Clin Exp Otorhinolaryngol.  2023 Feb;16(1):49-58. 10.21053/ceo.2022.00857.

Ghrelin Downregulates Lipopolysaccharide/ Leptin-Induced MUC5AC Expression in Human Nasal Epithelial Cells

• Choi YS
• Na HG
• Bae CH
• Song SY
• Kim YD

• KMID: 2539768
• DOI: http://doi.org/10.21053/ceo.2022.00857

Abstract

Objectives
. Obesity, which induces chronic low-grade systemic inflammation in the human body, is a known risk factor for various diseases. Recent studies have shown associations between various otorhinolaryngological diseases and obesity. In particular, inflammatory sinonasal diseases have been found to be strongly associated with obesity-related proinflammatory mediators. Many studies have been conducted to identify therapeutic agents for controlling obesity-related inflammatory airway diseases. Ghrelin, an endogenous peptide from the stomach, has anti-inflammatory and antioxidative effects in a wide range of tissues. However, the effect of ghrelin on the regulation of mucus secretion has not yet been studied in the human nasal mucosa. Therefore, we investigated the effects of ghrelin on lipopolysaccharide (LPS)/leptin-mediated MUC5AC expression and mechanisms involved in human nasal epithelial cells (HNEpCs).
Methods
. In HNEpCs, the effect and signaling pathways of ghrelin on LPS/leptin-induced MUC5AC expression were examined using reverse transcription polymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassays, Western blotting, and immunofluorescence staining.
Results
. Growth hormone secretagogue receptor 1a (GHSR1a) was expressed in the HNEpCs. Ghrelin downregulated LPS/leptin-induced MUC5AC expression, which was abolished by D-Lys-3-growth hormone-releasing peptide 6 (D-Lys-3-GHRP-6). Ghrelin significantly inhibited LPS/leptin-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs). These ghrelin-mediated changes in MAPK activation were abolished by D-Lys-3-GHRP-6. These results showed that ghrelin inhibits LPS/leptin-induced MUC5AC overexpression by modulating the ERK1/2 and p38 MAPK pathways in HNEpCs.
Conclusion
. These findings suggest that ghrelin is a potential therapeutic agent for treating obesity-related inflammatory sinonasal diseases.

Keyword

Obesity; Ghrelin; Lipopolysaccharide; Leptin; MUC5AC; Mitogen-Activated Protein Kinase; Nasal Mucosa

Figure

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  Fig. 1. Effect of ghrelin on lipopolysaccharide (LPS)-induced MUC5AC expression in human nasal epithelial cells. (A, B) The results of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR show that ghrelin significantly induced GHSR1a messenger RNA (mRNA) expression. (C) The results of RT-PCR and real-time PCR show that ghrelin significantly inhibited LPS-induced MUC5AC mRNA expression. LPS-induced MUC5AC expression started to be maximally suppressed at ta ghrelin concentration of 0.1 μM. (D) The results of real-time PCR show that the maximal inhibition of LPS-induced MUC5AC mRNA expression by ghrelin (0.1 μM) occurred after 2 hours. (E, F) The results of enzyme-linked immunosorbent assay also show that ghrelin significantly inhibited LPS-induced MUC5AC protein production. The images are representative of three separate experiments performed in triplicate. The bars indicate the mean±standard deviation of three independent experiments performed in triplicate. GHSR1a, growth hormone secretagogue receptor 1a; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a)P<0.05 compared to the baseline value. b)P<0.05 is compared with samples treated with LPS (1 μg/mL) alone.

• 

  Fig. 2. The regulatory mechanism of ghrelin on lipopolysaccharide (LPS)-induced MUC5AC expression in human nasal epithelial cells. (A, B) The results of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR show that ghrelin inhibited LPS-induced MUC5AC expression, and this inhibition was abolished by D-Lys-3-growth hormone-releasing peptide 6 (D-Lys-3-GHRP-6). (C, D) The results of enzyme-linked immunosorbent assay and immunofluorescence staining also show that ghrelin significantly inhibited LPS-induced MUC5AC protein production, while the inhibitory effect of LPS-induced MUC5AC protein production was abolished by D-Lys-3-GHRP-6. (E) Western blot results show that ghrelin significantly inhibited LPS-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs). These ghrelin-mediated changes in MAPK activation were also abolished by D-Lys-3-GHRP-6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4´,6-diamidino-2-phenylindole; p-ERK1/2, phosphorylated ERK 1/2; p-p38, phosphorylated p38. a)P<0.05 compared to the baseline value. b)P<0.05 compared with samples treated with LPS (1 μg/mL) alone. c)P<0.05 compared to samples treated with LPS (1 μg/mL) and ghrelin (0.1 μM).

• 

  Fig. 3. Effect of ghrelin on leptin-induced MUC5AC expression in human nasal epithelial cells. (A) The results of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR show that ghrelin significantly inhibited leptin-induced MUC5AC mRNA expression. Leptin-induced MUC5AC expression started to be maximally suppressed at a ghrelin concentration of 0.1 μM. (B) Real-time PCR results show that the maximal inhibition of leptin-induced MUC5AC mRNA expression by ghrelin (0.1 μM) occurred after 2 hours. (C, D) The results of enzyme-linked immunosorbent assay also showed that ghrelin significantly inhibited leptin-induced MUC5AC protein production. The images are representative of three separate experiments performed in triplicate. The bars indicate the mean±standard deviation of three independent experiments performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a)P<0.05 compared to the baseline value. b)P<0.05 compared with samples treated with lipopolysaccharide (LPS; 1 μg/mL) alone.

• 

  Fig. 4. The regulatory mechanism of ghrelin in leptin-induced MUC5AC in human nasal epithelial cells. (A, B) The results of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR show that ghrelin inhibited leptin-induced MUC5AC expression, and this inhibition was abolished by D-Lys-3-growth hormone-releasing peptide 6 (D-Lys-3-GHRP-6). (C, D) The results of enzyme-linked immunosorbent assay and immunofluorescence staining also show that ghrelin significantly inhibited leptin-induced MUC5AC protein production, while the inhibitory effect of leptin-induced MUC5AC protein production was abolished by D-Lys-3-GHRP-6. (E) Western blot results show that ghrelin significantly inhibited leptin-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs). These ghrelin-mediated changes in MAPK activation were also abolished by D-Lys-3-GHRP-6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4´,6-diamidino-2-phenylindole; p-ERK1/2, phosphorylated ERK 1/2; p-p38, phosphorylated p38. a)P<0.05 compared to the baseline value. b)P<0.05 compared to samples treated with leptin (0.1 μg/mL) alone. c)P<0.05 compared to samples treated with leptin (0.1 μg/mL) and ghrelin (0.1 μM).

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