Background
Epidemiologic studies have found a positive correlation between short-term exposure to elevated levels of airborne particulate matter having a mean diameter of less than 2.5 μm [
1] and increased morbidity and mortality in humans [
2,
3]. The pulmonary effects of urban particulate matter (UPM) exposure are associated not only with increased hospitalization and premature death but also cardiovascular disease [
4]. The extracellular side of the alveolar epithelium, which represents the largest surface area of the body in air-breathing animals and human [
5], is exposed to UPM, leading to generation of reactive oxygen species (ROS) [
6] and inflammatory cytokines [
7] in the epithelial cells. These factors can lead to disruption of epithelial barrier function and increase paracellular permeability by destabilizing tight junctions [
8]. Disruption of epithelial tight junctions followed by barrier dysfunction plays a critical role in the pathogenesis of a variety of gastrointestinal, hepatic, pulmonary, kidney, and ocular diseases [
9]. The disruption of epithelial tight junctions leads to accumulation of fluid and protein in the alveoli, causing asthma [
10] and acute respiratory distress syndrome [
11].
Induction of cytokines has been reported in in vivo [
12] and in vitro response to exposure to a variety of particles [
13,
14]. This response is related release by alveolar macrophages of inflammatory cytokines in the alveolar space [
7]. To evaluate the inflammatory response after UPM exposure, we determined the expression levels of tumor necrosis factor-α (TNF- α), interleukin-6 (IL-6), and IL-8 in alveolar epithelial cells. The degree of alveolar epithelial cell (AEC) injury and its recovery are pivotal determinants of the toxicity of UPM treatment [
15,
16]. However, little is known about protection by natural compounds against alveolar dysfunction induced by UPM. As a candidate, we used chebulic acid (CA) in this study, which was isolated from
Terminalia chebula [
17]. The fruit of this plant is used in Ayurvedic and Chinese folk medicine for a wide range of conditions, including asthma and heart disease [
18,
19].
T. chebular extract has been reported to neutralize free radicals [
20], and CA is one of its main active constituents [
17]. Recently, our group reported that CA inhibits advanced glycation end products (AGEs)-induced crosslinking of collagen [
21] and prevents collagen accumulation by ERK-phosphorylation-mediated Nrf2 nuclear translocation [
22]. Previous studies demonstrated that UPM can induce apoptosis in relevant lung cells [
23]. Therefore, we evaluated the effect of UPM on epithelial barrier integrity prior to damage of AECs and the ability of CA to prevent such injury. We estimated that the inflammatory response in AEC induced by oxidative stress caused damage to the tight junctions, leading to alveolar barrier dysfunction. Hence, the objective of this study was to evaluate the protective effect of CA against UPM-induced damage to AECs.
Methods
Materials
The medicinal dried ripe fruit of yellow myrobalan,
T. chebula (Kyungdong Herb Market, Seoul, Korea), was identified by Dr. B. W. Kang (College of Life and Environmental Sciences, Korea University). Voucher specimens were deposited in the Herbarium of the College of Life Sciences and Biotechnology, Korea University, register number H-358. CA was isolated from
T. chebula Retz. as reported previously [
17]. The purified compound was identified as CA using NMR spectra. CA (99.3% purity) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at −20 °C. This stock was further to a working concentration of 10 μM in culture media. A final concentration of no more than 0.1%
v/v DMSO was used. UPM standard reference material (SRM 1648a) was obtained from the National Institutes of Standards and Technology (Gaithersburg, MD, USA). Particles were collected in St. Louis, MO, over a 24-month period and combined into a single lot to be used as a standard reference. For some experiments, UPM was freshly dispersed in cell culture medium and treated at a concentration of 10–100 μg/mL for 12 h [
24,
25].
RPMI 1640 tissue culture medium, fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from Hyclone (Logan, UT), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), dexamethasone, rotenone (RO) fluorescein isothiocyanate (FITC)-dextran 4000 and phorbol 12-myristate 13-acetate were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The enhanced chemiluminescence (ECL) detection kit was purchased from Abclon (Seoul, Korea). Antibodies to zonula occludens (ZO-1) (sc-10,804), occludin (sc-5562), and α-tubulin (sc-586) were purchased from Santa Cruz Biotechnology Inc. (CA, USA). The secondary antibodies anti-rabbit IgG and Alexa Fluor 488-conjugated anti-rabbit IgG were purchased from Cell Signaling (Danvers, MA, USA).
Cell culture
The human pulmonary alveolar epithelial cell line NCI-H441 was obtained from ATCC (Manassas, VA, USA), and cultured in RPMI 1640 medium containing 2.5 g/L dextrose, 2.383 g/L HEPES, 0.11 g/L sodium pyruvate, 10% FBS (v/v), 2.2 g/L sodium bicarbonate, 100 units/mL of penicillin and streptomycin. Cells were plated at 2.4 × 105 cells/mL in RPMI 1640 medium. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Cell viability
MTT assay was used to determine the viability in order to process the next other experiments at a concentration of within a range that does not affect the cell death. NCI-H441 cells (2.4 × 105/well) were seeded in 24-well plates for 24 h. Cells were treated with 0.1, 1, and 10 μg/mL of UPM in serum-free medium for 24 h and with 10, 50, and 100 μg/mL of UPM for 6 h. Medium was changed to 5 mg/mL MTT reagent in modified RPMI 1640 medium, and the plate was incubated at 37 °C for 3 h. The medium was discarded, and 200 μL/well of DMSO were added to dissolve intracellular insoluble formazan crystals. The optical density at 540 nm was measured using a multiplate spectrometer, and the results were determined as percentages of the control.
Measurement of ROS
UPM-induced ROS formation was determined by monitoring conversion of DCF-DA to the fluorescent compound dichlorofluorescin (DCF). NCI-H441 cells were cultured at 2.4 × 104 and 1.0 × 104 cells/well in 96-well plates for 24 h with or without CA (0.5, 1, 5, and 10 μM) pretreatment. Then, the cells were incubated with 10 μM DCF-DA in conditioned medium for 30 min, followed by washing twice in warm phosphate-buffered saline (PBS; pH 7.4) and treatment with 10 μg/mL of UPM for 24 h. Then, the conditioned medium containing UPM was replaced with 100 μL of PBS per well. The DCF fluorescence intensity was measured at an excitation wavelength of 485 nm and emission wavelength of 535 nm using a fluorescence spectrophotometer (VICTOR3™ Perkin Elmer, Waltham, MA, USA).
RNA isolation and quantitative real-time PCR
NCI-H441 (2.5 × 10
5 cells/mL) cells were cultured in six-well plates and pretreated with CA (5 and 10 μM) or rotenone (5 μM) for 24 h, before being treated with UPM (100 μg/mL) in conditioned medium for 6 h. Total RNA was extracted from cells using RNAiso PLUS (TAKARA Korea Biomedical Co, Seoul, Korea). cDNA was generated using LeGene Premium Express First Strand cDNA Synthesis System (Legene, Sandiego, CA). Real-time PCR was performed with TOPreal™ qPCR 2× PreMIX SYBR green (Enzynomics, Seoul, Korea) and analyzed using an iQ5 Thermal Cycler (Bio-Rad, CA, USA). Primers are listed in Table
1. mRNA expression levels were normalized to that of β-actin. Products were analyzed in 1.0–1.5% agarose gels under UV light.
Table 1
Real-time PCR primer sequences
TNF-α | sense, 5’-CCCAGGGACCTCTCTCTAATCA-3’ antisense, 5’-GCTACAGGCTTGTCACTCGG-3’ | 80 bp |
IL-6 | sense, 5’-GGTACATCCTCGACGGCATCT-3’ antisense, 5’-GTGCCTCTTTGCTGCTTTCAC-3’ | 81 bp |
IL-8 | sense, 5’-AGAGTGATTGAGAGTGGACC-3’ antisense, 5’-ACTTCTCCACAACCCTCT-3’ | 118 bp |
ZO-1 | sense, 5’-GTGTTGTGGATACCTTGT-3’ antisense, 5’-GTGTTGTGGATACCTTGT-3’ | 92 bp |
Occludin | sense, 5’-GAAGCCAAACCTCTGTGAGC-3’ antisense, 5’-GAAGACATCGTCTGGGGTGT-3’ | 210 bp |
β-Actin | sense, 5’-AGCGAGCATCCCCCAAAGTT-3’ antisense, 5’-GGGCACGAAGGCTCATCATT-3’ | 285 bp |
Western blot analysis
Equal amounts of protein (65 μg) were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes for 12 h at 50 mA using tank transfer device (bio-rad, Hercules, CA, USA). The membranes were blocked with 5% fat-free dry milk in TBST buffer (25 mM Tris-HCl (pH 7.4), 0.1% Tween-20 and 150 mM NaCl) for 1 h at room temperature then incubated with primary antibodies at 4 °C overnight. After washing five times with TBST at 6 min, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature and washed in TBST five times. Blots were developed using the enhanced chemiluminescence (ECL) reagent (Abclon, Seoul, Korea). Band intensities were normalized to that of α-tubulin and presented as ratios. Band intensities were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
ZO-1 protein staining
To investigate the effect of UPM on a tight junction protein, ZO-1 of lung epithelial cells NCI-H441 cells (2.4 × 105 cells/well) were seeded on six-well culture plates and pretreated with CA (5 and 10 μM) or rotenone (5 μM). After 24 h, cells were treated with 100 μg/mL of UPM for 6 h. Cells were fixed in 3.7% paraformaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. PBS washing was conducted three times in each steps followed by blocking with 1% BSA in PBS for 1 h, and incubation with and anti-ZO-1 primary antibody in 1% BSA overnight at 4 °C. An anti-rabbit Alexa 488-conjugated secondary antibody was added, plates incubated for 2 h at room temperature, and nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI, 500 ng/mL) for 5 min. Stained cells were washed with 1% BSA and visualized using a Confocal Laser Scanning Microscope 700 (Carl-Zeiss, Oberkochen, BA, Germany).
Transepithelial electrical resistance (TEER) measurement
NCI-H441 cells were seeded onto a 12-mm Transwell with a 0.4 μm pore polyester membrane insert (Corning CoStar Corp., Cambridge, MA, USA) at a density of 2.0 × 104 cells/cm2. TEER was measured using EVOMX (World Precision Instruments, Sarasota, FL, UK) and expressed as Ohm*cm2 after subtracting the resistance of a blank Transwell insert.
Paracellular permeability assay
Fluorescein isothiocyanate (FITC)-dextran 4000 Da was used to determine epithelial barrier integrity. NCI-H441 cells were seeded in 12-mm transwell inserts in the presence of 1 μM dexamethasone, and fresh medium replaced every second day until formation of differentiated monolayers, and the maximum level of resistance was reached. At least 30 min before the experiments, medium in the basolateral and apical chambers was replaced with 1 mL and 250 μL of fresh medium, respectively, and then 50 μL of FITC-dextran was added to the apical chamber (final fluorescent tracer concentration 1–3 mg/mL). Plates were incubated at 37 °C in a humidified atmosphere containing 5% CO
2 for 3–4 h [
26]. The diffused FITC-dextran in the basolateral chamber was transferred to a black 96-well plate (100 μL/well). The fluorescence intensity was measured at an excitation wavelength of 485 nm and emission wavelength of 535 nm using a fluorescence spectrophotometer (VICTOR3™ Perkin Elmer, Waltham, MA, USA).
Statistical analysis
Results are expressed as mean values ± standard deviation (n = 3). Different letters indicate significant differences at p < 0.05 by Tukey’s multiple comparisons test. All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).
Discussion
The air quality in Seoul (South Korea) on most days is ‘good’ (0–30 μg/m
3 PM). However, the annual average inhalable PM concentration is 100 μg/m
3. PM causes chronic airway inflammation by which epithelial cells and alveolar macrophages secreted cytokine and chemokine. Many studies have shown a defense mechanism and response of alveolar macrophage against UPM lead to acute lung injury and chronic obstructive pulmonary disease [
35,
36]. Also, many studies have focused on the protective effect of food components against alveolar damage caused by UPM. Because we reported previously that CA isolated from
T. chebula, which has been used for treating asthma [
18], has potent antioxidant activity [
17], we hypothesized that CA could protect against the damage to human alveolar epithelial barrier caused by UPM and reduce inflammation in alveolar epithelial cells caused by UPM-induced ROS production. In this study we showed that CA affects epithelial monolayer integrity and paracellular permeability [
37]. In addition, the alveolar epithelium participates in immune responses to UPM [
38]. So, we focused on disruption of the alveolar epithelial barrier function caused by proinflammatory cytokines and intracellular ROS induced by UPM. RO, which is an inhibitor of electron transfer from ubiquinone binding site to complex I in the mitochondria [
39], was used as a positive control in this study and inhibited UPM-induced ROS production, suggesting that intracellular ROS generation by UPM in alveolar cells is mediated by electron transfer in mitochondria complex I. This indicated that damage to the mitochondrial electron transfer system by UPM, especially mitochondria complex I, could be a cellular source of ROS production.
Barrier integrity is critical for maintaining the physiological function of the epithelium in vivo [
40]. The alveolar epithelial barrier prevents leakage of intracellular fluids and controls ion transport to promote fluid absorption from the alveolar space [
41‐
43]. The main paracellular permeability characteristics of the epithelium are determined by tight junctions, which are formed by homotypic contacts of the transmembrane protein occludin with ZO-1 [
44]. Tight junction proteins are involved in the regulation of diffusion and allow alveolar epithelial cell layers to form selectively permeable cellular barriers that separate the apical and basolateral spaces [
44,
45]. In our study, the decreased expression levels of two typical tight junction proteins, ZO-1 and occludin, caused by treatment with 100 μg/mL of UPM were recovered by pretreatment with 5 μM CA. In addition, NCI-H441 cells are capable of forming monolayers of polarized cells and exhibiting a significant TEER [
46,
47]. In particular, increased expression of tight junction proteins was related to an increase in the TEER value of epithelial cells from ~45 to ~113 Ohm*cm
2 and an increase in paracellular permeability to a 4000 Da fluorescent tracer (Fig.
5). Pretreatment with CA prior to exposure to UPM ameliorated the decreased TEER value and reduced efflux of fluorescent tracer from the apical to the basolateral side, compared to treatment with UPM only.
Acknowledgements
This research was supported by a Korea University Grant (K1420071). This study was also supported by School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. The authors thank the Institute of Biomedical Science & Food Safety, Korea University Food Safety Hall, for providing the equipment and facilities. The funding source had no role in the design, conduct, or analysis of the study or in the decision to submit the manuscript for publication.