Investigating the Therapeutic Potential of Agarwood Nanoemulsion in Modulating TGF-β-Induced Airway Remodelling in BEAS-2B Cells

TL;DR

This study explores how Agarwood Nanoemulsion may help reduce airway changes caused by TGF-β in lung cells, offering a new approach to treating chronic respiratory diseases.

Contribution

The study introduces Agarwood Nanoemulsion as a novel therapeutic candidate targeting TGF-β-induced airway remodelling.

Findings

Agarwood Nanoemulsion reduces TGF-β-induced oxidative stress and restores nitric oxide production in BEAS-2B cells.

AW-NE inhibits TGF-β-induced cell migration and decreases key inflammatory proteins like MMP-9, angiogenin, and PTX-3.

The findings suggest AW-NE addresses underlying pathophysiology rather than just symptoms of respiratory diseases.

Abstract

Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis are significant global health concerns, characterised by inflammation, oxidative stress, and airway remodelling. These processes are driven by multiple cytokines, with transforming growth factor-beta (TGF-β) playing a central role in the remodelling process. TGF-β triggers pathways that promote epithelial-mesenchymal transition (EMT), excessive extracellular matrix deposition, and increased oxidative stress, all of which contribute to airway remodelling. Despite availability of therapies including corticosteroids and bronchodilators that offer symptomatic relief, these fail to address the underlying causes of oxidative damage, persistent inflammation, and fibrosis, limiting long-term effectiveness. This study investigates the effects of Agarwood Nanoemulsion (AW-NE) on TGF-β-induced oxidative stress, inflammation, and airway remodelling in BEAS-2B cells, in vitro. The results show that AW-NE significantly reduces oxidative stress and restores nitric oxide (NO) production, suppressed by TGF-β activation. AW-NE also inhibits TGF-β-induced cell migration, indicating potential in modulating TGF-β-induced airway remodelling. Additionally, AW-NE treatment decreased the expression of key inflammatory and pro-remodelling proteins, including MMP-9, angiogenin, and pentraxin-3 (PTX-3). These findings suggest that, while current treatments primarily manage symptoms, AW-NE shows potential in addressing the underlying pathophysiology. Further in vivo studies are required to confirm its therapeutic efficacy.

Figures

Effect of AW-NE on the cell viability of BEAS-2B Cells. BEAS-2B cells were incubated with increasing concentrations of AW-NE (0, 5, 10, 25, 50, 100, 150 and 200 µg/mL). After the treatment, the cell viability was measured *via* the MTT assay and normalised as a percentage compared to the untreated group. The results are expressed as mean ± SEM (*n* = 4, = *p* < 0.0001 with One-Way ANOVA)

Effect of AW-NE on the regulation of TGF-β stimulated NO generation. The BEAS-2B cells were pre-incubated for 24 h with or without 1 µg/mL of TGF-β. The cells were then incubated for further 24 h in the absence or presence of AW-NE (25 µg/mL). The NO production in the supernatant was measured by adding an equal volume of Griess reagent and measuring the absorbance with microplate reader. The data are represented as Mean ± SEM normalized by the untreated control (*n* = 3, ns: non-significant; *: *p* < 0.05; : *p* < 0.001, One-Way ANOVA test)

Effect of AW-NE on BEAS-2B cells migration (scratch wound assay) over a 48-h period. (A) Wound healing progress after 24 h (45% enhancement). (B) Wound healing progress after 48 h (60% enhancement). (C) Representative pictures of treatment of the AW-NE 2.5 µM and 5 µM and the wound closure over the 48 h. The data are represented as Mean ± SEM (One-way ANOVA, *n* = 3, *: *p* < 0.05; : *p* < 0.01; *: *p* < 0.001)

Effect of AW-NE on the regulation of TGF-β-induced remodeling cytokines. BEAS-2B cells were pre-incubated for 24 h with or without 1 µg/mL of TGF-β. The cells were then incubated for a further 24 h in the absence or presence of AW-NE (25 µg/mL). The relative expression levels of *MMP-9* (A), *Angiogenin* (B), and *PTX3* (C) were determined using the Human XL Cytokine Protein Array. The data are represented as Mean ± SEM (*n* = 4, *:*p* < 0.05; :*p* < 0.01 ^*#*^: *p* < 0.1;, One-Way ANOVA test)

Full text

Introduction

Chronic respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis, represent a significant global health burden, affecting an estimated 545 million people worldwide according to the World Health Organisation (WHO) [1, 2]. These conditions are responsible for approximately 3.23 million deaths annually, making COPD the third leading cause of death globally [1, 3]. Characterised by progressive airway inflammation, remodelling, and oxidative stress, chronic respiratory diseases contribute to high morbidity and mortality rates, significantly reducing the quality of life for affected individuals [2]. The intricate interplay between chronic inflammation, oxidative damage, and tissue remodelling underlies the pathophysiology of these diseases, with a central role played by the pleiotropic cytokine transforming growth factor-beta (TGF-β) [4, 5]. While current therapeutic strategies such as corticosteroids and bronchodilators provide symptomatic relief, they often fail to adequately address the underlying causes, leaving an unmet need for more targeted interventions [5–7].

TGF-β is a multifunctional cytokine that governs numerous physiological processes, including cell differentiation, proliferation, and tissue repair [8]. However, its dysregulation is implicated in various pathological conditions, especially chronic respiratory diseases. Within the respiratory system, TGF-β orchestrates epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, and angiogenesis—key processes in airway remodelling [8]. In asthma, TGF-β promotes mucus hypersecretion, subepithelial fibrosis, and airway hyperresponsiveness [9]. Similarly, in COPD, it contributes to emphysema and excessive ECM accumulation, leading to reduced airflow and irreversible structural changes [9, 10]. These pathological features underscore TGF-β’s central role in respiratory disease progression and highlight it as a potential therapeutic target.

One of the hallmarks of TGF-β-driven airway remodelling is oxidative stress, which arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defence mechanisms [11, 12]. ROS contribute to tissue damage, inflammation, and fibrosis, further exacerbating respiratory dysfunction. The production of NO, a critical signalling molecule in the respiratory epithelium, is also dysregulated in TGF-β-mediated processes [13]. TGF-β, in particular, suppresses NO production by downregulating endothelial nitric oxide synthase (eNOS), thereby promoting EMT and tissue remodelling [7, 14]. Restoring NO balance and mitigating oxidative stress are essential to counteract the deleterious effects of TGF-β in chronic respiratory diseases [12, 14].

In recent years, there has been growing interest in natural products and their derivatives as potential therapeutic agents due to their bioactive properties and relatively low toxicity [15–17]. Agarwood, a resinous wood derived from species of the Aquilaria genus, has been traditionally valued in Asian medicine for its anti-inflammatory, antioxidant, and antimicrobial properties [18–20]. Recent advancements in nanotechnology have enabled the formulation of agarwood-derived compounds into nanoemulsions, enhancing their bioavailability, stability, and cellular uptake [19, 20]. Preliminary studies have demonstrated the ability of an agarwood-based nanoemulsion (AW-NE) to modulate oxidative stress and inflammation, making it a promising candidate for respiratory disease therapy [20, 21]. However, its effects on TGF-β-induced processes in the respiratory epithelium remain largely unexplored. The current study investigates the therapeutic potential of AW-NE in modulating TGF-β-induced oxidative stress, inflammation, and remodelling in BEAS-2B human bronchial epithelial cells. Mechanistically, we examine its impact on NO production, cell migration, and the expression of remodelling-associated proteins such as MMP-9, angiogenin, and pentraxin-3 (PTX3).

MMP-9, an enzyme that degrades ECM components like type IV collagen, facilitates tissue remodelling and inflammatory cell recruitment [22]. Elevated levels of MMP-9 in the lung parenchyma of asthma and COPD patients lead to basement membrane degradation and abnormal airway remodelling [22, 23]. Angiogenin, a potent angiogenic factor, drives new blood vessel formation, exacerbating airway remodelling by increasing vascular permeability and supporting inflammatory cell infiltration [22, 24]. PTX3, a protein involved in innate immunity and inflammation, regulates ECM interactions and modulates the activation of the complement system, amplifying inflammatory responses [25]. These proteins act synergistically in TGF-β-mediated pathways. For example, TGF-β enhances MMP-9 expression, which breaks down ECM to release pro-angiogenic factors like angiogenin [26]. PTX3 further perpetuates inflammation and remodelling through its interactions with ECM components [27, 28].

In the present study, we demonstrated that AW-NE counteracts the effect of TGF-β on BEAS-2B human bronchial epithelial cells by (i) restoring baseline levels of NO; (ii) restoring physiological levels of cell migration, which is increased by TGF-β treatment; and (iii) reducing the TGF-β-induced overexpression of MMP-9, angiogenin, and PTX3.

By elucidating the effects of AW-NE on these critical processes, this study aims to provide valuable insights into its potential as a novel therapeutic agent for chronic respiratory diseases. Furthermore, the findings may contribute to the development of targeted interventions that address the underlying mechanisms of airway remodelling, offering a complementary approach to existing treatments. This research not only highlights the therapeutic promise of natural product-derived nanoemulsions but also underscores the importance of innovative strategies in tackling the global burden of chronic respiratory diseases.

Methods

Agarwood Nanoemulsion Formulation

The Agarwood Nanoemulsion was prepared and characterized as described in a previous study [29]. Briefly, 200 mg of agarwood oil was placed in a 50 mL conical tube. Separately, 50 mg of Poloxamer 407 was dissolved in approximately 10 mL of purified water and vortexed until fully solubilized. This aqueous solution was gradually added to the agarwood oil at room temperature and mixed for 1 min. The resulting coarse emulsion underwent probe sonication for 15 min at 80% amplitude with a 1 Hz pulse cycle to control heat generation. The final concentrated nanoemulsion was then diluted to a total volume of 20 mL using purified water.

Cell Culture

The BEAS-2B human bronchial epithelial cells (ATCC #CRL-9609) were generously provided by Professor Alaina Ammit from the Woolcock Institute of Medical Research, Sydney, Australia. These cells were maintained between passages 15 and 25 for the duration of the study and were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, Australia, Cat. #D6046), supplemented with 5% fetal bovine serum (Sigma-Aldrich, Australia, Cat. #F9423), 100 U/ml penicillin, and 100 µg/ml streptomycin (Pen-Strep solution, Sigma-Aldrich Australia, Cat. #P4333), in a 5% CO_2_ humidified incubator.

Cell Viability Assay - MTT

The effect of AW-NE on cell viability was evaluated using the MTT assay, as described in a previous study [20]. Briefly, 5,000 cells/well were seeded in a 96-well plate and allowed to attach overnight. The next day, cells were treated with various concentrations of AW-NE (0–200 µg/mL) for 24 h. After treatment, a concentration of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT, Sigma-Merck, Australia) was added to the cells, which were incubated for a further 4 h. Upon incubation, the supernatant was removed and the formazan crystals formed were dissolved in 100 µL dimethyl sulfoxide (DMSO, Sigma Merck, Australia). The resulting absorbance was read in a microplate reader (TECAN Infinite M1000) at a wavelength of 570 nm.

NO Levels Determination with Griess Reagent

The nitric oxide (NO) levels released by BEAS-2B cells were assessed using the modified Griess reagent (Sigma-Aldrich, Australia, Cat. #G4410). Cells were seeded at 100,000 cells per well in a 6-well plate and allowed to adhere overnight. The next day, they were treated with 5 ng/mL TGF-β1 for 24 h, followed by exposure to 25 µg/mL AW-NE for another 24 h. After incubation, the culture supernatants were collected, and 100 µL was mixed with an equal volume of Griess reagent in a 96-well plate. Following a 30-minute incubation in the dark at room temperature, NO levels were measured at 540 nm using a TECAN Infinite M1000 plate reader. Blank culture media served as the control, and NO levels were expressed as a percentage relative to untreated samples.

Wound Healing Assay

To evaluate the anti-migratory effects of Agarwood Nano-Emulsion (AW-NE) on TGF-β-stimulated BEAS-2B cells, a wound-healing assay was conducted. BEAS-2B cells (100,000 cells per well) were seeded in 6-well plates and allowed to grow until full confluence at 37 °C. Successively, a scratch was created in the cell monolayer using a sterile 200 µL pipette tip, followed by five washes with sterile PBS (Sigma-Aldrich, Australia, Cat. #P3813) to remove detached cells. The remaining cells were treated with 5 ng/mL TGF-β1 alone or in combination with 25 µg/mL AW-NE for up to 48 h. Scratch closure was monitored under a light microscope at 10× magnification at 0, 24, and 48 h. The extent of wound closure was calculated and expressed as a percentage relative to the untreated control group.

Human Cytokine Protein Array

The impact of AW-NE on the expression of cytokines and proteins in TGF-β-stimulated BEAS-2B cells was evaluated using a Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Australia). BEAS-2B cells (100,000 cells per well) were plated in 6-well plates and allowed to adhere overnight. The following day, the cells were treated with 5 ng/mL TGF-β1 and incubated at 37 °C for 24 h. Subsequently, the cells were treated with 25 µg/mL AW-NE for an additional 24 h. After incubation, the cells were lysed using RIPA buffer (ThermoFisher Scientific, Australia) supplemented with protease inhibitors. A total of 300 µg of protein from each group was loaded onto the cytokine arrays and incubated overnight at 4 °C. Additional steps involving antibody binding and chemiluminescence detection were performed according to the kit protocol. Images of the arrays were captured with a ChemiDoc system, and pixel density was analysed using ImageJ software.

Statistical Analysis

All data are expressed as mean ± SEM. Statistical comparisons were performed using ordinary one-way ANOVA followed by Tukey’s multiple comparison test, utilising GraphPad Prism software. A two-tailed p-value of < 0.05 was considered indicative of statistical significance for pairwise analyses.

Results

Effect of Agarwood Nanoemulsion on Cell Viability

The cytotoxicity of AW-NE on BEAS-2B cells was evaluated through the MTT assay. AW-NE was administered in various concentrations, ranging from 0 µg/mL to 200 µg/mL, to ascertain its effects on cell viability. Results indicated a concentration-dependent effect on cellular viability. At concentrations up to 25 µg/mL, AW-NE did not significantly impact cell viability compared to the control group. Conversely, higher concentrations, specifically from 50, 100, 150, and 200 µg/mL, resulted in a significant decrease in cell viability of 67.9%, 95.3%, 95.3%, and 95.3%. respectively, thereby establishing that the safe concentration threshold for AW-NE was 25 µg/mL (Fig. 1).

Fig. 1. Effect of AW-NE on the cell viability of BEAS-2B Cells. BEAS-2B cells were incubated with increasing concentrations of AW-NE (0, 5, 10, 25, 50, 100, 150 and 200 µg/mL). After the treatment, the cell viability was measured via the MTT assay and normalised as a percentage compared to the untreated group. The results are expressed as mean ± SEM (n = 4, **** = p < 0.0001 with One-Way ANOVA)

Effect of Agarwood Nanoemulsion on NO Production

The production of NO was evaluated under conditions of TGF-β exposure alone and in conjunction with AW-NE treatment. Exposure to TGF-β resulted in a significant 33.3% reduction in NO production compared to the control samples (Fig. 2). Notably, the administration of AW-NE alongside TGF-β reinstated the levels of NO production that had been diminished by TGF-β to levels corresponding to 93.1% compared to the untreated control, indicating a potential anti-fibrotic function of AW-NE in counteracting TGF-β-altered NO modulation (Fig. 2).

Fig. 2. Effect of AW-NE on the regulation of TGF-β stimulated NO generation. The BEAS-2B cells were pre-incubated for 24 h with or without 1 µg/mL of TGF-β. The cells were then incubated for further 24 h in the absence or presence of AW-NE (25 µg/mL). The NO production in the supernatant was measured by adding an equal volume of Griess reagent and measuring the absorbance with microplate reader. The data are represented as Mean ± SEM normalized by the untreated control (n = 3, ns: non-significant; *: p < 0.05; **: p < 0.001, One-Way ANOVA test)

Effects of Agarwood Nanoemulsion on Cell Migration in TGF-β-Stimulated BEAS-2B Cells

Furthermore, the effect of AW-NE on cell migration was investigated using the scratch wound assay. The presence of TGF-β notably enhanced cell migration at both 24 h (45%, Fig. 3A) and 48 h (60%, Fig. 3B) compared to the control, respectively. Interestingly, the addition of AW-NE significantly curtailed this TGF-β-enhanced cell migration at both time intervals. These findings indicate that AW-NE may possess the capacity to influence dysregulated wound-healing processes in respiratory epithelial cells.

Fig. 3. Effect of AW-NE on BEAS-2B cells migration (scratch wound assay) over a 48-h period. (A) Wound healing progress after 24 h (45% enhancement). (B) Wound healing progress after 48 h (60% enhancement). (C) Representative pictures of treatment of the AW-NE 2.5 µM and 5 µM and the wound closure over the 48 h. The data are represented as Mean ± SEM (One-way ANOVA, n = 3, *: p < 0.05; **: p < 0.01; ***: p < 0.001)

Effect of Agarwood Nanoemulsion on the expression of Inflammation and Remodelling-associated Proteins

Through protein array analysis, we investigated the impact of AW-NE on proteins associated with the remodelling in BEAS-2B cells stimulated by TGF-β. The treatment with TGF-β indicated an upward trend in MMP-9 expression compared to the control, as it increased the expression of MMP-9 by 52% (Fig. 4A). Interestingly, simultaneous treatment with AW-NE significantly diminished the TGF-β-induced MMP-9 expression by 68% compared to the TGF-b-only treated group (Fig. 4A). Furthermore, while TGF-β treatment led to an increase in angiogenin levels, this increase was statistically insignificant after 24 h (Fig. 4B). Treatment with AW-NE significantly reduced the angiogenin levels. In conducting parallel analyses with remodelling-associated proteins, we also explored AW-NE’s impact on inflammatory markers on the same protein array. Post TGF-β administration, we observed increased Pentraxin-3 (PTX3) levels, though statistical significance was not attained. In contrast, a co-treatment with AW-NE resulted in a downward trend in PTX3 levels, although without statistical significance (Fig. 4C).

Fig. 4. Effect of AW-NE on the regulation of TGF-β-induced remodeling cytokines. BEAS-2B cells were pre-incubated for 24 h with or without 1 µg/mL of TGF-β. The cells were then incubated for a further 24 h in the absence or presence of AW-NE (25 µg/mL). The relative expression levels of MMP-9 (A), Angiogenin (B), and PTX3 (C) were determined using the Human XL Cytokine Protein Array. The data are represented as Mean ± SEM (n = 4, :p < 0.05; **:p < 0.01 ^#*^: p < 0.1;, One-Way ANOVA test)

Discussion

The current study demonstrated the impact of Agarwood Nano-Emulsion (AW-NE) on TGF-β-stimulated BEAS-2B cells by investigating its effect on critical experimental endpoints such as cell viability, NO synthesis, and cellular migration. Furthermore, the study also revealed the protective effects of the AW-NE on the TGF-β-induced expression of remodelling proteins like Angiogenin, MMP-9 and pentraxin-3, a marker of inflammation. These findings offer significant insights into the prospective therapeutic applications of AW-NE for treating fibrotic lung diseases and chronic respiratory conditions.

TGF-β is integral to airway remodelling, a fundamental process in advancing chronic respiratory illnesses. In asthma, it contributes to increased mucus secretion and heightened airway hyperresponsiveness [30]. Similarly, in COPD, TGF-β initially facilitates tissue repair; however, its dysregulation—often due to extrinsic factors such as smoking—leads to excessive deposition of extracellular matrix and, consequently, to the development of emphysema [31]. As a multifunctional cytokine, TGF-β is instrumental in various cellular processes, including inflammation, fibrosis, and tissue repair [31]. In the context of lung pathology, TGF-β-driven remodelling is characterised by subepithelial fibrosis, leading to airway wall thickening, reduced elasticity, and subsequent airflow limitation seen in respiratory diseases like asthma and COPD [32, 33]. Furthermore, the epithelial-mesenchymal transition (EMT) prompted by TGF-β results in epithelial cells adopting mesenchymal characteristics, thereby augmenting their migratory and invasive potential [32, 33]. This transition markedly contributes to the fibrotic modifications within the airway structure and is critical in COPD pathogenesis. Additionally, TGF-β influences epithelial-mesenchymal interactions, impairing repair mechanisms and thereby exacerbating fibrosis and other structural changes in the airways, ultimately causing airflow obstruction and worsening respiratory symptoms [34].

The current study demonstrates that AW-NE mitigates specific remodelling traits provoked by TGF-β within BEAS-2B human bronchial epithelial cells, as assessed through a wound healing assay. Importantly, in TGF-β stimulated BEAS-2B cells subjected to AW-NE treatment, a marked decrease in enhanced cell motility was noted up to 48 h following treatment, with migration levels comparable to those of the control group. To our knowledge, this is the first study that has proven the efficacy of AW-NE by inhibiting cell migration in the wound healing assay, a key step in airway remodelling [35, 36].

Furthermore, stimulation of BEAS-2B cells with TGF-β also showed a significant reduction in nitric oxide (NO) production, which is aligned with the work of Vyas-Read et al., who demonstrated that TGF-β facilitates the epithelial-mesenchymal transition (EMT) in alveolar epithelial cells by decreasing endogenous NO levels through the downregulation and suppression of endothelial nitric oxide synthase (eNOS) [30]. Moreover, administration of AW-NE to TGF-β-stimulated BEAS-2B cells markedly reversed the TGF-β effects, reinstating nitric oxide production to levels comparable to those observed in the control group. Contrary to our earlier assessments, where a similar formulation of AW-NE exhibited significant anti-inflammatory and antioxidant capabilities that effectively reduced elevated nitric oxide (NO) levels in LPS-stimulated RAW264.7 mouse macrophages, primarily by downregulating inducible NO synthase (iNOS) expression [20], these current findings reveal the intricate and context-dependent biological effects of agarwood oil emulsion. This highlights its ability to sustain physiological nitric oxide concentrations while fostering a balanced, healthy phenotype, which can be attributed to its pronounced anti-inflammatory, antioxidant, and anti-fibrotic properties.

Airway remodelling involves several processes, with angiogenesis being particularly important and involving the expansion of microvasculature that can exacerbate inflammation and airway hyperresponsiveness in chronic respiratory diseases like asthma and COPD [32, 33]. Managing the intricate signalling pathways in angiogenesis and airway remodelling demands precise coordination of treatments to prevent unwanted side effects [33]. Several growth factors significantly influence inflammation and angiogenesis by affecting the infiltration of inflammatory cells or promoting neovascularisation [31]. Angiogenin, a powerful angiogenesis stimulator initially identified from human adenocarcinoma cell lines, is vital in various physiological and pathological conditions [31]. Although structurally distinct from heparin-binding growth factors, angiogenin fosters neovascularisation and plays a key role in the EMT process [37]. Increased angiogenin levels have been observed in the bronchial mucosa of individuals with asthma, particularly during exacerbation episodes, highlighting its involvement in the disease’s pathophysiology [37]. Although direct studies on the increased expression of angiogenin are lacking, stimulating epithelial cells with TGF-β can indeed boost angiogenesis by upregulating angiogenic factors [34]. This aligns with our observations, as we didn’t find increased protein expression of angiogenin following TGF-β stimulation. Interestingly, when TGF-β stimulated BEAS-2B cells were treated with AW-NE, there was a noteworthy downregulation of angiogenin expression. This finding suggests that AW-NE may inhibit TGF-β-induced angiogenesis by suppressing various angiogenic factors, with angiogenin likely playing an indirect role in this process.

Airway remodelling is also intricately linked to the degradation of the extracellular matrix (ECM) and modulation of inflammation, with MMP-9 serving as a pivotal mediator in this process that influence respiratory pathologies such as asthma, COPD, and infections [38]. Its activity is modulated by a variety of signalling pathways and external stimuli, notably increasing in response to pathogens like Mycoplasma pneumoniae and respiratory syncytial virus (RSV), primarily through the activation of Toll-like receptor pathways and the MAPK/NF-κB/AP-1 signalling cascades [38]. This results in inflammation and significant alterations in ECM composition [39]. Furthermore, MMP-9 enhances EMT via the EGFR/Akt/GSK3β/β-catenin pathway when exposed to stimuli such as cigarette smoke extract, contributing to structural modifications in the airways [40]. Elevated levels of MMP-9 are frequently associated with the severity of asthma and COPD, potentially leading to airway obstruction [41, 42]. Aligned with previous research results, an elevation in MMP-9 expression was identified in cell lines following the stimulation by TGF-β. Consistent with our prior studies on AW-NE as an anti-inflammatory agent, a notable decrease in MMP-9 expression was recorded, indicating its possible role in preventing ECM degradation while also inhibiting the activation of numerous inflammatory and EMT-related pathways.

The promising anti-remodelling activity of AW-NE confirms the enormous potential of phytoceuticals as therapeutic options specifically targeting the process of airway remodelling [43]. In a previous study from our team, we employed the same model showing that liquid crystalline nanoparticles loaded with berberine, a benzylisoquinoline alkaloid, significantly counteract TGF-β-induced remodelling features by restoring NO production, decreasing cell migration, and modulating the expression of endoglin, thrombospondin-1, basic fibroblast growth factor, vascular-endothelial growth factor, myeloperoxidase, and cystatin C [44]. In another study, Zhang and colleagues showed that artesunate, a semi-synthetic water-soluble derivative of artemisinin, significantly inhibited airway remodelling in an in vivo model of asthma by targeting the MAPK pathway [45]. Furthermore, a recent study by Cai et al. showed that a Hyssopus cuspidatus extract prevented airway remodelling in a mouse model of bronchial asthma by inhibiting proliferation and migration of airway smooth muscle cells and the release of inflammatory factors and metalloproteinases [46].

Conclusion

This study highlights the promising therapeutic potential of AW-NE in addressing the pathophysiological mechanisms underlying chronic respiratory diseases. By modulating key processes such as oxidative stress, inflammation, and airway remodelling in TGF-β-stimulated BEAS-2B cells, AW-NE demonstrated its ability to restore NO production, reduce cell migration, and suppress the expression of critical remodelling and inflammatory proteins such as MMP-9, angiogenin, and PTX3. These findings suggest that AW-NE offers a novel approach to mitigate airway remodelling and inflammatory processes, which are inadequately addressed by current therapies.

While this in vitro study underscores AW-NE’s potential as a multifaceted therapeutic agent, further research is required to validate these findings. Future directions should include in vivo studies to confirm its efficacy and safety in relevant animal models of chronic respiratory diseases such as asthma, COPD, and pulmonary fibrosis. Additionally, mechanistic investigations exploring its molecular targets and pathways in detail would provide deeper insights into AW-NE’s mode of action. These efforts can pave the way for clinical trials, ultimately determining its translational applicability as a treatment for respiratory diseases.

Beyond therapeutic application, the versatility of nanoemulsion technology offers opportunities to enhance drug delivery, stability, and bioavailability. Further exploration into optimising AW-NE formulations for targeted pulmonary delivery via inhalation could maximise its efficacy while minimising systemic exposure and potential side effects. Such advancements could redefine treatment paradigms for chronic respiratory disorders, offering patients not only symptom relief but also a means to address the disease at its roots.