Introduction
Acute respiratory distress syndrome (ARDS) continues to be major causes of mortality in the intensive care units, even though mechanical ventilation with low tidal volume [
1], early neuromuscular blockade [
2], and prone positioning [
3] have been shown to reduce mortality from ARDS. No pharmacotherapy has proven effective in decreasing mortality in adult patients with ARDS [
4]. The pathophysiology of ARDS involves inflammation with diffuse alveolar damage, formation of hyaline membranes, increased capillary permeability, interstitial edema, and influx of circulating inflammatory cells [
5]. Although neutrophil influx and activation within the lung are important factors in the pathogenesis of ARDS, alveolar macrophages (AMs) and alveolar and bronchial epithelial cells are also involved in the disease process [
5],[
6]. In particular, increasing evidence demonstrates that AMs contribute to the modulation of inflammatory responses and the resultant lung injury [
7],[
8]. Pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, secreted by AMs stimulate neutrophils, and these activated neutrophils release oxidants, proteases, leukotrienes, and platelet activating factors, resulting in the development of ARDS [
5].
Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, can induce inflammatory responses and disturb immune system function [
9]. Intratracheal administration of LPS has gained wide acceptance as a clinically relevant model of ARDS in mice [
10].
Quercetin is one of the most abundant dietary flavonoids and is found in a broad range of fruits, vegetables, and beverages. Quercetin has been demonstrated to have potent anti-inflammatory and anti-oxidant activities [
11],[
12]. We reported that quercetin exhibited cytoprotective effects through the induction of heme oxygenase (HO)-1 [
11],[
13],[
14]. HO-1 is a stress-inducible protein and catalyzes the rate-limiting step in the degradation of heme to biliverdin, carbon monoxide (CO), and ferrous iron [
15].
ARDS often develops in intubated patients. During intubation management, the antioxidants supplied from a regular diet cannot be taken in. Therefore, we first focused on the prophylactic effects of quercetin to prevent the development of ARDS in high-risk patients. To qualify as a prophylactic treatment, it must be harmless, relatively inexpensive, and easily and widely applicable. It is thought that quercetin fulfills these conditions. Moreover, we considered that local administration to the lung could more effectively reveal the effects of quercetin, which is not easily absorbed, in the lung.
In this study, we investigated whether the intratracheal administration of quercetin could suppress LPS-induced acute lung injury (ALI).
We also investigated the involvement of HO-1 in the suppressive effects of quercetin.
Materials and methods
Reagents
LPS from Klebsiella pneumoniae LEN-1 (O3:K1−) was kindly donated by Prof. T. Hasegawa (Aichi Medical University School of Medicine, Aichi, Japan). Quercetin was obtained from Sigma (St. Louis, MO). Tin protoporphyrin IX (SnPP) was obtained from Frontier Scientific (Carnforth, UK).
Cell culture
The mouse AM cell line, AMJ2-C11, and the mouse alveolar epithelial cell line, LA-4, were purchased from the American Type Culture Collection (Manassas, VA). The
cdk4/hTERT-immortalized normal human bronchial epithelial cell line, HBEC4 [
16] was obtained from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX). AMJ2-C11 and LA-4 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 100 U/ml penicillin, 0.1 U/ml streptomycin, 2.5x10
−4 U/ml amphotericin B, 1 mM sodium pyruvate, Minimum Essential Medium (MEM) non-essential amino acids, and 10% Fetal bovine serum (FBS). HBEC4 cells were cultured in keratinocyte serum-free medium (Life Technologies, Gaithersburg, MD) supplemented with 50 ng/ml bovine pituitary extract and 5 ng/ml epidermal growth factor. Cells were grown under standard conditions in a humidified incubator at 37°C and 5% CO
2.
Animals
Wild-type BALB/c mice were purchased from SLC (Shizuoka, Japan). 8- to 12-week-old mice (weight: 18–25 g) matched for age and weight were used for the studies. Animals were maintained in a temperature (22-24°C), humidity (55 ± 5%), and light (12 hours light–dark cycle; lights on at 8:00) regulated room with access to food and water ad libitum. All procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagoya University.
Mouse model of ALI
Mice were randomly divided into 4 groups (each group: n = 3 ~ 5): control (Phosphate buffered saline (PBS))-treated, quercetin-treated, LPS-treated, and quercetin + LPS-treated. We made a small incision on the neck of mouse’s skin and exposed a trachea under sodium pentobarbital anesthesia. Then the mice were challenged intratracheally with either 50 μl PBS alone or 50 μl PBS of 1.25 μg LPS by stabbing the trachea with a microsyringe with a 22-gauge needle. In some experiments, mice were administered 50 μl of 0.1% propylene glycol (vehicle) or 10 μM quercetin in 0.1% propylene glycol intratracheally 6 hours before LPS challenge. The bronchoalveolar lavage fluid (BALF) and lungs were collected 24 hours after LPS administration in separate experiments. The severity of lung injury was assessed by wet lung-to-body weight ratio, pathological changes in lung tissues, and cellular profiles in BALF.
Collection of BALF
BALF were collected as previously described [
7]. Briefly, mice were exsanguinated by aortic perforation under pentobarbital anesthetization. The trachea was cannulated, and the lungs were lavaged six times with PBS (0.5 ml each time). Collected BALF were centrifuged at 1,200 rpm for 3 minutes, and the pelleted cells were then re-suspended in RPMI-1640 medium supplemented with 100 U/ml penicillin, 0.1 U/ml streptomycin, 2.5x10
−4 U/ml amphotericin B, 1 mM sodium pyruvate, MEM non-essential amino acids, 50 μM 2-mercaptoethanol, and 10% FBS.
Leukocyte counts in BALF
The number of leukocytes was enumerated with a hemocytometer. For differential counts, smears of BALF cells from each mouse were prepared with centrifugation using Cytofuge2 (StatSpin, Norwood, MA) at 1,000 revolution per minute for 2 minutes and then stained with May-Grünwald and Giemsa solutions.
Wet lung-to-body weight ratio
The lungs were removed from the thoracic cavity and cleared of extraneous tissue. Each lung was weighed, and the wet lung-to-body weight ratio was then calculated to assess lung inflammation.
Histological study
For histological examination, paraffin sections (6 μm thick) were stained with hematoxylin and eosin (H&E). For immunohistochemical examination, 3-μm sections were treated with 0.3% hydrogen peroxide, and then treated with 10% goat serum (Nichirei Biosciences, Tokyo, Japan) prior to incubating with a primary antibody against HO-1 (Enzo, Lausen, Switzerland) followed by the secondary antibody, Simple Stain Mouse MAX-PO (R) (Nichirei Biosciences). The slides were then visualized with DAB chromogen (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin.
Cell activation
As for HO-1 mRNA and protein expression in AMJ2-C11, LA-4, and HBEC4 cells, cells were cultured with various concentrations (0, 5, 10, 20 μM) of quercetin for 4 or 8 hours, respectively. As for the cytokine expression and production in AMJ2-C11 cells or BALF cells, cells were cultured with quercetin (AMJ-2C11 cells; 20 μM, BALF cells; 10 μM) for 1 hour and then stimulated with LPS (5 μg/ml) for 2 hours or 18 hours, respectively. In some experiments, AMJ-2C11 cells were treated with SnPP (20 μM) for 30 minutes before quercetin treatment. As for the MMPs activity and cytokine production in activated BALF cells in advance, BALF were collected 24 hours after an intratracheal LPS challenge and cultured with quercetin or vehicle for 1 hour. After the medium was changed, BALF cells were cultured with vehicle or quercetin for another 18 hours and the supernatant was collected.
Western blotting
Western blotting was performed as described previously [
14]. For analysis of HO-1
in vivo, lungs were collected 6 hours after intratracheal administration of vehicle or quercetin. For analysis of HO-1
in vitro, AMJ2-C11, LA-4, and HBEC4 cells were cultured with various concentrations (0, 5, 10, 20 μM) of quercetin for 8 hours.
Reverse transcription (RT) - polymerase chain reaction (PCR), quantitative real-time PCR
Total ribonucleic acid (RNA) was isolated using ISOGEN II (Nippon Gene, Toyama, Japan) and reverse-transcribed to cDNA using PrimeScript RT MasterMix (Takara Bio, Shiga, Japan). Quantitative real-time PCR was performed on a Thermal Cycler Dice Real Time System II (TaKaRa Bio). Primers and probes for Hmox1, Tnfa, Il1b, Il6, Gapdh, HMOX1, and GAPDH were obtained from Nippon EGT (Toyama, Japan). Transcripts of Gapdh or GAPDH, as a house-keeping gene, were quantified as endogenous reference RNA to normalize each sample. Relative quantities of expression were estimated by the standard curve method. The results were normalized as relative expression, in which the average value of Hmox1, Tnfa, Il1b, Il6, or HMOX1 was divided by the average value of Gapdh or GAPDH. The ratio was calculated by dividing the normalized values of stimulated cells by the values in control cells.
Enzyme-linked immunosorbent assay (ELISA)
The cytokine production of TNF-α, IL-1β, and IL-6 in cell culture supernatants was quantified using a murine ELISA development kit (PEPROTECH, Rocky Hill, NJ) according to the manufacturer’s recommendations.
Gelatin zymography
Gelatin zymography to determine matrix metalloproteinase (MMPs) activity was performed as previously described with some modifications [
7]. The molecular weights of the gelatinolytic bands were estimated using Precision Plus Protein marker (BIO-RAD, Carlsbad, CA). The intensities of MMP-9 bands were estimated using Scion Image (Scion, Fredrick, MD).
Statistical analysis
Statistical comparisons among the groups were assessed by one-way analysis of variance (ANOVA). When F ratios were significant (p < 0.05), Tukey-Kramer’s post-hoc test (between group comparison) were performed, and p < 0.05 was considered a statistically significant. Statistical analysis was performed with StatView (Abacus Concept Inc., Gloucestershire, UK).
Discussion
In the current study, we first demonstrated that the intratracheal administration of quercetin attenuated LPS-induced ALI in mice and that quercetin suppressed LPS-induced pro-inflammatory cytokine production via an HO-1-dependent pathway. Furthermore, quercetin decreased the activity of MMP-9 and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance.
Intragastric administration of quercetin has been demonstrated to show preventive effects in LPS-induced sepsis in mice [
17]. Although the daily dietary intake of quercetin has been estimated as 5–40 mg/day in humans [
18], total quercetin derived from the diet was reported to be in the nanomolar range (<100 nM) in plasma as a result of its poor absorption and metabolism [
19]. Furthermore, daily supplementation with 1 g of quercetin for 4 weeks increased plasma concentrations only up to 1.5 μM [
20], which could not provide adequate amounts of quercetin to produce anti-inflammatory and anti-oxidant effects [
19]. In consideration of these reports on the low bioavailability and the inability of patients with serious diseases, such as ARDS, to take quercetin orally, we administered quercetin intratracheally to maintain quercetin at high local concentrations.
We demonstrated that intratracheal prophylactic quercetin treatment suppressed the LPS-induced increase in the wet lung-to-body weight ratio. The wet lung-to-body weight ratio, an index of pulmonary edema, is correlated with the severity of lung injury, as previously reported [
21]. Pulmonary edema, which is a major feature of ARDS, is associated with the malfunction of two lung cellular barriers: epithelial and endothelial cells [
22]. Quercetin has been shown to improve the barrier function of both epithelial and endothelial cell [
23],[
24]. Considering our results and previous reports together, the improvement of barrier function might be involved in the mechanism of suppression by quercetin on LPS-induced inflammation. Moreover, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were reported to be increased in patients with ARDS, and play an important role in the initiation and propagation of the inflammatory cascade in ARDS [
5],[
25]. Suppression of pro-inflammatory cytokine production by quercetin pretreatment may also have contributed to the attenuation of LPS-induced ALI in mice.
Lung protection as a consequence of HO-1 induction has been demonstrated in a number of lung injury models
in vitro and
in vivo [
26]. HO-1 is an essential enzyme that catalyzes the degradation of heme to ferrous iron, CO, and biliverdin, which is subsequently converted to bilirubin [
15]. The protective effects of HO-1 against inflammation are considered not only to decrease harmful heme, but also to produce the metabolites CO and bilirubin, which have the cytoprotective effects [
27],[
28]. Importantly, quercetin induced the significant upregulation of HO-1 expression [
13],[
14]. In fact, in this study quercetin induced HO-1 in AMs, alveolar and bronchial epithelial cells in mouse lung. Our results are consistent with previous reports that HO-1 expression was detected in AMs and epithelial cells in lungs with LPS stimulation or oxidative stress [
29],[
30]. Furthermore, we confirmed the induction of HO-1 expression in these types of cells using cell lines. AMs and lung epithelial cells may produce pro-inflammatory cytokines locally in the lung in ARDS [
5], so it makes sense that HO-1, which has anti-inflammatory effects, is expressed on these cells.
We investigated the activities of MMPs other than on the secretion of cytokines. MMP-9 is mainly produced by inflammatory cells, such as neutrophils and macrophages, and activation mechanisms of MMP-9 are involved in other MMPs, such as MMP-3, and neutrophil elastase [
31],[
32]. The levels of MMP-9, which is a major factor in neutrophil migration across basement membranes [
33], have been reported to be increased in patients with ARDS [
34]. We observed that quercetin decreased the activity of MMP-9, the inhibition of which could mitigate the inflammatory response in lungs by suppressing the production of MMP-3 and neutrophil elastase [
35],[
36]. In fact, mice lacking MMP-9 and the inhibition of MMP-9 have shown less lung injury [
37],[
38]. Taken together, it is suggested that quercetin could suppress LPS-induced inflammation and have beneficial effects on lungs not only as a prophylactic treatment but also a supportive therapeutic drug.
Acknowledgement
This study was supported in part by a Grant-in-Aid for Scientific Research form the Japan Ministry of Education, Culture, Sports, Science and Technology, and a Grant-in-Aid from the Nagono Medical Foundation, Nagoya, Japan.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conception and design: KT, MM, TK. Acquisition of the data: KT, MM, KH, HN, MS. Analysis and interpretation: KT, MM, TK. Drafting the manuscript for important intellectual content: KT, MM, NH, YH, TK. All authors read and approved the final manuscript.