In the pulmonary system, mucus is a thin gel-like layer that covers the luminal surface of the airway. It contains various molecules, ions, and water, and its components have antioxidant and antimicrobial properties (Kim et al., 2023). The primary biochemical macromolecules in mucus are mucins, which confer the mucus with viscoelasticity. Among the various subtypes of human mucins, MUC2, MUC5AC, MUC5B, and MUC6, are the four gel-forming mucins. MUC2 is the major intestinal mucin but is also expressed at low levels in the lungs. MUC5AC and MUC6 are gastric mucins; MUC5AC is abundant in the pulmonary system, whereas MUC6 is not. MUC5AC is the predominant mucin secreted by the airways. MUC5B is a glandular mucin present in the tracheobronchial submucosal glands (Evans and Koo, 2009). Under normal physiological conditions, airway mucus plays a crucial role in protecting the pulmonary epithelium from damage caused by viruses, bacteria, inhaled particles, and irritating gases (Mann et al., 2022). However, excessive production or secretion of mucus due to alterations in mucin quantity or quality can impair the host defense system and contribute to increased morbidity and mortality in diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF) (Ryu et al., 2023). In the human lung, MUC5AC localizes to the epithelial cells at the airway surface. It is expressed within goblet cells on the luminal surface of healthy airways and is the most abundant gel-forming mucin in asthma, COPD, and CF. Although MUC5AC levels are low in patients with stable disease, they increase approximately tenfold during disease exacerbation (Evans and Koo, 2009; Kim et al., 2023).

Mucolytics, glucocorticoids, and expectorants are commonly used to manage abnormal mucus secretion; however, they can cause airway irritation and other adverse effects, including rebound mucus hypersecretion (Li et al., 2020).

There is significant potential for the development of novel agents that regulate mucin production and secretion by targeting mucin degradation and biosynthesis. Natural products from medicinal plants traditionally used to treat inflammatory pulmonary diseases could be particularly promising for managing abnormal mucin production. Previous studies have demonstrated that various natural compounds can influence the production of airway mucus glycoproteins (mucins) by altering gene expression (Kim et al., 2012; Ryu et al., 2013, 2014; Seo et al., 2014; Sikder et al., 2014; Lee et al., 2015; Kim et al., 2016; Li et al., 2020; Hossain et al., 2022a, 2022b; Kim et al., 2023; Ryu et al., 2023). These findings highlight the potential of natural products for the development of new therapeutics to control mucin production in the airways under inflammatory conditions.

Galangin (Fig. 1), a natural flavonol found in honey and in the medicinal plants Alpinia officinarum and A. galanga, has shown anticancer (Rampogu et al., 2021), anti-inflammatory (Lee et al., 2018), antioxidant (Aloud et al., 2017), and antifibrotic (Wang et al., 2020) activities. Research indicates that galangin reduces respiratory inflammation via the nuclear factor-κB (NF-κB) signaling pathway (Zha et al., 2013) and may regulate pulmonary fibrosis and airway remodeling by decreasing reactive oxygen species production and mitogen-activated protein kinase (MAPK)/Akt phosphorylation in asthmatic animal models (Liu et al., 2015). However, no studies have explored the effects of galangin on mucin gene expression in airway epithelial cells.

Figure 1. Chemical structure of galangin.

Therefore, in this study, we investigated the effects of galangin on MUC5AC mRNA expression and glycoprotein production induced by phorbol esters in NCI-H292 cells. The NCI-H292 human pulmonary mucoepidermoid cell line is commonly used to study the signaling pathways involved in airway mucin gene expression (Li et al., 1997; Takeyama et al., 1999; Shao et al., 2003). Phorbol esters are known to stimulate MUC5AC gene expression via a pathway involving intracellular NF-κB signaling (Ishinaga et al., 2005; Laos et al., 2006; Wu et al., 2007; Fujisawa et al., 2009; Kim et al., 2012; Kurakula et al., 2015; Garvin et al., 2016; Choi et al., 2019; Li et al., 2020). Therefore, to elucidate the mechanisms through which galangin exerts its effects, we examined whether galangin affected activation of the NF-κB signaling pathway induced by phorbol esters in NCI-H292 cells.

Materials

Anti-β-actin (cat. no. sc-8432), anti-NF-κB p65 (cat. no. sc-8008), and anti-NF-κB inhibitor α (IκBα; cat. no. sc-371) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-inhibitor of κB kinase (IKK) α/β (Ser176/180; cat. no. 2687), phospho-specific anti-IκBα (serine 32/36; cat. no. 9246), and phospho-specific anti-p65 (serine 536; cat. no. 3036S) antibodies were sourced from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-nuclear matrix protein p84 (cat. no. ab-487) antibodies were purchased from Abcam (Cambridge, MA, USA). Goat anti-mouse IgG (cat. no. 401215) and goat anti-rabbit IgG (cat. no. 401315) secondary antibodies were purchased from Calbiochem (Carlsbad, CA, USA). Galangin (purity: 98.0%; Fig. 1) and other chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Culture of NCI-H292 cells

NCI-H292 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and HEPES (25 mM) at 37°C in a humidified incubator with 5% CO₂ and 95% air. For serum deprivation, confluent cells were washed twice with phosphate-buffered saline (PBS) and then cultured in RPMI 1640 medium supplemented with 0.2% fetal bovine serum for 24 h.

Treatment of cells with galangin

Following serum deprivation, cells were pretreated with varying concentrations of galangin for 30 min and then exposed to phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) for 24 h in serum-free RPMI 1640. Galangin was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium at a final DMSO concentration of 0.5%. The pH of these solutions ranged from 7.0 to 7.4. Both the culture medium and 0.5% DMSO did not affect mucin gene expression or NF-κB signaling in NCI-H292 cells. After 24 h, cells were lysed using a buffer solution containing 20 mM Tris, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, and a protease inhibitor cocktail (Roche Diagnostics, IN, USA) to measure MUC5AC glycoprotein production (in 24-well culture plates). Total RNA was extracted to assess MUC5AC mRNA expression (in 6-well culture plates) using reverse transcription polymerase chain reaction (RT-PCR). For western blot analysis, the cells were treated with galangin for 24 h and then with PMA for 30 min.

Quantitative analysis of MUC5AC

Airway MUC5AC mucin production was assessed by enzyme-linked immunosorbent assay (ELISA). Cell lysates were prepared in PBS at a 1:10 dilution, and 100 μL of each sample was incubated at 42°C in a 96-well plate until dry. The plates were washed three times with PBS and blocked with 2% bovine serum albumin (fraction V) for 1 h at room temperature. After additional washes with PBS, the plates were incubated with 100 μL of 45M1, a mouse monoclonal anti-MUC5AC antibody (1:200; NeoMarkers, CA, USA), diluted in PBS containing 0.05% Tween 20. After 1 h, the wells were washed three times with PBS, and 100 μL horseradish peroxidase-goat anti-mouse IgG conjugate (1:3,000) was added to each well. Following another hour of incubation and washing, the color reaction was developed using 3,3′,5,5′-tetramethylbenzidine peroxide solution and stopped with 1 N H₂SO₄. The absorbance was measured at 450 nm.

Isolation of total RNA and RT-PCR

Total RNA was extracted using an Easy-BLUE Extraction Kit (INTRON Biotechnology, Inc., Seongnam, Korea) and reverse-transcribed using AccuPower RT Premix (BIONEER Corporation, Daejeon, Korea), according to the manufacturer’s instructions. Two micrograms of total RNA was mixed with 1 μg of oligo(dT) in a final volume of 50 μL for the RT reaction. Two microliters of the RT product was PCR-amplified in a 25 μL mixture using ThermoPrime Plus DNA Polymerase (ABgene, Rochester, NY, USA). The MUC5AC primers used were as follows: (forward) 5′-TGATCATCCAGCAGGGCT-3′ and (reverse) 5′-CCGAGCTCAGAGGACATATGGG-3′. The Rig/S15 rRNA primers, which served as a quantitative control, were as follows: (forward) 5′-TTCCGCAAGTTCACCTACC-3′ and (reverse) 5′-CGGGCCGGCCATGCTTTACG-3′. The PCR conditions included an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. PCR products (5 μL) were analyzed using 1% agarose gel electrophoresis and visualized using ethidium bromide under a transilluminator.

Preparation of nuclear and cytosolic extracts

NCI-H292 cells (grown to confluence in a 150-mm dish) were pretreated with 1, 5, 10, or 20 μM galangin for 24 h at 37°C, then stimulated with PMA (50 ng/mL) for 30 min in serum-free RPMI 1640. Following galangin treatment, cells were harvested with trypsin-EDTA solution and centrifuged in a microcentrifuge (1,200 rpm for 3 min at 4°C), and the cell pellet was washed with PBS. Cytoplasmic and nuclear protein fractions were extracted using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. The extracts were stored at −20°C, and protein content was quantified using the Bradford method.

Western blotting for protein detection

Cytosolic and nuclear protein extracts (50 μg each) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and probed with the appropriate primary antibody in blocking buffer overnight at 4°C. After washing with PBS, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized using an enhanced chemiluminescence kit (Pierce ECL western blotting substrate; Thermo Scientific, Waltham, MA, USA).

Statistical analysis

Data are expressed as means ± standard errors of the means, with individual group means converted to percent control. Differences between groups were analyzed using one-way analysis of variance with Holm-Sidak post-hoc testing. Statistical significance was set at p<0.05.

Effects of galangin on PMA-induced mRNA expression and MUC5AC glycoprotein production

Our results showed that galangin suppressed PMA-induced MUC5AC glycoprotein production in a dose-dependent manner. The amounts of MUC5AC in the cells of galangin-treated cultures were 100% ± 9% (control), 359% ± 53% (10 ng/mL PMA alone), 262% ± 29% (PMA plus 1 μM galangin), 195% ± 16% (PMA plus 5 μM galangin), 188% ± 23% (PMA plus 10 μM galangin), and 181% ± 11% (PMA plus 20 μM galangin), as shown in Fig. 2. PMA-induced MUC5AC mRNA expression was also inhibited by galangin pretreatment (Fig. 3). Cytotoxicity was checked using methyl thiazolyl diphenyl tetrazolium assays, and the results showed that galangin did not have cytotoxic effects at 1, 5, 10, or 20 µM (data not shown).

Figure 2. Effects of galangin on phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC glycoprotein production in NCI-H292 cells. NCI-H292 cells were pretreated with varying concentrations of galangin for 30 min and then stimulated with PMA (10 ng/mL) for 24 h. Cell lysates were collected to measure MUC5AC glycoprotein production by enzyme-linked immunosorbent assay (ELISA). Each bar represents the mean ± standard error of the mean of three culture wells compared with that of the control set at 100%. Three independent experiments were performed, and representative data are shown. *Significantly different from the control (p<0.05). ^†Significantly different from PMA treatment alone (p<0.05). cont: control, G: galangin, concentrations are shown in μM.

Figure 3. Effects of galangin on phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC mRNA expression in NCI-H292 cells. NCI-H292 cells were pretreated with varying concentrations of galangin for 30 min and then stimulated with PMA (10 ng/mL) for 24 h. Cell lysates were collected for measurement of MUC5AC mRNA expression using RT-PCR. Three independent experiments were performed, and representative data are shown. *Significantly different from the control (p<0.05). ^†Significantly different from PMA treatment alone (p<0.05). cont: control, G: galangin, concentrations are shown in μM.

Effects of galangin on PMA-induced phosphorylation and degradation of IκBα

To activate NF-κB, PMA induces the phosphorylation of IKK, and phosphorylated IKK sequentially phosphorylates IκBα. Phosphorylated IκBα then dissociates from NF-κB and is degraded. Thus, we next checked whether galangin affected the phosphorylation and degradation of IκBα induced by PMA. As shown in Fig. 4, PMA increased IκBα phosphorylation, whereas galangin suppressed this effect. Similarly, the PMA-induced increase in IκBα degradation was suppressed by galangin (Fig. 4).

Figure 4. Effects of galangin on phorbol 12-myristate 13-acetate (PMA)-induced IκBα phosphorylation and IκBα degradation in NCI-H292 cells. NCI-H292 cells were incubated with varying concentrations of galangin for 24 h and then treated with PMA (50 ng/mL) for 30 min. Cytosolic extracts were fractionated and then subjected to western blot analysis using a phospho-specific anti-IκBα (Ser 32/36) antibody or an antibody against IκBα. Equal protein loading was evaluated by analysis of β-actin levels. *Significantly different from the control (p<0.05). ^†Significantly different from PMA treatment alone (p<0.05). cont: control, G: galangin, IκBα: NF-κB inhibitor α, concentrations are shown in μM.

Effects of galangin on PMA-induced phosphorylation and nuclear translocation of NF-κB p65

After NF-κB activation, the protein translocates from the cytosol to the nucleus, where it binds to specific sites on DNA. This assembly of NF-κB/DNA recruits RNA polymerase, and the resulting mRNA is translated into specific proteins, including MUC5AC. The transcriptional activity of NF-κB p65 is phosphorylation-dependent. As shown in Fig. 5, PMA increased the phosphorylation of p65, whereas galangin suppressed this phosphorylation event. Overall, galangin blocked the PMA-induced nuclear translocation of NF-κB p65.

Figure 5. Effects of galangin on phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation and translocation of NF-κB p65 in NCI-H292 cells. Nuclear protein extracts were prepared and subjected to western blotting using a phospho-specific anti-p65 (Ser 536) antibody and an antibody against p65. p84 levels were analyzed as a loading control. The results are representative of three independent experiments. *Significantly different from the control (p<0.05). ^†Significantly different from PMA treatment alone (p<0.05). cont: control, G: galangin, concentrations are shown in μM.

Currently, no specific medications are available to regulate the production or secretion of MUC5AC in airway mucus. Although various treatments, such as hypertonic saline, N-acetyl L-cysteine, glyceryl guaiacolate, bromhexine, glucocorticoids, ambroxol, azithromycin, dornase-α, erdosteine, letocysteine, 2-mercaptoethane sulfonate sodium, mannitol, S-carboxymethyl cysteine, myrtol, and sobrerol, are used to treat pulmonary diseases characterized by mucus hypersecretion, they have not demonstrated significant clinical efficacy and often produce various side effects (Li et al., 2020).

Therefore, the development of novel agents capable of controlling the production and/or secretion of pulmonary mucins by targeting their degradation or biosynthesis holds great promise. The primary aim of treatment should be to effectively manage inflammatory responses in patients with pulmonary disease. Natural products derived from medicinal plants that are traditionally used for the treatment of inflammatory conditions of the pulmonary airways are particularly promising. These natural compounds may provide novel approaches to regulate abnormal mucin production and secretion, thereby offering new therapeutic options for these diseases (Kim et al., 2012; Ryu et al., 2013, 2014; Seo et al., 2014; Sikder et al., 2014; Lee et al., 2015; Kim et al., 2016; Li et al., 2020; Hossain et al., 2022a, 2022b; Kim et al., 2023; Ryu et al., 2023).

In our study, galangin reduced PMA-stimulated MUC5AC mRNA expression and MUC5AC glycoprotein production (Fig. 2, 3). These results suggest that galangin modulates the expression of pulmonary mucin genes by directly acting on airway epithelial cells. To the best of our knowledge, this is the first report highlighting the regulatory effects of galangin on pulmonary mucin gene expression at both the transcriptional and translational levels.

Flavonoids, including galangin, are polyphenolic compounds found in edible plants and fruits. They exhibit pharmacological properties, such as antioxidant, anticancer, antimicrobial, anti-inflammatory, and radical-scavenging activities. The anti-inflammatory effects of flavonoids often involve suppression of the synthesis and activity of various pro-inflammatory mediators, including cytokines, eicosanoids, C-reactive protein, and adhesion molecules. Their molecular action may be mediated through the regulation of transcription factors like activating protein-1 and NF-κB (Serafini et al., 2010).

NF-κB, a family of inducible transcription factors, includes five structural subunits: NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel. These proteins regulate the transcription of target genes by binding to specific DNA elements, called κB enhancers, via the formation of various dimers. Inhibitory proteins, including members of the IκB family, typically sequester NF-κB proteins in the cytosol. Among these, IκBα is a key focus. In the canonical pathway, NF-κB or Rel proteins are inhibited by IκB. Various regulators, such as inflammatory mediators, growth factors, bacterial stimulants, and antigen receptors, activate the IκK complex, leading to IκB phosphorylation, proteasomal degradation, and release of NF-κB/Rel complexes. The NF-κB signaling pathway is crucial for mediating immune responses and inflammation (Huang et al., 2018; Lee et al., 2019; Shang et al., 2019). Under pathological conditions, NF-κB expressed in airway epithelial cells is involved in mucin hypersecretion and cytokine regulation (Liu et al., 2020), and its activation can be abnormal in asthma (Huang et al., 2018). The expression of MUC5AC can be triggered by inflammatory mediators that activate various transcription factors, including NF-κB (Fujisawa et al., 2009; Kurakula et al., 2015; Garvin et al., 2016). PMA has been shown to induce MUC5AC expression via a pathway involving NF-κB signaling (Ishinaga et al., 2005; Laos et al., 2006; Wu et al., 2007; Kim et al., 2012).

Our study demonstrated that galangin inhibited the phosphorylation and nuclear translocation of NF-κB p65 by affecting IκBα phosphorylation and degradation in human airway epithelial cells. These results suggested that the effects of galangin on MUC5AC gene expression may be at least partly due to its ability to reduce IκBα degradation and NF-κB p65 nuclear translocation (Fig. 4, 5, Supplementary Fig. 1). However, galangin may also affect MUC5AC gene expression through intracellular signaling pathways other than the NF-κB pathway. For example, we investigated whether galangin influenced MUC5AC gene expression through signaling pathways mediated by epidermal growth factor (EGF) receptor. The results indicated that galangin did not affect EGF-induced MUC5AC mRNA expression or glycoprotein production in NCI-H292 cells (Supplementary Fig. 2). Moreover, galangin did not affect the EGF-induced MAPK signaling pathway, including EGF receptor phosphorylation, p38 MAPK phosphorylation, extracellular signal-regulated kinase 1/2 phosphorylation, or the nuclear expression of specificity protein-1 (Supplementary Fig. 3).

Overall, the inhibitory effect of galangin on airway MUC5AC expression appeared to be primarily mediated through the modulation of PMA-stimulated IκBα degradation and NF-κB p65 nuclear translocation (Fig. 6). These findings highlighted the potential of galangin as a novel mucoregulator in inflammatory pulmonary diseases. Future research should focus on modifying and optimizing the chemical structure of galangin, a flavonol with anti-inflammatory, antioxidant, and anti-fibrotic activities found in the medicinal plants A. officinarum and A. galanga, using medicinal chemistry and bioinformatics technologies to enhance its regulatory effects on pulmonary mucus secretion and production.

Figure 6. A potential mechanism of action of galangin.

This work was supported by BK21 FOUR Program by Chungnam National University Research Grant, 2024.

The authors declare no conflicts of interest.