HX110B Alleviates Excessive Cough and Sputum Overproduction via Downregulation of MUC5AC and MUC5B

Abstract

Introduction

HX110B is an herbal formulation that contains Taraxacum officinale, Dioscorea batatas, and Schizonepeta tenuifolia. Each plant is known to relieve respiratory symptoms. In our previous study, administering HX110B ameliorated acute lung injury (ALI) caused by lipopolysaccharide (LPS) in mice. Herein, we investigated the antitussive and expectorant activities of HX110B in vitro and in vivo.

Methods

The antitussive effects of HX110B were tested in citric acid-induced guinea pig model. The expectorant activities of HX110B were examined by phenol red secretion assay. Additionally, it was evaluated whether HX110B regulates mucin viscosity and affects the expression of MUC5AC and MUC5B in A549 cells.

Results

The frequency of cough evoked by exposure to citric acid was effectively suppressed by HX110B. Next, we observed an increase in the phenol red secretion after administering HX110B, indicating that it stimulates mucus expectoration. In addition, the viscosity of mucus was reduced and the expression of MUC5AC and MUC5B was inhibited after treatment with HX110B.

Conclusion

These data indicate that HX110B could be used in the treatment of excessive and productive cough and sputum, providing new insights for its application.

Keywords

HX110B cough sputum MUC5AC MUC5B

Introduction

Cough is the most prevalent symptom of acute or chronic respiratory diseases, with an annual financial burden of approximately 3.6 billion dollars in the USA.¹ Coughing is a reflex that protects the respiratory tract from foreign irritants.² However, excessive coughing not only exacerbates symptoms of respiratory diseases, but also adversely affects the quality of life of patients.³ Currently, natural opiates and opiate derivatives (codeine and dextromethorphan) are widely used to relieve cough. However, these opioid alkaloids have side effects such as constipation, sedation, and addiction tendencies.⁴ Thus, there is a huge unmet need for safe antitussives without those side effects.

Sputum is a type of mucus that has been expectorated by coughing. Airway mucus serves as a physical barrier that protects the lungs from inhaled foreign materials.⁵ However, overproduction of sputum may block mucociliary clearance and cause airway obstruction, leading to respiratory distress and excessive coughing.^6,7 Therefore, agents exhibiting expectorant activities such as N-acetylcysteine (NAC) and bronchodilators are prescribed to treat chronic cough.⁸ Drugs that influence the secretion, quality, and clearance of sputum are known as mucoactive agents. They include expectorants, mucolytics, mucoregulators, and mucokinetics. They reduce mucus viscosity to ease expectoration.⁹ However, mucoactive agents, such as NAC and guaifenesin, have side effects such as stomach discomfort, diarrhea, nausea, vomiting, and headache.^7,10 Taken together, there is still a high unmet need for safer mucoactive agents.

Mucus is a viscoelastic colloid primarily produced by cells that line the lumen of the respiratory, gastrointestinal, and genitourinary systems. It contains water, salts, lipids, globular proteins, and mucin glycoproteins.¹¹ There are two main subfamilies of mucin proteins: gel-forming (secretory) and membrane-bound mucin.^12,13 Among the airway mucins, gel-forming mucins 5AC and 5B (MUC5AC and MUC5B) are the most common, and they contribute to the viscosity and viscoelasticity of mucus.^14,15 MUC5B is mainly produced in submucosal gland cells, and MUC5AC is produced in goblet cells which are located in airway epithelium.^16,17 Although MUC5AC and MUC5B are involved in mucociliary clearance of sputum under normal conditions, their overexpression correlates with the onset of many muco-obstructive diseases.¹⁸ Hypersecretion of MUC5AC leads to the progressive accumulation of mucus and airway plugging in asthma and chronic obstructive pulmonary disease.^19,20 Overexpression of MUC5B leads to mucosal dysfunction associated with idiopathic pulmonary fibrosis.²¹ Additionally, mucus plugging induced by excessive secretion of MUC5AC and MUC5B is known to increase airway hypersensitivity, which causes cough.^22,23 Therefore, developing effective therapeutic agents that control mucus production under abnormal conditions assumes significance.

Traditional herbal formulas are used to treat many respiratory diseases worldwide. HX110B is an example and it contains three plants: Dioscorea batatas, Taraxacum officinale, and Schizonepeta tenuifolia.²⁴ These plants are dietary supplements in Korea and are known to possess many therapeutic effects. D. batatas possesses antioxidant and anti-inflammatory properties.^24,25 T. officinale exerts antioxidant properties as they relieve patients of cough and fever.^26,27 S. tenuifolia possesses antimicrobial effects against respiratory infections, as well as anti-inflammatory effects by inhibiting TLR4 signaling.^28,29 Whereas the effects of HX110B on productive cough is still unvailed.

Herein, we demonstrated that HX110B ameliorates cough- and sputum-related symptoms. It equally decreased the frequency of cough responses caused by citric acid in guinea pigs. Moreover, HX110B enhanced phenol red secretion, indicating its expectorant activity. The mucolytic and mucoregulatory effects of HX110B were evaluated by measuring the viscosity of mucin solution and expression levels of airway mucins. HX110B decreased the viscosity and inhibited the expression of gel-forming mucins. These findings indicate that HX110B could be used in the treatment of excessive and productive cough and sputum.

Material & Methods

Preparation of HX110B Extract

HX110B was obtained as previously mentioned.²⁴ Dioscorea batatas, Taraxacum officinale and Schizonepeta tenuifolia were purchased from Humanherb Co., Ltd (Gyeongsan, Korea). Their species were determined through DNA sequencing analysis (miDNA Genome Research Institute, Kunsan, Korea). TADIOS was prepared by mixing three dried herbs in a ratio of 1:1:1, and then extracting the mixture with 25% ethanol. The voucher specimens used in this study were kept at the herbarium of Helixmith Co., Ltd (Seoul, Korea). Voucher Specimen No: S14161213 for Dioscorea batatas; F02170525 for Taraxacum officinale; H2719121 for Schizonepeta tenuifolia.

Experimental Animals

Male Hartley guinea pigs (8 weeks old) and C57BL/6N mice (6 weeks old) were obtained from Orient Bio (Korea) and Koatech (Korea). All animals were kept at constant temperature (22°C ± 2°C) under a 12 h light/dark cycle and housed in certified standard laboratory cages. All animal experiments were conducted following the guidelines of Knotus Co. Ltd The protocol was accepted by the Institutional Animal Care and Use Committee of Knotus Co., Ltd (approval number for guinea pigs: 21-KE-717; approval number for mice: 21-KE-724).

Antitussive Activity Evaluation

The guinea pigs were separated into six groups: Normal control (NC) group, cough-induction group, Theobromine (50 mg/kg) group, HX110B (25 mg/kg) group, HX110B (50 mg/kg) group, and HX110B (100 mg/kg) group (n = 10 per group). The test substances were given orally to animals at a dose of 10 mL/kg, daily for 8 days. Theobromine (Sigma, USA) and HX110B were dissolved in 2% hydroxypropyl methylcellulose (HPMC; Shin-Etsu Chemical, Japan) and filtered. On the last three days, the animals were placed in a glass chamber one hour after they ingested the test substances. They were inhaled with citric acid (0.2 M, Sigma) for 10 min and the frequency of coughs per 15 min was noted. For the normal control group, phosphate-buffered saline (PBS) or distilled water (DW) was exposed to the same conditions. The rate of inhibition of cough was calculated as follows: inhibition rate (%) = [(C₀-C_t)/C₀× 100]. (C0: Frequency of cough in the cough induction group, Ct: Frequency of cough in the treatment group).

Expectorant Activity Evaluation

The animals were separated into six groups: Normal control (NC) group, phenol red induction group, ambroxol (10 mg/kg) group, HX110B (100 mg/kg) group, HX110B (200 mg/kg) group, and HX110B (400 mg/kg) group (n = 15 per group). The test substances were given orally to animals at a dose of 10 mL/kg, daily for 8 days. Ambroxol hydrochloride (Sigma) and HX110B were dissolved in 2% HPMC (Sigma) and filtered. On the last day, 500 mg/kg phenol red was intraperitoneally injected 30 min after administering the test substances. 30 min after phenol red injection, the trachea was prepared. The trachea was washed in saline, and then centrifuged. Moreover, we added 1 N NaOH to the supernatant obtained (0.1 mL of 1 N NaOH per 1 mL of supernatant), and we measured the absorbance at 546 nm. In the normal control group, normal saline was injected instead of phenol red.

Viscosity Measurement

Mucin (Sigma) was prepared by mixing 1 M NaOH (Sigma) at a ratio of 1:2 and 40 mg/mL (4%) NAC (N-acetylcysteine; Sigma). Different concentrations of HX110B (2, 4, 8 mg/mL) were then added. Seven hours later, the viscosities of all samples were obtained using a rotational viscometer (AMETEK Brookfield, Middleboro, USA).

Cell Culture and Reagents

We obtained A549 cells from the ATCC (USA). Moreover, we maintained A549 cells using RPMI1640 culture medium (Invitrogen, USA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 unit/mL of penicillin, and 0.1 mg/mL of streptomycin. We purchased PMA from Sigma.

qRT-PCR Analysis

RNA extraction from cells was performed using TRIzol reagent (Invitrogen). Oligo dT primers (QIAGEN, Germany) and Reverse Transcriptase XL (avian myeloblastosis virus (AMV)) (Takara, Japan) were used to synthesize cDNA. The real-time quantitative PCR was conducted with SYBR Premix (Takara), and detailed PCR conditions are as follows: denaturation: 95 °C, 5 s; annealing and extension: 60 °C, 30 s. Table S1 presents the primer sequences (Bioneer, Korea) synthesized for PCR.

Statistical Analysis

All parameters are shown as mean ± SEM We performed an unpaired Student's t-test or one-way ANOVA with Tukey correction, using GraphPad Prism software (GraphPad, USA).

Result

HX110B Suppresses Citric Acid-Induced Cough in Guinea Pigs

The antitussive effects of HX110B were tested in a citric acid-induced cough guinea pig model. Guinea pigs were inhaled with citric acid, and the frequency of cough reflex in 15 min was recorded. 50 mg/kg of theobromine or HX110B (25, 50, or 100 mg/mL) was administered 30 min before the cough reflex was induced. Cough responses were triggered more than 20 times in the control group; however, this change in frequency was dose-dependently reduced after administering HX110B (Figure 1).

Figure 1.

Antitussive effects of HX110B on citric acid-induced cough reflex. The number of citric acid-mediated cough (A) and cough reflex inhibition rate (B) following HX110B administration in Guinea pigs. ####p < 0.0001 versus the NC group; ****p < 0.0001 versus the cough-induction group.

HX110B Enhances Expectorant Activity in Mice

The expectorant activity of HX110B was tested using tracheobronchial secretion assay in mice. Thirty minutes after administering ambroxol or HX110B (100, 200, and 400 mg/mL), we injected phenol red solution intraperitoneally, and the concentration of the injected secretion from the bronchi was measured. In the HX110B -treated group, phenol red secretion rate was significantly increased compared to the vehicle and ambroxol-treated group (Figure 2). This reveals that HX110B affects mucus secretion.

Figure 2.

Expectorant activity of HX110B in phenol red-treated mice. The amount of tracheal phenol red secretion was measured after HX110B treatment. ###p < 0.001 versus the NC group; *p < 0.05, ****p < 0.0001 versus the vehicle-treated group.

HX110B Decreases Viscosity of Mucin Solution

Mucolysis may contribute to the expectoration of excessive sputum by reducing its viscosity. To investigate the dose-related effects of HX110B, multiple concentrations of HX110B were treated to the mucin solution, and the change in viscosity was measured after 7 h. As a result, significant dose-dependent inhibition of the viscosity was observed in the HX110B -treated group (Figure 3A).

Figure 3.

Mucolytic effects of HX110B to reduce mucin viscosity. (A) Evaluated dose-dependent mucolytic effects of HX110B (2, 4, and 8 mg/mL), (B) time kinetics of the mucous viscosity after 8 mg/mL of HX110B treatment. ###p < 0.001 versus the TDW group; ****p < 0.0001 versus the vehicle-treated group.

To test the mucolytic effects of HX110B, NAC (8 mg/mL) was treated to the mucin solution. After 6 h, the viscosity increased up to 300 cP in the vehicle or NAC-treated group. However, the HX110B-treated group exhibited a lower viscosity than other groups that lasted for 72 h (Figure 3B).

HX110B Inhibits the Expression of Mucin Genes in Alveolar Epithelial Cells

MUC5AC and MUC5B are the most common mucins, and they contribute to the viscosity of airway mucus.^14,15 Calcium signaling is vital in the expression of MUC5AC and MUC5B.^30–33 To investigate whether HX110B affects the expression level of MUC5AC and MUC5B, cells were co-cultured with PMA and various concentrations of HX110B (0.5, 1, and 2 mg/mL). As shown in Figure 4A and B, HX110B considerably downregulated the PMA-induced expression of MUC5AC and MUC5B. Similarly, the amount of MUC5AC and MUC5B protein levels were dose-dependently decreased following HX110B treatment (Fig. S1).

Figure 4.

Effects of HX110B on the expression of MUC5AC and MUC5B in A549 cells. (A, B) mRNA expression levels of MUC5AC and MUC5B in PMA-treated A549 cells. #p < 0.05, ###p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the PMA group.

Discussion

In this study, we showed that HX110B relieves excessive cough and sputum-related symptoms. It inhibited citric acid-induced cough reflex in vivo. Moreover, it promoted phenol red secretion in mice, indicating its expectorant ability. The viscosity of the mucin solution significantly decreased after HX110B treatment. In addition, HX110B significantly downregulated the expression of MUC5AC and MUC5B. Thus, HX110B is a potential treatment option for chronic cough and overproduction of sputum that aggravates various respiratory diseases.

The stimuli for cough reflex include foreign irritants, respiratory inflammation, and histamine-induced allergic responses. HX110B attenuated the inflammatory reactions in the experimental mouse model of ALI induced by LPS.²⁴ Furthermore, it significantly suppressed the expression of allergic mediators that induce coughing in an OVA-induced asthmatic mouse model (unpublished data). Therefore, HX110B suppresses the cough reflex and factors that promote cough in many respiratory diseases.

The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are closely involved in the calcium-induced expression of MUC5AC and MUC5B.^34–36 Intracellular calcium influx activates the MAPK^37,38 and NF-κB signaling pathways.^39,40 All three constituents of HX110B are known to suppress the MAPK or NF-κB signaling pathways and therefore ameliorate inflammatory diseases. D. batatas also inhibited the NF-κB signaling pathway, which leads to anti-inflammatory effects.^41,42 T. officinale alleviated oxidative stress-induced cytotoxicity through Nrf2 signaling.⁴³ S. tenuifolia inhibits MAPK signaling as it exerts anti-inflammatory effects.⁴⁴ Therefore, we hypothesized that HX110B regulates the expression of MUC5AC and MUC5B in a similar manner. Studies to explore the mechanisms of action of HX110B in regulating calcium-activated MAPK and NF-κB signaling pathways are ongoing presently.

Transient receptor potential ankyrin-1 (TRPA1) evokes the cough reflex by stimulating sensory neurons in the vagal nerve of the airway as it enhances calcium influx through it. TRPA1 channels are activated by lower temperature, inflammatory mediators, and environmental irritants and send sensory information to the central nervous system.^45,46 Citric acid-induced cough reflex is caused by calcium influx via TRPA1 receptor.⁴⁷ TRPA1 also regulates the expression of MUC5AC and MUC5B via the stimulation of airway sensory nerve fibers. For example, activation of TRPA1 by particulate matter exaggerates the overproduction of MUC5AC,^48,49 and TRPA1 agonists induce MUC5B secretion.⁵⁰ Based on the mechanisms of TRPA1, several trials to treat various respiratory diseases by blocking TRPA1 are ongoing.^46,47,50,51 Therefore, one of the key mechanisms of action of HX110B that exerts therapeutic effects on various respiratory diseases may be the modulation of the TRPA1 activity. Interestingly, caffeic acid contained in HX110B has been reported to inhibit AITC-induced TRPA1 activation.⁵² A binding assay and molecular docking simulation study for TRPA1 is ongoing to elucidate these effects.

Herbal extracts have been used to relieve patients of productive cough for thousands of years. For example, Synatura^® syrup, which consists of ivy leaf and Coptis rhizome, has been approved for treatment of chronic cough by the Korea Food and Drug Administration (KFDA). As a mixture of various chemical compounds having properties of multi-component and multi-target, their antitussive and expectorant abilities are controlled by many complex factors. According to the results of our recent phytochemical analysis, HX110B contains chlorogenic acid, caffeic acid, coumaric acid, rutin, hesperidin, chicoric acid, rosmarinic acid, and luteolin 3'-glucuronide.⁵³ These components have been reported to have various activities related to the alleviation of excessive cough and sputum. Chicoric acid, which is contained in the highest proportion in HX110B, reduces inflammatory cytokines and inflammatory cell infiltration in bronchoalveolar lavage fluid, suggesting its anti-inflammatory effects.⁵⁴ Chlorogenic acid, which belongs to the caffeoylquinic acid group, effectively inhibits ammonia-induced cough and also increases phenol red secretion through expectorant activity.³ Caffeic acid and rosmarinic acid dose-dependently inhibit the expression of MUC5AC induced by PMA in NCI-H292 cells.⁵⁵ In addition, rosmarinic acid has been reported to effectively suppress airway hypersensitivity and improve OVA-induced asthma.^56,57 Luteolin attenuates mucus overproduction by downregulating the expression of MUC5AC mucin.⁵⁸ Thus, the various active compounds contained in HX110B are expected to show a synergistic effect on improving cough and sputum when mixed with each other. Interestingly, it has been reported that when caffeic acid, coumaric acid, and chlorogenic acid contained in HX110B are mixed, the activity in inhibiting the expression of MUC5AC is much higher than when treated alone.⁵⁹ Therefore, HX110B is predicted to have a greater effect on chronic cough and sputum than other regimens that regulate only a single target. Identifying the optimal mixture composition of the active compounds contained in HX110B is considered to be a very important process in the efficacy evaluation and quality control process related to the future pharmaceutical development of HX110B. To this end, we plan to analyze the change in bioactivity after mixing the components of HX110B under various conditions considering the actual contents in the extract.

The limitation of this study is that although it suggests the possibility that HX110B may improve excessive cough and sputum by regulating the expression of MUC5AC and MUC5B, it has not been completely elucidated. It is well known through many studies that increased expression of MUC5AC and MUC5B causes chronic cough and sputum, which worsens respiratory diseases such as asthma. Therefore, since HX110B reduces the expression of these mucin genes in alveolar epithelial cells, it is highly likely that this result will improve cough and sputum. In order to clearly prove this possibility, it will be necessary to first evaluate what changes in the expression of MUC5AC and MUC5B in the trachea and lung tissue occur when HX110B is administered to an animal disease model. We plan to conduct follow-up studies on this and report separately.

Conclusion

Taken together, we elucidated the therapeutic potential of HX110B as an antitussive and expectorant agent. HX110B is safe as it has been used in treating other diseases. In addition, it showed no adverse effects in acute or repeated-dose toxicity studies. Thus, a clinical trial is ongoing to demonstrate the therapeutic potential of HX110B against chronic cough and phlegm.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251318123 - Supplemental material for HX110B Alleviates Excessive Cough and Sputum Overproduction via Downregulation of MUC5AC and MUC5B

Supplemental material, sj-docx-1-npx-10.1177_1934578X251318123 for HX110B Alleviates Excessive Cough and Sputum Overproduction via Downregulation of MUC5AC and MUC5B by Eujung Kim, Jisun Song, Jungkyu Lee, Sooyeon Choi, Yoonseon Jeong, Jong-Hyung Park, Chang Hyung Lee, In-Jeong Nam, Hyun Myung Lee, Seungyeon Jeong, Soojin Lee, Doo Suk Lee and Wonwoo Lee in Natural Product Communications

Footnotes

Acknowledgements

Authors gratefully acknowledge animal experiment support from Knotus Co., Ld

Declaration of Conflicting Interest

All authors are the employees of Helixmith Co., Ld

Ethical Approval

The animal experimental protocol was accepted by the Institutional Animal Care and Use Committee of Knotus Co., Ltd (approval number for guinea pigs: 21-KE-717; approval number for mice: 21-KE-724).

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Wonwoo Lee

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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