Combination of green tea catechins, polysaccharides, and flavonols against urban fine dust particle-induced bronchial damage mediated by modulation of inflammation factors and airway cilia CURRENT STATUS:

Many studies have reported that environmental pollution has become a serious public health issue. Airborne fine dust particles (FDPs) have been identified as major toxins in air pollution that threaten human respiratory health. Developing strategies for defense against FDPs is one of the primary goals of air pollution research. While searching for an anti-FDP reagent, we found that green tea extract (GTE) and green tea fractions rich in flavonol glycosides (FLGs) and crude green tea polysaccharides (CTPs) had protective effects against FDP-stimulated cellular damage in the BEAS-2B airway epithelial cell line. The results demonstrated that GTE, FLGs, and CTPs significantly increased viability and lowered oxidative stress levels in FDP-treated cells. Major catechin and flavonol compounds increased cell survival, and (-)-epigallocatechin gallate (EGCG) and myricetin exerted synergistic effects on cell survival under FDP stimulation. Combined treatment with GTE, FLGs, and CTPs also exerted synergistic protective effects on cells, and the green tea components attenuated FDP-induced elevations in inflammatory gene expression. Moreover, the green tea components increased the proportion of ciliated cells and upregulated ciliogenesis in the airway in FDP-stimulated BEAS-2B cells. Our findings provide insights into how natural phytochemicals protect the airway and suggest that green tea could be used to reduce FDP-induced airway damage as an individual therapeutic agent or as an ingredient in pharmaceutical and cosmeceutical products. airway that develops after inhalation of FDPs disrupts immune modulation. To determine whether green tea and the combination of GTE, FLGs, and CTPs attenuated FDP-induced effects on the airway immune system, we investigated the expression of known bronchial inflammatory genes. Upon Our results demonstrated that FDPs induced cytotoxicity by increasing intracellular oxidative stress levels, reducing cell viability, increasing inflammatory gene expression, and attenuating airway ciliogenesis in BEAS-2B cells. Green tea components including catechins, flavonols, and polysaccharides exerted protective effects individually and in combination against FDP-induced cellular damage. Specifically, GTE, FLGs, and CTPs lowered intracellular ROS levels, attenuated the hyperimmune response and increased ciliated cell beating rates and ciliogenesis. Amelioration of acute oxidative stress, proper modulation of the immune response and activation of MCC are critical strategies for protection against air pollution-induced airway/lung damage. Based on these results, we suggest that green tea catechins, flavonols, and polysaccharides are promising reagents for protection against FDP-induced airway damage and are candidates for use in the pharmaceutical and cosmeceutical fields.


Background
Many studies have reported that environmental pollution has become a serious public health issue.
Airborne fine dust particles (FDPs) have been identified as major toxins in air pollution that threaten human respiratory health. Developing strategies for defense against FDPs is one of the primary goals of air pollution research.

Results
While searching for an anti-FDP reagent, we found that green tea extract (GTE) and green tea

Conclusions
Our findings provide insights into how natural phytochemicals protect the airway and suggest that green tea could be used to reduce FDP-induced airway damage as an individual therapeutic agent or as an ingredient in pharmaceutical and cosmeceutical products. Background Ambient air pollution is composed of gaseous constituents and particulate matter (PM). PM can be classified on the basis of the particle size, also known as the aerodynamic equivalent diameter, as PM 10 , PM 2.5 , or ultrafine PM [1]. PM10 reaches the lower airways and accumulates in the more proximal conducting airways [2,3]. PM is naturally inhaled as a particulate suspension in the air and is deposited in the airway as it passes through the respiratory tract, after which it interacts with airway cells. Human bronchial epithelia are invariably exposed to toxic factors such as ambient fine dust particles (FDPs), which can cause acute and chronic pulmonary infections and respiratory diseases [4,5]. In addition, toxic pollutants induce oxidative stress and imbalance between reactive oxygen species (ROS) production and scavenging [6,7]. Elevated oxidative stress causes airway epithelial barrier dysfunction, airway inflammation, infection, and mitochondrial dysfunction [8,9,10].
Airway defense strategies include coughing, anatomical barrier-mediated blockade, immune mechanisms, and primary defense via mucociliary clearance (MCC). MCC enables the efficient clearance of inhaled particles, and in human studies, MCC has been found to remove inhaled particles larger than 6 µm from the airway within 24 h [11,12,13]. Because of the toxic effects of FDPs, removal of air pollutants via activation of MCC and attenuation of FDP-induced oxidative stress in airway cells via ROS scavenging are important. Moreover, FDP-generated ROS cause airway cilia dysfunction [14], and applied ROS scavengers may show positive effects in reducing oxidative stress and activating MCC.
In this study, we focused on the potent activities of natural phytochemicals in green tea. Green tea is an important dietary product that contains antioxidative molecules with cytoprotective and antiinflammatory activities that protect cells from oxidative stress-induced apoptosis [15,16,17].
Catechin compounds, such as (-)-epigallocatechin-3-gallate (EGCG), act as radical scavengers and metal-chelating agents and play roles in various cellular processes; for example, they exert neuroprotective functions, regulate blood pressure, and protect against cardiovascular disease [18,19,20]. Previous studies have reported the protective effects of plant extracts and phenolic compounds against oxidative stress and inflammation induced by FDPs [21,22,23,24], and we hypothesized that green tea components could exert potent protective effects against FDP-induced toxicity. Similar to N-acetyl cysteine [25], EGCG exhibits scavenging efficacy against FDP-induced ROS [26]. EGCG also reduces skin inflammation and asthma in rats caused by FDP stimulation [26,27,28].
Despite exerting these positive effects, green tea flavonols and polysaccharides, unlike catechins, have received little attention with respect to their use and functionality.
In the present study, we investigated the protective effects of green tea catechins, flavonols, and polysaccharides against FDP-induced airway cellular toxicity, oxidative stress, and cilia dysfunction using human bronchial epithelial cells (BEAS-2B cells) as an experimental model.

Results
FDPs induce oxidative damage and cell death in airway/lung cells Following inhalation, the primary sites of air pollution exposure are respiratory tract cells, including bronchial and lung cells. Inhaled FDPs can interact with the epithelial cells lining the airway and with lung cells. Since FDPs are known to cause toxicity in various individual cell types [43,44], we attempted to identify the cellular toxicity and oxidative stress levels in airway and lung cells exposed to FDPs. We evaluated cell survival rates and intracellular ROS levels under FDP treatment to examine the cellular damage induced by FDPs. We first performed the experiments using the bronchial epithelial cell line BEAS-2B, the lung fibroblast line IMR90, and the adenocarcinomic alveolar epithelial cell line A549 to assess the toxicity of FDPs on the overall respiratory tract. Treatment with FDPs at concentrations in the range of 1-100 µg/ml resulted in significant concentration-dependent reductions in the survival rates of the cells (Fig. 1a) and increases in the cellular ROS levels (Fig. 1b). The morphology of individual cells was also examined (Fig. 1c). The viability of cells treated with 100 µg/ml FDPs was lower than 50%, and the ROS levels in these cells were nearly 2-fold those in the control cells. As the damaging effects of FDPs on viability and ROS levels were similar among the tested respiratory tract cell lines, we proceeded to perform further experiments using BEAS-2B cells, the first airway cells exposed to air pollution. because green tea polysaccharides exhibit beneficial antioxidant [50], antitumor [36], and antiaging properties [51], we hypothesized that green tea polyphenols and polysaccharides could also ameliorate FDP-induced cellular damage in the airway. Our results revealed that GTE, FLGs, and CTPs attenuated the cellular toxicity induced by FDPs in BEAS-2B cells in a concentration range of 10-50 ppm (Fig. 2a). These bioactive green tea ingredients also lowered oxidative stress levels in FDPtreated cells (Fig. 2b).
The chemical properties of GTE, FLGs, and CTPs were investigated. As shown in Table 1, the total catechins in GTE amounted to 36.52 ± 1.6% of the dry matter and included EGCG (16.8 ± 0.8%), EGC (11.8 ± 2.0%), EC (3.68 ± 0.8%), and ECG (2.94 ± 0.2%). On the other hand, catechins, a major group of phenolics in green tea, were not detected, and the FLGs quercetin, kaempferol, and myricetin were contained mainly in the FLG fraction [46]. In addition, the CTPs included mainly pectic substances and glucosidic macromolecules found in unlignified cell walls, major components of the middle lamellae in plants [45]; these pectic polysaccharides have various pharmacological properties [30,52,53]. We next examined the effects of higher levels of GTE and FLGs on FDP-induced cellular toxicity in BEAS-2B cells. The epicatechins EGCG, EGC, EC, and ECG effectively reduced FDP-induced cytotoxicity when applied at concentrations ranging from 1-10 µM (Fig. 2c). The flavonols quercetin, kaempferol, and myricetin also showed protective effects on cell viability in the 1-100 µM concentration range (Fig. 2d). Among these active compounds, EGCG and myricetin, which were found in the GTE and FLGs, respectively, showed the most potent protective efficacy against FDP.

Combined effects of the green tea components on FDP-induced cellular damage
According to the cell survival rates under FDP treatment, the catechin EGCG and the flavonol myricetin exhibited potent and concentration-dependent protective effects against FDP-induced toxicity ( Fig. 2c-d). EGCG (Fig. 3a) was the most abundant catechin in GTE and is commonly used in cosmetics, functional foods, and dietary supplements due to its health benefits [54,55]. EGCG has also been shown to reduce FDP-induced skin inflammation in epidermal keratinocytes and dermal fibroblasts [26,28]. Myricetin (Fig. 3b) is a flavonol that is present in vegetables, fruits, nuts, berries, and tea. Like many other flavonols, myricetin shows antioxidant, antiviral, and anti-inflammatory effects [21, 31,56]. Interestingly, among polyphenolic compounds, myricetin and EGCG have been shown to exhibit inhibitory effects against house dust-induced allergic reactions [57]. According to a previous report, among polyphenols (including catechins and flavonols), myricetin and EGCG effectively inhibit the release of kinin by house dust mites.
We tested the effects of EGCG and myricetin on cellular toxicity and ROS levels to investigate whether these active compounds in green tea have synergistic effects. Administration of 10 µM EGCG or myricetin reduced toxicity and intracellular ROS levels in cells under FDP treatment ( Fig. 3c-d).
Surprisingly, co-treatment with 5 µM EGCG and myricetin exhibited synergistic effects, enhancing cell viability and attenuating oxidative stress levels in FDP-treated cells ( Fig. 3c-d). The effects of representative catechins and flavonols on FDP-induced morphological changes are depicted in Fig. 3e.
We next tested the combined preventative effects of GTE and the FLG and CTP green tea fractions against FDP-induced cellular damage. Combined treatment with the green tea components was more effective than GTE treatment alone in preventing FDP-induced cell survival impairment and oxidative stress ( Fig. 4a-c). Taken together, these data suggest that supplementation with whole green tea rather than with a specific fraction may enable useful synergy between catechins and flavonols to combat FDP-induced cellular damage.
Protective effects of the green tea components against the FDP-induced immune response Following inhalation, the primary site of exposure to FDPs is the airway tract. Inhaled PM directly affects the immune processes of airway epithelial cells, which can be stimulated by airborne materials in the environment. FDP-stimulated airway cells act as components of multicellular immune responses and trigger cellular signaling pathways. Because improper and excessive immune reactions can result in serious infections, malignancies, and autoimmune conditions, proper regulation of the effects of air pollution on the immune system is important [44,58]. The proinflammatory cytokine milieu in the airway that develops after inhalation of FDPs disrupts immune modulation. To determine whether green tea and the combination of GTE, FLGs, and CTPs attenuated FDP-induced effects on the airway immune system, we investigated the expression of known bronchial inflammatory genes. Upon sensing of toxic particles, bronchial epithelial cells produce many proinflammatory cytokines [59,60].
These molecules are well known asthma and chronic obstructive pulmonary disease (COPD) markers [61,62]. Treatment with 100 µg/ml FDPs greatly increased the expression of the inflammatory marker genes IL-4, IL-13, IL-17A, CCL-11, CCL-17, and MMP-12. Combined treatment with GTE, FLGs, and CTPs largely reduced this hyperimmune response to a greater extent than treatment with GTE alone (Fig. 5). This pollution-induced cytokine response is clinically relevant [63], and as IL-17A is also known as a COPD marker, the green tea components exhibit potential for the prevention of airway disease. Moreover, given that IL-4, IL-13, CCL-11, and CCL-17 regulate lung cell tight junctions, these results suggest that the green tea components can be used for lung health applications.

Protective effects of the green tea components on airway cilia
Bronchial epithelial cells create a physical barrier at the airway lumen, form the mucous lining of the respiratory tract, sense toxic biological and anthropogenic PM accumulation on the airway wall and help to remove PM from the airway via ciliary action. MCC plays a crucial role in the airway defense machinery, as it involves secretion of antimicrobials, fluids, and anti-inflammatory proteins [64]. The mucociliary system removes PM and pathogens mechanically via the actions of cilia and coughing [65]. MCC abnormalities related to ciliary dysfunction can result in chronic pulmonary disorders, including asthma and COPD. In patients with primary ciliary dyskinesia, airway clearance of particles is impaired and prolonged, which allows longer residence times for bacteria, viruses, and toxins in the airway [66]. Disruption of ciliated cell functions has been detected in many chronic lung diseases and contributes to morbidity, mortality, and infection in individuals with these disorders [67,68,69,70].
To determine the effects of the green tea components on ciliogenesis in bronchial epithelial cells, we treated FDP-treated BEAS-2B cells with GTE or with GTE, FLGs, and CTPs. FDP treatment greatly suppressed cilium formation (Fig. 6a-b) and cilium length (Fig. 6c). Cilia are specialized microtubulebased cellular organelles that beat in metachronal waves to facilitate the expulsion of inhaled particles and pathogens trapped in the mucus layer from the airway [71]. To detect cilia, immunofluorescence was performed for acetylated α-tubulin and the ARL13B protein [72,73]. Ciliated cells are defined by their multiple motile apical cilia and by the presence of motor proteins that mediate directional beating, which is critical for MCC [74]. Although the biomechanical actions of cilia in epithelial cells are well identified as essential building blocks of MCC [66], recent studies have revealed that airway ciliated cells sense and respond to mechanical and irritant stimulation [13,74].
In our study, we found that GTE increased the proportion of ciliated cells by approximately 3-fold under FDP treatment conditions; moreover, the combined green tea components increased the ciliated cell proportion by over 4-fold (Fig. 6a-b). Additionally, GTE, FLG, and CTP application protected cilia against FDP-induced declines in length (Fig. 6c), indicating that these green tea components activate airway cilia and can upregulate MCC of inhaled pollutants such as FDPs.

Discussion
We depicted here the protective effects of green tea catechins, polysaccharides, and flavonols against airborne particles. Compared to green tea catechins, flavonols and polysaccharides from green tea have received little attention. Green tea contains a many of polyphenols, especially monomeric flavonols [75,76]. Since there are very limited studies for investigating protective effects of green tea polyphenols against particulate pollutants [77,78], we examined green tea flavonols rich fraction FLGs and its representative compounds, quercetin, kaempferol, and myricetin for damage attenuation by FDP. Among these compounds, myricetin have shown potent positive effects of cell survival under FDP-stimulation dose-dependent manner (Fig. 2d). Moreover, myricetin represented synergetic effects against FDP with EGCG. Combination of myricetin with EGCG effectively lowered cellular ROS level and increased cell survival under FDP treatment (Fig. 3c-d).
Along with green tea catechins and flavonols, polysaccharides are also gaining attention due to their health benefits, especially immune response [40,79]. Air pollution has increased concern about its inhalation toxicity. Inadequate or excessive immune reactions by FDP could result in serious infection, metastatic malignancies, and auto-immune state. FDP stimulates cells through ROS sensing pathways and activate pro-inflammatory signaling cascades such as MAPK pathways [80,81]. The affection of FDP to stimulate cells may due to the particles containing microbial molecules and also pollutants inducing host-derived molecules production. FDP could stimulate airway epithelial cells and generate ROS in cellular and acellular systems [82,83]. FDP also cause oxidative stress by both the heavy metal and organic compounds and can directly reduce endogenous antioxidants [84,85]. In our results, combination of green tea components effectively inhibited hyper-immune responses by FDPstimulation (Fig. 5). These pro-inflammatory cytokine milieus after ambient pollutant inhalation may be important for perturbing immune regulation and actually closely related with airway and pulmonary dysfunction, such as asthma and COPD. Treatment of whole green tea component could be more promising application to attenuate immune dysfunction more than green tea catechin only.
Although in vitro experiments are important for understanding the toxicity of air pollution and its therapeutic treatment, pollution exposure in cell culture system has limitation of actual inhalation.
However, relationship between airway defenses and epithelial cells gives a clue to protect airway against FDP. FDP in naturally inhaled as a particulate matter in the air, deposit in the airway, passing through respiratory tract before interacting with airway cells. The bronchial airway cells form a physical barrier, sense dangerous biological and toxic particles, and deposit on the airway wall by ciliary action to clear particles from the airway. The airway cilia are an organelle producing from the cell body that senses external stimulations and removes harmful particles by its movements. Mucociliary clearance is an integral part of airway-lung defense methods, enabling efficient clearance of inhaled particles from the respiratory tract [4,12]. The activity and efficacy of clearance closely related with ciliated cells proportion and ciliogenesis in airway epithelial cells [13,71]. As shown in our results, application

Chemicals and reagents
The airborne PM reference material ERM-CZ100 (PM10-like) and the pure catechin compounds EGCG, epigallocatechin (EGC), epicatechin (EC), and epicatechin gallate (ECG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenolic compounds, quercetin, kaempferol, and myricetin were also obtained from Sigma-Aldrich. An alcoholic water extract and crude tea polysaccharides from green tea leaves were prepared according to previously described methods [45]. All other chemicals used in this study were of analytical grade.
ERM-CZ100 was suspended in serum-free DMEM and homogenized by sonication to make a 10 mg/ml stock solution. Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and the fluorescent probe
Determination of the effects of the reagents on cell survival after exposure to fine dust Cells were incubated in a 12-well plate for 24 h and then treated with different reagents. After 2 days of treatment, FDPs (100 mg/ml) were added to the cells. After 24 h of incubation, MTT solution (2 mg/mL) was added to each well, and the cells were further incubated for 1 h. The cell viability was determined by MTT assay [87] with a microplate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). After washing, the coverslips were mounted onto glass slides and visualized using a confocal laser scanning microscope (LSM800, Carl Zeiss, Germany). DAPI was used to counterstain the cell nuclei.

Determination of intracellular ROS levels
The acquired images were analyzed using ZEN software (Carl Zeiss).

Statistical analysis
The data are expressed as the mean ± SD. The normality of the data was analyzed using the Shapiro-Wilk test, and the results between different groups were compared using one-way ANOVA (followed by Dunnett's post hoc test) or Student's t-test. All statistical tests were two-sided, with the level of significance established at p < 0.05. SPSS software (ver. 21, SAS Institute, NC, USA) was used for statistical analyses.

Consent for publication
Not applicable.

Availability of data and materials
The raw data used and/or analyzed in current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no conflicts of interest.

Funding
This research was part of a basic bioscience research project funded by the Amorepacific R&D Center.

Authors' contributions
J. K., H. C., and M. P. have full access to all data from the study and take responsibility for the integrity of the data as well as for the manuscript. J. K., H. C., H. K., and M. P. conceived, designed and performed most of the experiments and data analyses and prepared the manuscript. D. C. and K. P. performed the immunofluorescence assay. J.K., H.K., and M.P. conceived and designed the research, analyzed and interpreted data, wrote the manuscript, and administered the project.