Naringin as a natural bitter tastant promotes proliferation of cultured human bronchial epithelial cells via activation of TAS2R signaling


 Bitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate the relaxation of airway smooth muscle cells, which have great potential in the development of novel asthma therapy. However, recent study shows that canonical bitter substance denatonium induces apoptosis of bronchial epithelial cells (BECs), indicating the toxic effect of bitter tastants on airways. Considering the diversity of bitter tastants in nature and TAS2Rs expressed in airway cells, it is thus necessary to carefully evaluate the bitter tastant for its effect on the proliferation of BECs, if aimed to treat airway disease. Here we first screened a group of bitter flavonoids, including apignenin, hespretin, kaempferol, naringenin, naringin and quercetin which are commonly used in food and traditional medicine, and then quantitatively evaluated the effects of this group of bitter flavonoids on the proliferation of BECs (i.e. 16HBE14o- cells) cultured in vitro. The results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of 16HBE14o- BECs in vitro at the dose of a few hundred micromoles. Furthermore, the naringin-promoted cell proliferation was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling. Inhibition of the TAS2R signaling pathways with specific blockers attenuated the naringin-enhanced cell proliferation, cyclin E expression and calcium signaling/ERK activation. Taken together, these findings indicate that although many bitter flavonoids may inhibit the proliferation of BECs, naringin emerges as one of the kind that promotes the proliferation of BECs via cell cycle progression and TAS2R-activated intracellular signaling. Only such bitter tastant proven to be unharmful to the epithelial structure and function could be further developed as safe and effective TAS2Rs-based bronchodilator in asthma therapy.


Introduction
Bitter taste receptors (TAS2Rs) are usually considered to be expressed in gustatory tongue to mediate bitter taste sensing to prevent toxic damage from foods [1]. Interestingly, in the last decade, it has be veri ed that TAS2Rs are also widely expressed in non-gustatory cells including airway smooth muscle cells (ASMCs) and bronchial epithelial cells (BECs), and consequently mediate bene cial functions such as relaxation of ASMCs and the ciliary beating of BECs when the cells are exposed to bitter substances [2,3]. Subsequently, there has been a great surge of interest in investigation of bitter tastants for their potential as novel drug candidates for bronchodilation in asthma therapy because of their alternative pharmacological mechanisms as opposed to the conventional β2-adrenergic agonists, and their wide availability in nature [4].
However, a recent study has reported that the canonical bitter substance such as denatonium can induce apoptosis of BECs [5]. Since BECs form the rst-line physical barrier in the bronchial airways to protect them from harmful insults of inhaled pathogens and pollutants such as airborne particulates, the healthy integrity of BECs plays a vital role in maintaining the airway health [6][7][8], and otherwise the accumulation of BECs damage may lead to asthma-associated pathologies including airway in ammation, airway hyper-responsiveness and airway narrowing. For example, it has been well known that asthma occurrence and severity is directly attributable to the shedding of BECs [9,10]. It is also known that local injury of BECs can cause global airway smooth muscle contraction and airway constriction, which may be detrimental to asthma symptoms [11]. Therefore, it is very important to examine the cytotoxic effect on BECs of bitter tastants when they are considered to be used as bronchodilators in asthma therapy.
It is fortunate, however, that there are at least 25 human TAS2Rs bitter taste receptors known in existence and each of them can recognize a variety of bitter tastants [12]. On the other hand, there are thousands of bitter tastants that have been well characterized and documented in data bases [13]. Of particular interest are the numerous natural bitter substances used in food and traditional medicine that provides a gold mine to search for potent bronchodilators [14]. Combining such diverse resources of TAS2Rs and bitter tastants makes it highly possible to nd bitter tastants that are not only potent in inducing relaxation of ASMCs but also nontoxic to the normal function of BECs. With a literature review following this principle, a group of bitter avonoids including apigenin, hesperetin, kaempferol, naringenin, naringin and quercetin emerges as the rst group of bitter tastants that may meet the requirement of being bene ciary to the functioning of both ASMCs and BECs in asthma therapy, as suggested by their reported alleviating effects on experimental models of asthma [15].
However, none of these bitter tastants has been well evaluated in regards of its cytotoxicity to BECs. In view of the lack of such critical knowledge for drug development of TAS2Rs-based bronchodilators, we aimed to screen these six bitter avonoids in this study for their effects on the speci c functions of BECs related to cell viability and cell cycling. We demonstrate that at least in this group of avonoids the majority of the bitter tastants inhibited the proliferation of BECs, but only naringin turned out to promote the proliferation and cell cycle via canonical signaling pathway correlated with TAS2Rs.

Cell Cultures And Drug Treatment
The human BEC 16HBE14o-cells, one kind of basal cell featured with functional tight junction and welldeveloped barrier properties [16], were obtained as a gift from Prof. Gruenert of University of California (San Francisco) [17]. They were maintained in Dulbecco's modi ed Eagle medium (DMEM) supplemented with 10% FBS, L-glutamine and antibiotics at 37 °C in a 5% CO 2 humidi ed incubator. Cells were passaged at 90% con uence using 0.05% trypsin in 1:3 or 1:4 proportions. The healthy cells with cubiclike morphology were used for the following experiments.
Stock solutions (1 M) of apigenin, hesperetin, kaempferol, naringin, naringenin, and quercetin were prepared in DMSO. The stock solution (1 M) of denatonium was dissolved in phosphonate buffer solution (PBS). All those stock solutions were kept frozen at -20 °C. The cells cultured in basal DMEM medium without FBS were exposed to different concentrations of bitter tastants, with or without inhibitors (6Met, U73122, and PD) for TAS2R signaling. For each experiment, an appropriate vehicle control was run in parallel, with the nal concentration of DMSO at 0.1% (v/v) to avoid the cytotoxicity of DMSO.

Cell Morphological Assessment
To directly present the effect of six bitter avonoids on cell morphology, the cells were treated with different concentrations of bitter avonoids for 48 h and then imaged by phase contrast microscopy using an inverted optical microscope with a 20x objective (Primovert, Carl Zeiss, Jena, Germany). The view-eld for imaging was chosen randomly to prevent subjective bias.

Cell Proliferation Assay
The cell proliferation was evaluated by using trypan blue exclusion cell counting method. To this end, 16HBE14o-cells were seeded at the density of 2 × 10 4 cells/cm 2 into 6-well plates for 24 h. Then the medium was replaced with fresh serum free DMEM containing bitter tastant at different concentrations.
After the cells were incubated for 48 h with/without inhibitors for TAS2Rs signaling or ERK activity, cell number was counted by using cytometer after staining with 0.4% trypan blue for living cells. The cell proliferation is presented as a ratio of cell number in experimental groups to that of the vehicle group (% of control).

Cell Viability Assay
To verify the cell proliferation analysis, the cell viability of 16HBE14o-cells were tested by using ATP luminescence assay, which can e ciently measure metabolic activity in the cells [18]. Brie y, subcon uent, exponentially growing 16HBE14o-cells at the density of 1 × 10 4 cells/cm 2 were seeded in 96-well plates and cultured for 24 h. Then the medium was replaced with fresh serum free DMEM containing bitter tastant at different concentrations. After the cells were incubated in this medium for 48 h, the medium was replaced again with fresh serum free DMEM so that the ATP secreted from the cells into the culture medium during culture was excluded from the cell viability measurement. Then the cell viability was determined using ATP luminescent cell viability assay (#C0065M, Beyotime Biotechnology, China), which directly presents the metabolic activity of cells based on the quantitation of the amount of ATP in cells. The cell viability is expressed as the percentage of luminescence (RLU) in experimental groups to that of the vehicle group (% of control).

Cell Cycle Analysis
Cells were seeded in 6-well plate at a density of 2 × 10 4 cells/cm 2 and then exposed to 0.1, 0.5 and 1 mM naringin with/without inhibitors (6Met, U73122, and PD) for 48 h. Cells were collected and xed with 70% ethanol. Following incubation on ice for 30 min, samples were washed with ice-cold phosphate-buffered saline (PBS) and then centrifuged at 1200 rpm for 5 min. Pellets were resuspended in PBS with RNase A (#ST578, Beyotime) (100 µg/mL) and propidium iodide (#ST511, Beyotime) (40 µg/mL). Samples were kept at 37 °C for 30 min in the dark. The cell cycle pro le was analyzed using a ow cytometer (BD Accuri C6, BD Biosciences, San Jose, CA) and analyzed with ModFit software (Verity Software House, Topsham, ME).

Assessment Of Intracellular [Ca 2+ ] In Cultured BECs
Intracellular calcium signals were visualized as described previously, using the membrane permeable

Real-time qPCR Assessment Of Cyclin Protein Expression
Cyclin proteins, including Cyclin A, B, D and E are molecules that regulate cell proliferation and cell cycle.
In order to determine whether naringin in uences these molecules in bronchial epithelial cells, we used real-time qPCR to measure the mRNA expression level of Cyclin A to E in 16BEC14o-cells with/out exposure to naringin at 01 or 1 mM for 24 h. Total RNA from cultured BECs was extracted using the TRI Reagent RNA Isolation Reagent (#T9424, Sigma). 500 ng total RNA was used to generate 1st strand cDNA using the Revert Aid First Strand cDNA Synthesis Kit (#K1622, Thermo, MA, USA). Primers for Cyclin A, Cyclin B, Cyclin D, and Cyclin E as target genes and GADPH as reference gene were purchased from General Biosystems (Anhui, China). PowerUp SYBR Green Master Mix (#A25742, Applied Biosystems, CA, USA) was used. The reaction was run in the Real-time PCR system (StepOnePlus, Applied Biosystems) using 1 µl of the cDNA in a 10 µl reaction according to the manufacturer's instructions in triplicate. Calibrations and normalizations were done using the 2 −∆∆CT method, where ∆∆CT = C T (target gene) -C T (reference gene). Fold changes in mRNA expression were calculated from the resulting CT values from three independent experiments.

FRET Analysis
The FRET biosensor ERK, EKAREV, was prepared as reported previously [19]. Cells were seeded in 6-well plates and grown to reach 40% of con uency. Cells were then transfected with 2.5 µg of DNA per well using Lipofectamine 3000 transfection kit (#L3000-008, Thermo). After 12 h, the medium was replaced with 10% FBS DMEM without antibiotics overnight. After transfection for 24 h, cells were seeded on 10 µg/ml bronectin-coated glass-bottom dishes in 10% FBS DMEM for 12 h. Before imaging on microscope, cells were starved in DMEM without FBS overnight. Live cell imaging was performed as previously reported [19,20]. By using a live cell image system (Cell Observer 1A, Carl Zeiss), cells were excited at a 436 nm wavelength, and the uorescence of the CFP (emission at 480) channel and YFP channel (emission at 535) were recorded, respectively. FRET/CFP, was used to represent the level of FRET ON state.
Images were acquired every 2 min. After 10 min, drugs were added to the cell medium by gentle pipetting. The ECFP and FRET images were processed and quanti ed by Fluocell, a MATLAB-based Open Source image analysis software package(http://github.com/lu6007/ uocell [21,22]).

Statistical analysis
Statistical analysis was performed by using GraphPad Prism (Graph Pad Software, La Jolla, CA). Unless stated otherwise, data were reported as means ± s.e.m, and n represents the number of samples. One-way analysis of variance (ANOVA) followed by Post Hoc test was carried out for multiple comparisons. The signi cance level was set at p < 0.05 (*p < 0.05; **p < 0.01).

Naringin promoted the proliferation of 16HBE14o-cells
We rst analyzed the effect of denatonium on the proliferation of 16HBE14o-cells by cell counting (Figures S1A) and ATP luminescent assay (Figures S1B). The result shows that denatonium inhibited the proliferation and decreased cell activity of the epithelial cells, which con rmed the data reported by Wen et al. [5]. To explore bitter tastant that is not only useful for relaxation of ASMCs but also unharmful for proliferation of BECs, we rst focused on six bitter avonoids that are commonly used in both food and TCM and also known to alleviate asthma symptoms (Table S1). We then analyzed these bitter avonoids for their effect on cell proliferation at the concentration of a few hundred micromoles that is considered as maximal safe dosage for effective bronchodilation via activating TAS2Rs [14]. As shown in Fig. 1A, we found that like denatonium the majority of these bitter avonoids including apigenin, hesperetin, kaempferol, naringenin, and quercetin dramatically decreased proliferation of 16HBE14o-cells cultured in DMEM without FBS. Only naringin appeared to enhance the proliferation of 16HBE14o-cells cultured in DMEM without FBS. To clarify the abovementioned effect, we quanti ed the cell activity of 16HBE14ocells treated with these bitter avonoids for 48 h by using cAMP luminance assay. As shown in Fig. 1B, we also found that only naringin enhanced the cell activity of 16HBE14o-cells while apigenin, hesperetin, kaempferol, naringenin, and quercetin all inhibited it. Morphological observation under light microscope further con rmed this phenomenon by showing cellular injury in 16HBE14o-cells treated with apigenin, hesperetin, kaempferol, naringenin, and quercetin, but not in those treated with naringin ( Figure S2).

Naringin Promoted The Cell Cycle Progression Of 16HBE14o-Cells
Since naringin emerged from this group of avonoids as the only bitter tastant to promote proliferation of 16HBE14o-cells which were cultured with basal DMEM medium without FBS, we then investigated whether naringin promotes cell cycle progression of the cells. Representative cell cycle pro les of 16HBE14o-cells treated with naringin in the presence or absence of TAS2R inhibitor as shown in Figure  S3 clearly indicate that naringin caused dose dependent decrease in the percentage of G0/G1 phase and increase in the percentage of S phase and G2/M phase. Figure 2A shows the quanti ed percentage of cell cycle. Compared with the vehicle control group, a 10% reduction of G1 phase was got in group incubated with the dose of 1 mM naringin. However, cells in S phase increased from 9-14% and cells in G2 phase increased from 20-25%.
The real-time qPCR results show that except for cyclin A, naringin seemed to in uence cyclin B, D, E in a dose dependent manner (Fig. 2B). At 0.1 mM, exposure to naringin increased the mRNA expression of cyclin B and E, but decreased that of cyclin D in the 16HBE14o-cells, as compared to the untreated cells. On the other hand, exposure to 1 mM naringin only increased the mRNA expression of cyclin E but not cyclin B while maintaining the decreased mRNA level of cyclin D, as compared to the untreated cells. Considering the Cyclin proteins promote cell cycle transition through checkpoint, these results indicate that naringin may promote the cell cycle progression in cultured 16BEC14o-cells through in uence on the mRNA expression of cyclin B and cyclin E, and the latter may be more signi cant.

Naringin Evoked Calcium Signaling And Downstream ERK Signaling
Ca 2+ signaling is known to be evoked by the activation of TAS2Rs with bitter tastant in ASMCs, wh{ich plays an essential role in regulation of ASMC function. To ascertain whether Ca 2+ signaling is also involved in naringin-induced BEC response, the [Ca 2+ ] i of 16HBE14o-cells was evaluated with Fluo-4/AM before and after exposure to naringin at 0.01, 0.1, 1 mM, respectively. As shown in Fig. 3A, calcium signaling, as indicated by the fold change of uorescence intensity of Fluo-4, was activated instantaneously upon exposure to naringin in all cases, but the peak magnitude increased from ~ 1.5 tõ 3.0 fold as the concentration of naringin increased from 0.01 to 1 mM.
ERK is another downstream signaling of TAS2Rs, which also plays essential role in regulating animal cell proliferation [23,24]. Figure 3B shows that naringin could induce remarkable activity of ERK phosphorylation in 16HBE14o-cells, as assayed with FRET analysis. When 16HBE14o-cells expressing ERK FRET biosensor EKARE V were stimulated with 1 mM naringin and imaged by time lapse These data indicate that naringin evoked Ca 2+ signaling/ERK activation in cultured 16HBE14o-cells.

Naringin promoted proliferation of 16HBE14o-cells via activation of TAS2Rs
To further determine whether naringin-enhanced proliferation in cultured 16HBE14o-cells did involve TAS2Rs signaling, we cultured the 16HBE14o-cells either in the absence or presence of TAS2Rs inhibitors (6Met, U73122, PD, respectively) and subsequently evaluated the cell proliferation, calcium signaling, ERK activity, the mRNA expression of cyclin E in response to 1 mM. The results show that the 6Met (TAS2R inhibitor) and U73122 (PLCβ inhibitor) blocked naringin-enhanced calcium signaling (Fig. 4A) and ERK activation (Fig. 4B). Similarly, 6Met, U73122, and PD (ERK inhibitor) blocked naringin-enhanced mRNA expression of Cyclin E (Fig. 4C) and proliferation (Fig. 4D) of 16HBE14o-cells. Thus, the naringin-induced proliferation and related Ca 2+ signaling/ERK activation were indeed dependent on the activation of TAS2Rs.

Discussion
The primary nding of this study is that although the majority of the tested bitter avonoids including apigenin, hesperetin, kaempferol, naringenin, and quercetin inhibited proliferation of the BECs cultured in vitro, naringin emerged as the only one to promote proliferation of the BECs. This effect of naringin on proliferation of BECs was mediated at least partially via enhancing the cyclin E expression and intracellular calcium level that are all dependent on TAS2Rs signaling. We further veri ed that the naringin-enhanced proliferation of BECs may be owing to the acceleration of cell cycle transition.
Our data on the inhibitory effect of the majority of the tested bitter avonoids on the proliferation of BECs are consistent with that of the canonical bitter tastant denatonium, as previously reported by Wen et al. [5]. Additionally, these bitter avonoids exhibited inhibitory effect on the proliferation of BECs at a dose of a few hundred micromoles, which falls within the dosage level for the bitter tastant to be used to relax ASMCs [25]. Thus, many bitter avonoids may be pharmacologically harmful to the integrity of BECs if used at the concentration effective for ASMC relaxation. Therefore, for any bitter tastant to be considered in the development of TAS2Rs-based bronchodilators, it must be carefully evaluated for its potential not only to relax ASMCs but also to proliferate BECs.
Fortunately, however, it is well-known that BECs express diverse TAS2Rs and there are numerous bitter avonoids in the natural world [13]. It is highly possible that these different TAS2Rs and agonists may mediate very different pharmacological reactions in BECs. Indeed, we found in this study that naringin, a natural bitter tastant mainly from grapefruits, could enhance the proliferation of BECs, at least in the 16HBE14o-cells cultured in vitro. Considering that naringin has already been shown in vitro and in vivo to relax ASMCs in culture and dilate bronchial airways in ovalbumin-induced asthmatic mice, respectively [26], these bene cial effects of naringin on the proliferation of BECs as presented in this report indicate that naringin may be the one promising bitter substance that can be practically used as drug agent of bronchodilator in asthma therapy.
The airways are lined by three types of cells in the epithelium including ciliated cells, secretory cells, and basal cells [27,28]. Damages to these cells due to environmental hazards can disrupt the barrier integrity of the bronchial airways and lead to various respiratory diseases. In particular, the basal cells appear to be pluripotent stem cells which are important in maintaining the structural and functional integrity of bronchial epithelial layers [28]. However, there is currently a lack of therapies for promoting the recovery of damaged BECs, especially those aiming at regulating basal cell proliferation to help repairing the damaged airway epithelium and restore its normal phenotype. In this study, naringin promoted the proliferation of 16HBE14o-cells, which suggests that naringin may help repair the damaged basal cells, and if true in vivo it can be used as a drug agent to protect human airways from potential damages.
In addition to the effects on ASMC relaxation and BEC proliferation, previous studies have demonstrated that naringin possesses several other therapeutic properties for the pulmonary airways, including antioxidant, anti-in ammatory, antitussive and expectorant effects, as well as protection against lipopolysaccharide (LPS)-induced acute lung injury [29][30][31][32][33]. These therapeutic properties combined together indicate that naringin may be a favorable bitter tastant used in asthma therapy to alleviate multiple symptoms through various mechanisms.
It should be noted that naringin and its aglycon naringenin are two main bitter avanones and very common dietary constituents in grapefruit, citrus fruits, and many other dietary products such as beans, cherries and tomatoes of which they are responsible for the bitter taste. Although naringin and naringenin may have the same origin, we found in this study that it was naringin but not naringenin promoted the proliferation of BECs. Others have found that orally administrated naringin is hydrolyzed to its aglycon naringenin by lactase-phlorizin hydrolase and intestinal micro ora, turning the BEC bene cial naringin to BEC harmful naringenin [34]. Therefore, when developing naringin-based asthma therapy, it should always be considered as preparations for nebulized inhalation instead of oral administration.
Furthermore, naringin may also promote cell proliferation through other pathways alternative to TAS2Rs signaling. For example, naringin can up-regulate the expression of the growth factors such as vessel endothelial growth factor and transforming growth factor-β during angiogenesis and wound healing, which may stimulate the proliferation of BECs [35]. It is also reported very recently that naringin can enhance the proliferation of endothelial progenitor cells that promote endothelial tube formation via CXCR4/PI3K signaling pathway [36]. Whethere these signaling pathways are involved in the naringinenhanced proliferation of BECs remains to be investigated in future study.

Conclusion
The present study demonstrate that different bitter tastants could induce different or even opposite consequences in BECs in terms of cell proliferation. For the majority of the bitter avonoids, at least those tested in this study, exposure to the bitter tastant inhibited the proliferation of BECs cultured in vitro. In contrast, exposure to naringin showed promotive effect on the proliferation of the cells, which was dependent on TAS2R signaling. Since naringin is also capable of relaxing ASMCs, it may be considered a favorable drug agent candidate for the development of both effective and safe bronchodilator and bronchoprotector in asthma therapy.

Availability of data and materials
The dataset supporting the conclusions of this article is included within the article (and its additional le).   The effect Naringin promotes the proliferation of cultured 16BEC14o-cells. (A) Bar graph shows the proliferation of 16BEC14o-cells treated with or without six bitter avonoids (0.1, 0.5, and 1 mM) for 48 h, analyzed with cell number counting. (B) Bar graph shows the cell activity of 16BEC14o-cells treated with or without six bitter avonoids (0.1, 0.5, and 1 mM) for 48 h, analyzed with ATP luminescent assay.