/K+-ATPase β subunit level. H+/K+-ATPase upregulation in laryngeal epithelial cells in response to acidified pepsin was essential for the mitophagy-promoting effect of acidified pepsin. H+/K+-ATPase knockout or inhibition further reduced MMP in the presence of acidified pepsin.
Conclusions: Our findings suggest that in an acidic environment, pepsin promotes laryngeal epithelial cell growth and survival by upregulating H+/K+-ATPase and activating mitophagy, potentially leading to laryngeal precancerosis.
Keywords
Laryngopharyngeal reflux, gastroesophageal reflux, pepsin, autophagy, mitophagy, H+/K+-ATPase
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This is a list of supplementary files associated with this preprint. Click to download.
- S1.tif
Figure S1. Effect of pepsin on H+/K+-ATPase expression. (A, B) An esophageal acid perfusion mouse model was established by perfusing 0.15 mL HCl (1 M, pH=3) with or without 2.5 mg/g pepsin. The levels of the H+/K+-ATPase α (A) and β (B) subunits in laryngeal mucosal tissues were determined by IHC. (C, D) The expression levels of the H+/K+-ATPase α (C) and β (D) subunits in cells treated with acidic medium or acidified pepsin were determined by IF staining. (E, F) The mRNA (E) and protein (F) levels of H+/K+-ATPase α and β subunits in cells treated with acidic medium or acidified pepsin were determined by qRT-PCR or Western blotting, respectively. The right panel shows the relative expression levels of H+/K+-ATPase α and β subunits. ** indicates significance at P<0.01.
- S2.tif
Figure S2. Effect of H+/K+-ATPase α subunit depletion on laryngeal epithelial cell viability. (A) The efficiency of H+/K+-ATPase-α-subunit KO in laryngeal epithelial cells was assessed by Western blotting. The relative levels of the H+/K+-ATPase α subunit in different groups are also shown. **, ##, and ^^ indicate significance at P<0.01. (B) Wild-type and H+/K+-ATPase-α-subunit-KO laryngeal epithelial cells were treated with acidic medium or acidified pepsin. Cell proliferation was assessed by CCK-8 assay. (C) Cell apoptosis was assessed by flow cytometry. (D) The level of cleaved caspase-3 was determined by Western blotting. The ratio of cleaved caspase-3 to procaspase-3 levels is also shown.
- S3.tif
Figure S3. Effect of different PPIs on the viability of laryngeal epithelial cells. Cells were treated with 10 or 50 µg pantoprazole, omeprazole, lansoprazole, rabeprazole, esomeprazole, or SCH-28080 at pH 3. Cell viability was assessed by CCK-8 assay. * and ## indicate significance at P<0.05 and P<0.01, respectively.
- S4.tif
Figure S4. Effects of pantoprazole on acidified pepsin-mediated induction of autophagy and mitophagy. (A) Cells were treated with acidic medium or acidified pepsin in the presence or absence of pantoprazole. LC3 and p62 protein levels were determined by Western blotting. The relative p62 levels and LC3II/LC3I ratio are also shown. (B) Mitophagy induction was assessed by IF using a fluorescent mitophagy dye.
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Shui-Hong Zhou
Zhejiang University School of Medicine First Affiliated Hospital
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
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History
CURRENT STATUS: Posted
Version 1
Posted 28 Dec, 2020
No community comments so far
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Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Although laryngopharyngeal reflux (LPR) has been implicated in various upper aerodigestive tract and laryngeal diseases, the underlying mechanisms remain elusive. In this study, we investigated the role of gastric acidified pepsin in laryngeal precancerosis.
Results: Acidified pepsin (pH=3) enhanced the growth and survival of mouse laryngeal epithelial cells in vitro and promoted laryngeal mucosal thickening and laryngeal epithelial cell growth in vivo. Furthermore, acidified pepsin promoted autophagy/mitophagy induction, accompanied by a significant decrease in mitochondrial membrane potential (MMP). Inhibition of autophagy by chloroquine abolished the ability of acidified pepsin to promote mitophagy and cell growth in laryngeal epithelial cells. Additionally, chloroquine promoted cell apoptosis and further reduced MMP in laryngeal epithelial cells treated with acidified pepsin. The expression levels of pepsin and H+/K+-ATPase α and β subunits in 31 human laryngeal mucosa specimens were 51.6%, 48.4%, and 48.4%, respectively. Importantly, the pepsin level was correlated with the H+/K+-ATPase β subunit level. H+/K+-ATPase upregulation in laryngeal epithelial cells in response to acidified pepsin was essential for the mitophagy-promoting effect of acidified pepsin. H+/K+-ATPase knockout or inhibition further reduced MMP in the presence of acidified pepsin.
Conclusions: Our findings suggest that in an acidic environment, pepsin promotes laryngeal epithelial cell growth and survival by upregulating H+/K+-ATPase and activating mitophagy, potentially leading to laryngeal precancerosis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- S1.tif
Figure S1. Effect of pepsin on H+/K+-ATPase expression. (A, B) An esophageal acid perfusion mouse model was established by perfusing 0.15 mL HCl (1 M, pH=3) with or without 2.5 mg/g pepsin. The levels of the H+/K+-ATPase α (A) and β (B) subunits in laryngeal mucosal tissues were determined by IHC. (C, D) The expression levels of the H+/K+-ATPase α (C) and β (D) subunits in cells treated with acidic medium or acidified pepsin were determined by IF staining. (E, F) The mRNA (E) and protein (F) levels of H+/K+-ATPase α and β subunits in cells treated with acidic medium or acidified pepsin were determined by qRT-PCR or Western blotting, respectively. The right panel shows the relative expression levels of H+/K+-ATPase α and β subunits. ** indicates significance at P<0.01.
- S2.tif
Figure S2. Effect of H+/K+-ATPase α subunit depletion on laryngeal epithelial cell viability. (A) The efficiency of H+/K+-ATPase-α-subunit KO in laryngeal epithelial cells was assessed by Western blotting. The relative levels of the H+/K+-ATPase α subunit in different groups are also shown. **, ##, and ^^ indicate significance at P<0.01. (B) Wild-type and H+/K+-ATPase-α-subunit-KO laryngeal epithelial cells were treated with acidic medium or acidified pepsin. Cell proliferation was assessed by CCK-8 assay. (C) Cell apoptosis was assessed by flow cytometry. (D) The level of cleaved caspase-3 was determined by Western blotting. The ratio of cleaved caspase-3 to procaspase-3 levels is also shown.
- S3.tif
Figure S3. Effect of different PPIs on the viability of laryngeal epithelial cells. Cells were treated with 10 or 50 µg pantoprazole, omeprazole, lansoprazole, rabeprazole, esomeprazole, or SCH-28080 at pH 3. Cell viability was assessed by CCK-8 assay. * and ## indicate significance at P<0.05 and P<0.01, respectively.
- S4.tif
Figure S4. Effects of pantoprazole on acidified pepsin-mediated induction of autophagy and mitophagy. (A) Cells were treated with acidic medium or acidified pepsin in the presence or absence of pantoprazole. LC3 and p62 protein levels were determined by Western blotting. The relative p62 levels and LC3II/LC3I ratio are also shown. (B) Mitophagy induction was assessed by IF using a fluorescent mitophagy dye.
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