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Anti-inflammatory Effects of Resveratrol on Hypoxia/ Reoxygenation–induced Alveolar Epithelial Cell Dysfunction Po-Len Liu, Inn-Wen Chong, Yi-Chen Lee, Jong-Rung Tsai, Hui-Min Wang, ChongChao Hsieh, Hsuan-Fu Kuo, Wei-Lun Liu, Yung-Hsiang Chen, and Hsiu-Lin Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01168 • Publication Date (Web): 15 Oct 2015 Downloaded from http://pubs.acs.org on October 15, 2015

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Journal of Agricultural and Food Chemistry

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2

(jf-2015-01168g.R3)

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Anti-inflammatory Effects of Resveratrol on Hypoxia/ /Reoxygenation– –

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induced Alveolar Epithelial Cell Dysfunction

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Po-Len Liu,1,# Inn-Wen Chong,1,2 Yi-Chen Lee,1 Jong-Rung Tsai,1,2 Hui-Min

8

Wang,1 Chong-Chao Hsieh,2 Hsuan-Fu Kuo,3 Wei-Lun Liu,4 Yung-Hsiang

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Chen,5,6,7,# and Hsiu-Lin Chen1,2,*

10 11 12 13 14 15 16 17 18 19 20 21 22 23

1

Department of Respiratory Therapy, Department of Fragrance and Cosmetic Science, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan

2

Department of Pediatrics, Department of Internal Medicine, Department of Chest Surgery, Division of Cardiovascular Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan

3

Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 801, Taiwan

4

Department of Intensive Care Medicine, Chi Mei Medical Center, Tainan 736, Taiwan

5

Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan

6

Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan

7

Department of Psychology, College of Medical and Health Science, Asia University, Taichung 413, Taiwan

24 25

#

P.-L. Liu and Y.-H. Chen contributed equally to this study.

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*Correspondence should be addressed to Hsiu-Lin Chen; Tel: +886-4-22053366#3501.

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Fax: +886-2-33663462. E-mail: [email protected]

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Short title: Resveratrol Inhibits Pneumocyte Dysfunction and Inflammation

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ACS Paragon Plus Environment


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ABSTRACT Reducing

oxidative

stress

is

crucial

to

prevent

hypoxia-reoxygenation

34

(H/R)-induced lung injury. Resveratrol has excellent antioxidant and anti-inflammatory

35

effects, and this study investigated its role in H/R-induced type II pneumocyte

36

dysfunction. H/R conditions increased expression of inflammatory cytokines including

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interleukin (IL)-1β (142.3±21.2%, P < 0.05) and IL-6 (301.9±35.1%, P < 0.01) in a type

38

II alveolar epithelial cell line (A549) while the anti-inflammatory cytokine IL-10

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(64.6±9.8%, P < 0.05) and surfactant proteins (SPs) decreased. However, resveratrol

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treatment effectively inhibited these effects. H/R significantly activated an

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inflammatory transcription factor, nuclear factor (NF)-κB,

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significantly inhibited H/R-induced NF-κB transcription activities. To the best of our

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knowledge, this is the first study showing resveratrol-mediated reversal of H/R-induced

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inflammatory responses and dysfunction of type II pneumocyte cells in vitro. The

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effects of resveratrol were partially mediated by promoting SP expression and inhibiting

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inflammation with NF-κB pathway involvement. Therefore, our study provides new

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insights into mechanisms underlying the action of resveratrol in type II pneumocyte

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dysfunction.

while resveratrol

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Keywords: Resveratrol, inflammation, hypoxia, reoxygenation, surfactant 2


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INTRODUCTION Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenol compound that is found in

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many plants and abundantly in grapes and red wine.1,

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quantified as glucoside and aglycone forms of resveratrol, has been determined. The

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average between ratio glucoside/aglycone forms of resveratrol in these wines was

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considerably high, ranging from 82 to 91% of resveratrol in its glycosidic form.3 A

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growing number of in vivo studies indicate that resveratrol has protective effects in

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various oxidative stress and disease conditions.4 It has been previously reported that

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resveratrol lowers blood pressure, plasma free fatty acids, cholesterol, triacylglycerol,

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and glucose.5 Resveratrol has been suggested to have a wide range of beneficial health

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effects including cardioprotective, neuroprotective, antioxidant, anti-inflammatory,

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anti-proliferative, and anti-angiogenic effects.6

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Oxidative

stress

plays

an

important

role

2

in

Total resveratrol content,

the

pathogenesis

of

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ischemia-reperfusion injury. For example, hypoxia-reoxygenation (H/R)-induced

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pulmonary injury leads to increased mortality and morbidity in neonates and patients

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who have received lung transplants.7 Neonatal pulmonary physiology studies show that

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approximately 10% of newborns require some assistance to begin breathing at birth,8

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i.e., an intervention is required to facilitate the transition from intrauterine to 3



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extrauterine conditions. Approximately 1% of newborns require extensive resuscitative

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measures including the establishment of the airway, provision of oxygen, and

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positive-pressure ventilation.9 The lung epithelial cells of these newborns are exposed to

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hypoxia before and during resuscitation and then to reoxygenation afterward. There are

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adaptive strategies for cells to cope with hypoxia;10 however, subsequent reoxygenation

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leads to cellular dysfunction, which subsequently influences the clinical outcomes for

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newborn babies. This suggests that even if there is successful resuscitation in hypoxic

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newborns, reoxygenation afterward could cause injury by increasing free radical

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generation, elevating levels of inflammation-related cytokines, and decreasing the

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expression of lung surfactants.11

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Previous reports showed that hypoxia leads to injury of the lung epithelial cells,

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thickened respiratory membranes, and formation of a hyaline membrane as well as

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increase in inflammatory cell adhesion molecules, inflammatory cytokines, and growth

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factors.12, 13 In addition, there is a reduction in activity of and deactivation of surfactants,

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which results in alveolar edema and cellular apoptosis or necrosis following hypoxia.14,

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15

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Therefore, the reduction of neonatal morbidity after H/R, essentially requires a

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preventive strategy to decrease inflammation-related lung epithelial cell dysfunction

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after resuscitation. Despite its well-established antioxidant and beneficial health effects,

Lung H/R injury involves inflammation caused by innate immune responses.13

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there is very little information available on the application of resveratrol in the

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prevention and treatment of H/R-caused lung injury.

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Reducing oxidative stress is crucial to preventing H/R-induced lung injury.

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Therefore, in the present study, we focused on type II lung alveolar epithelial cells and

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explored the potential benefits of the antioxidative and anti-inflammatory cytoprotective

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effects of resveratrol in the prevention and treatment of H/R-induced inflammation and

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alveolar epithelial cell dysfunction.

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MATERIALS AND METHODS

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Chemicals. Antibodies for interleukin (IL)-1β, IL-6, and IL-10 were from

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GeneTex (Irvine, CA, USA). Antibodies for α-tubulin, IκB, phospho-p65 (Ser276), and

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phospho-p65 (Ser536) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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Resveratrol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

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were from Sigma (St. Louis, MO, USA). The electrophoretic mobility shift assay

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(EMSA) kit for nuclear factor (NF)-κB was from Roche (Indianapolis, IN, USA) and

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the nuclear protein extraction kit was from Millipore (Temecula, CA, USA).16, 17 The

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anaerobic chamber was from Don Whitley Scientific (Shipley, West Yorkshire, UK).

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Cell Culture. Since human primary alveolar epithelial cells are not commercial

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available, A549 cells (an adenocarcinomic human alveolar basal epithelial cell line)

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(BCRC, Hsinchu, Taiwan) were used in the present study. In nature, these cells are

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squamous and responsible for the diffusion of some substances, such as water and

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electrolytes, across the alveoli of lungs. A549 cells grow as monolayer cells, adherent or

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attaching to the culture flask in vitro. Another characteristic of these cells is that they are

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able to synthesize lecithin and contain high level of unsaturated fatty acids, which are

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important to maintain the membrane phospholipids in cells.12, 18, 19 A549 were cultured

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in the base medium for this cell line is American Type Culture Collection-formulated

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F-12K Medium (Invitrogen, Grand Island NY, USA) supplemented with 5% fetal 6


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bovine serum (FBS), 100 units/mL penicillin, and 100 pg/mL streptomycin (Invitrogen,

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Grand Island, NY, USA) in a humidified incubator with 5% CO2 at 37°C.

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H/R Model. Cultured cells were exposed to H/R, as described previously.20 The in

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vitro H/R model was created by the Whitley anaerobic and microaerophilic jar gassing

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system (Don Whitley Scientific, Shipley, West Yorkshire, UK). Briefly, confluent

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beating A549 monolayer cells were co-incubated or pretreated with resveratrol (50 µM

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for 12 h) and then exposed to anoxia (0.33% O2, 5% CO2, 95% N2) for 0.5, 1, 2, or 3 h

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in FBS-free F12K medium and then reoxygenated (normoxia: 20% O2, 5% CO2, 80%

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N2) for 6 h in FBS-free F12K medium.

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Cell Viability Assay. Cell viability was measured using the MTT assay. Briefly,

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cells were incubated with or without resveratrol for various doses and times, and 100 µL

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of MTT (0.5 mg/mL in medium) was then added to each well. After 4 h of incubation

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with MTT at 37°C, the medium was carefully removed and cells were washed twice

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with phosphate-buffered saline (PBS). The metabolized MTT was solubilized with 100

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µL of dimethyl sulfoxide, and the absorbance of the solubilized blue formazan was read

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at 530 nm with 690 nm as reference using a DIAS microplate reader (Dynex

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Technologies, Chantilly, VA, USA).21, 22 The cells incubated with control medium were

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considered 100% viable.

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Western Blot Analysis. Cells were washed with phosphate-buffered saline (PBS), 7



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pH 7.4, centrifuged for 10 minutes at 4°C at 1200 × g, and lysed for 1 h at 4°C with

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lysis buffer (NaCl 0.5 M, Tris 50 mM, EDTA 1 mM, 0.05% SDS, 0.5% Triton X-100,

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PMSF 1 mM, pH 7.4. The cell lysates were centrifuged at 4000 × g for 30 minutes at

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4°C. Protein concentrations in the supernatants were measured using a Bio-Rad protein

138

determination kit (Bio-Rad, Hercules, CA, U.S.A.). Cytoplasm protein extracts (40

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µg/lane) were separated on 10% SDS–PAGE and then transferred to polyvinylidene

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difluoride membranes for 1 h at room temperature.23-25 The membranes were treated

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with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room

142

temperature. They were then incubated separately with various primary antibodies and

143

then secondary antibodies. The protein bands were detected using an enhanced

144

chemiluminescence kit (PerkinElmer, Waltham, MA, USA) and exposure to Biomax

145

MR Film (Kodak, Rochester, NY, USA). Data were quantified using ImageQuant 5.2

146

(Healthcare Bio-Sciences, Philadelphia, PA, USA).

147

RNA Extraction and Real-time PCR. Total RNA was isolated from cells and

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subsequently analyzed by real-time PCR. The primers were designed using Primer

149

Express software (RealQuant, Roche) based on published sequences (Table 1). PCR

150

conditions included an initial denaturation at 94°C for 180 s, followed by 40 cycles at

151

95°C for 30 s, 60°C for 25 s, 72°C for 30 s, and 1 cycle at 72°C for 7 minutes.

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Fluorescence data were acquired after the final extension step. A melt analysis was 8


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conducted for all products to determine the specificity of the amplification. In addition,

154

PCR products were run on 1 % agarose gels to confirm their correct sizes.

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Nuclear Extract Preparation and EMSA. Nuclear protein extracts were prepared

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as previously described. Briefly, after washing with PBS, the cells were scraped off the

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plates in 0.6 mL of ice-cold buffer A (HEPES [pH 7.9], 10 mM KCl, 1 mM DTT, 1 mM

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PMSF, 1.5 mM MgCl2, and 2 mg/mL each of aprotinin, pepstatin, and leupeptin). After

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centrifugation at 300 × g for 10 minutes, the cells were resuspended in buffer B (80 mL

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of 0.1% Triton X-100 in buffer A), left on ice for 10 minutes, and centrifuged at 12,000

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× g for 10 minutes. The nuclear pellets were resuspended in 70 mL of ice-cold buffer C

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(20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.42 M NaCl, 1 mM DTT, 0.2 mM EDTA, 1

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mM PMSF, 25% glycerol, and 2 mg/mL each of aprotinin, pepstatin, and leupeptin) and

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incubated for 30 minutes, followed by centrifugation at 15,000 × g for 30 minutes. The

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resulting supernatant was as the nuclear extract. Protein concentrations were determined

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by the Bio-Rad method. The NF-κB probe was used in the gel shift assay was a 31-mer

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synthetic double-stranded oligonucleotide (5’-ACA AGG GAC TTT CCG CTG GGG

168

ACT TTC CAG G-3’; 5’-CCT GGA AAG TCC CCA GCG GAA AGT CCC TTG T-3’)

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containing a direct repeat of the κB site.

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Statistical Analyses. Data are presented as mean ± standard deviation (SD) for

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each group. Data were analyzed by ANOVA and subsequently by Dunnetts’ test. All 9



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statistics were calculated using SigmaStat version 3.5 (Systat Software Inc., Chicago, IL,

173

USA), and a P value of less than 0.05 was considered statistically significant.

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RESULTS

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H/R Increased Inflammatory Response in Alveolar Epithelial Cells. IL-1β and

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IL-6 are pro-inflammatory cytokines, whereas IL-10 is an anti-inflammatory cytokine.

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Previous study showed that, after lung reperfusion, a decrease in IL-10 mRNA

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expression was observed and markedly increased the expression of the proinflammatory

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cytokines.26 We determined the effect of H/R on cytokine expression in A549 type II

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alveolar epithelial cells by exposing them to hypoxia for varying amounts of time (0.5,

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1, 2, and 3 h) and then reoxygenating them for 6 h. Western blot and real-time PCR

182

analyses (Figure 1A and 1B, respectively) showed that H/R induced a higher

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expression of the inflammatory cytokines IL-1β and IL-6 during a 0.5–3 h exposure to

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hypoxia than was induced under normoxia conditions. Furthermore, exposure to H/R for

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up to 3 h decreased the protective cytokine IL-10 expression in the A549 cells.

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H/R Reduced Surfactant Protein (SP) Levels in Alveolar Epithelial Cells.

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Alveolar type II cells are also distinguished by the presence of lamellar bodies and

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intracellular organelles that store and secrete SPs.27 We evaluated the H/R-induced

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changes in SP levels of A549 cells exposed to hypoxia for varying amounts of time (0.5,

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1, 2, and 3 h) and then reoxygenated for 6 h. The western blot analysis demonstrated

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that H/R exposure for up to 3 h decreased SP-A and SP-D protein levels

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time-dependently. Cells exposed to transient hypoxia treatment (0.5 or 1 h) and then 11



Page 12 of 35

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reoxygenated for 6 h showed increased SP-B and SP-C protein expression. In contrast,

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prolonged hypoxia treatment (2–3 h) decreased SP-B and SP-C protein levels in A549

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cells (Figure 2).

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Effect of Resveratrol on Viability of Alveolar Epithelial Cells. To evaluate a

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optimize dose of resveratrol, the cytotoxicity of varying concentrations of resveratrol

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(12.5–100 µM) applied to A459 cells for 6–48 h was initially determined using the

199

MTT assay. Figure 3 shows that resveratrol at 12.5–100 µM concentration range (for

200

24 h) exhibited no significant cytotoxic effects. Also, 50 µM resveratrol exhibited no

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significant cytotoxic effects for 6–48 h. Therefore, the 50 µM concentration of

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resveratrol and a 12 h treatment period, which showed no cytotoxicity against the A549

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cells, were used for subsequent experiments. We also performed a study of survival rate

204

of

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hypoxia/reoxygenation condition. Figure 3 shows that hypoxia/reoxygenation condition

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(hypoxia for 3 h and reoxygenation for 6 h, respectively) did not reduce cell viability

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even pre-treatment with or without resveratrol. The conditions that hypoxia for 3 h and

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reoxygenation for 6 h were used for further experiments.

A549

cells

which

was

pre-treated

with

or without resveratrol

under

209

Resveratrol Attenuated H/R-caused Inflammatory Responses in Alveolar

210

Epithelial Cells. We explore the potential anti-inflammatory effects of resveratrol,

211

using a Western blot assay. Pretreatment of cells with resveratrol significantly reduced 12


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H/R-induced IL-1β and IL-6 expression. In contrast, pretreatment of cells with

213

resveratrol

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co-incubation with resveratrol in H/R conditions only significantly decreased IL-1β

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expression (Figure 4).

significantly

prevented

H/R-induced

IL-10

downregulation

while

216

Resveratrol Reversed H/R-induced SP Downregulation in Alveolar Epithelial

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Cells. We next explore the effect of resveratrol on SP expressions. Pretreatment as well

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as co-incubation of cells with resveratrol significantly reversed H/R-induced

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downregulation of SP-A, SP-D, SP-B, and SP-C (Figure 5).

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Resveratrol Inhibited H/R-induced NF-κ κB Activation in Alveolar Epithelial

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Cells. Subsequently, we examined whether NF-κB activation is associated with

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increased inflammation in cells after H/R. The results showed that H/R significantly

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decreased IκB, p65, and p50 in the cytosolic cellular fractions (Figure 6A), whereas the

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phospho-p65 (Ser276) and levels of p65 and p50 were significantly increased in the

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nuclear cellular fractions (Figure 6B). Additionally, Western blot (Figure 6A and 6B)

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and EMSA (Figure 6C) showed that pretreatment or co-incubation of cells with

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resveratrol significantly inhibited H/R-induced NF-κB transcription activities.

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DISCUSSION

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In the present study, our results are the first to show that resveratrol attenuates

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H/R-induced inflammatory responses in alveolar epithelial cells. Furthermore,

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resveratrol treatment reversed H/R-induced SP downregulation. Our data also suggest

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that NF-κB activation may be involve to play a pivotal role in the anti-inflammatory

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effects of resveratrol on type II lung alveolar epithelial cells.

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The present study showed that the protected effects exerted by the

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resveratrol-mediated inhibition of inflammation in type II lung alveolar epithelial cells

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exposed to H/R might be associated with NF-κB signaling pathway.28 We found that

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H/R induced NF-κB nuclear translocation in an IκB-dependent manner and enhanced

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the production of inflammatory cytokines in lung alveolar epithelial cells. This result

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implies that NF-κB may play an important role in triggering the inflammatory response

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that results from pulmonary H/R. Additionally, it has been shown that IL-10 exerts its

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anti-inflammatory activity in part through the inhibition of NF-κB. In the absence of an

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activating stimulus, IL-10 specifically induces the nuclear translocation of repressive

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p50/p50 homodimers, which compete with pro-inflammatory p65/p50 heterodimers for

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DNA-binding to NF-κB promotor sites on inflammatory genes such as IL-6. In the

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presence of a stimulus, IL-10 can suppress nuclear translocation and DNA-binding of

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p65/p50 heterodimers by inhibiting IκB kinase (IKK) activity and thus delaying 14


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degradation of IκBα. These findings suggest that the selective induction of nuclear

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translocation and DNA-binding of the repressive p50/p50 homodimer is an important

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anti-inflammatory mechanism utilized by IL-10 to repress inflammatory gene

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transcription.29

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Evidence shows that at a lower dose, resveratrol acts as an anti-inflammatory and

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cytoprotective agent by increasing the expression of cell survival proteins and, thereby,

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improves post-ischemic recovery. However, at a higher dose resveratrol acts as a

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pro-apoptotic compound and induces apoptosis by activating cell death signaling.30

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Consistent with a previous study,31 our data demonstrate that resveratrol protected lung

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alveolar epithelial cells at a relatively low dose (

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