It is reported that chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide, and mortality rates will become the third leading cause of death by 2030 [1
]. Oxidative stress, protease–antiprotease imbalance and inflammation are important in the pathogenesis of COPD [2
]. Lipopolysaccharide (LPS) and cigarette smoke (CS) have become the preferred stimuli for researching COPD [3
]. CS is a mixture of oxidant radicals and different chemical compounds, which cause oxidative stress in the lungs. LPS is an endotoxin of gram negative bacteria, which can induce lung injury. CS and LPS were combined to replicate severe inflammation and oxidative stress in rat lungs in this study. The role of cell signaling pathway dysfunction and oxidative stress in COPD were recognized.
CS augments the production of reactive oxygen species (ROS) and numerous pro-inflammatory cytokines such as IL-6 and IL-8 [4
].The lipid peroxidation product malondialdehyde (MDA) is the most commonly measured indicator of oxidative damage to membrane lipids [5
]. Superoxide dismutase (SOD) eliminates superoxide and reduces oxidative stress and tissue damage. There is believed to be a need for a new therapy for candidates exhibiting anti-oxidant and anti-inflammatory properties for COPD. SIRT1 is a well-known longevity gene, which regulates stress resistance and inflammation by deacetylation of intracellular signaling molecules and histones [6
], which probably acts via different mechanisms to regulate age-related changes including increasing mitochondriogenesis via modulating PGC-1α deacetylation [7
Resveratrol is a phytoalexin found in the skin and seeds of grapes, recent studies have demonstrated the role of resveratrol in lung injuries induced by different xenobiotics via the antioxidant and anti-inflammatory pathway [5
]. Resveratrol restored ovarian function through diminishing ovarian inflammation, predominantly via upregulation of SIRT1 expression leading to the inhibition of inflammatory cytokines [8
]. Resveratrol could also inhibit COPD-associated cytokines/chemokines such as IL-6 and IL-8 from releasing from the human airway smooth muscle cells (HASMCs) through the activation of SIRT1 [9
However, to our knowledge, it is unknown whether resveratrol reduces inflammation and oxidative stress in the lungs of COPD via the SIRT1/PGC-1α signaling pathway. This study was designed to evaluate the effects of resveratrol on COPD in rats. It was hypothesized that upregulation of SIRT1/PGC-1α expression might represent essential regulatory mechanisms implicated in the effects of resveratrol.
In the present study, we demonstrated that treatment with resveratrol attenuated CS- and LPS-induced lung inflammation in a rat model of COPD. Resveratrol administration significantly decreased levels of pro-inflammatory and oxidative stress [5
]. In the study, the levels of pro-inflammatory cytokines such as IL-8 and IL-6 were increased after CS and LPS exposure. The resveratrol therapy significantly impeded the levels of these inflammatory cytokines (IL-8 and IL-6) and inflammatory cells in BALF, which may explain the anti-inflammatory activity of resveratrol. Resveratrol reduced MDA content and increased SOD, which may explain the anti-oxidative stress of resveratrol. For the first time, our results showed that SIRT1 and PGC-la expression were reduced by cigarette smoke and LPS, and elevated by resveratrol. These results imply that the effect of resveratrol on airway inflammation induced by cigarette smoke exposure might act through the SIRT1/PGC-lα pathways.
Inflammation and oxidative stress are thought to play a pivotal role in the pathogenesis of COPD [10
]. The inflammatory environment in COPD leads to oxidative stress, as activated cells recruited to the airways produce excessive quantities of ROS [11
]. IL-6 and IL-8 play key roles in the pathogenesis of stable and exacerbated COPD [12
]. SOD is an anti-inflammatory enzyme as well as a major anti-oxidant [13
] and MDA, a by-product of polyunsaturated fatty acid peroxidation, may be a reliable marker of oxidative stress in COPD [14
]. Resveratrol has a cardiac protective effect against smoking and lipopolysaccharides (SM/LPS)-induced oxidative stress through upregulation of SOD activity [15
]. Our results showed that treatment with resveratrol significantly improved the histological structure in the lung tissue. It also reduced in the extent of cellular infiltration, and the number of inflammatory cells. Our study also demonstrated that resveratrol significantly reduced pulmonary inflammation as determined by the numbers of total cells and the proportion of neutrophils in BALF. Resveratrol reduced the levels of IL-6 and IL-8, which may be potentially related to its anti-inflammatory property. We discovered that resveratrol has a protective effect through upregulation of SOD activity and regulation of MDA activity against CS/LPS induced oxidative stress.
SIRT1 has broad biological effects, which involve both oxidative stress and cell metabolism [16
]. It has strong antioxidative stress and anti-apoptosis effects in the heart [17
]. SIRT1, a longevity associated protein, is important in maintaining mitochondrial function [18
]. Previous studies have demonstrated that the anti-inflammatory action of resveratrol may be mediated through enhancing SIRT1 expression [8
]. A study demonstrated that IL-8 and IL-6 expressions were attenuated in cells pretreated with SIRT1 activators, and proinflammatory effects were exacerbated by the knockdown of SIRT1 expression [19
]. Since resveratrol acts as a SIRT1 mimetic [15
], we investigated the effects of resveratrol treatment on lung SIRT1 expression in COPD. The expression of SIRT1 was detected by western blotting and real-time PCR and immunohistochemical staining. Our results showed that SIRT1 was decreased in the lung of COPD rats and was reversed in COPD rats with resveratrol treatment. These findings suggest that resveratrol plays an important role in the regulation of inflammation and oxidants by SIRT1 in COPD.
More importantly, SIRT1 has been demonstrated to interact directly with PGC-1 to increase PGC-1α expression and mitochondria biogenesis [20
]. SIRT1 was expressed in both the cytoplasm and nuclei in many metabolically active tissues, such as the PPAR-γ receptor and its coactivator PGC-1α [21
]. PGC-1α suppresses ROS production in cells through the induction of ROS detoxifying enzymes. It has been demonstrated that decreased PGC-1α expression increases oxidative stress and neurodegeneration [22
]. PGC-1α may not only stimulate mitochondrial biogenesis, but also protect neurons from oxidative injury through the induction of several ROS-detoxifying enzymes [23
]. PGC-1α is also a master regulator of ROS-scavenging enzymes including Mn-SOD2, catalase and GSH-Px [24
]. Resveratrol can stimulate SIRT1 and PGC-1α activation, which in turn may promote the expression of the slow, oxidative myogenic program in mdx mouse muscle [25
]. ROS play important roles in COPD, which is regulated by inflammation mechanisms, and excessive ROS can directly initiate inflammatory responses [26
]. PGC-1α null have reduced expression of the anti-ROS genetic program [22
]. The SIRT1-PGC-1α signaling pathway can up-regulate the expression of antioxidant enzymes, scavenge free radicals, reduce intracellular oxidative stress levels, and reduce the degeneration of cells [27
]. SIRT1/PGC-1α could be related to an ROS-initiated signaling cascade which requires further study in COPD. The present study revealed that the expression of PGC-1α was upregulated by the SIRT1 activator, resveratrol, in the COPD rats. The results suggested that the SIRT1/PGC-1α signaling pathway was inhibited in COPD. Further experiments found that, resveratrol significantly increased SOD and reduced MDA, enhanced the expression of PGC-1α mRNA and protein levels. SIRT1 could regulate transcription of PGC-lα expression. It was possible to enhance the activity of the SIRT1-PGC-lα signaling pathway, which could alter the oxidative stress reaction in COPD. Resveratrol was expected to become one of the new drugs in COPD.
Taken together, our results reveal that resveratrol has a therapeutic effect, which can regulate inflammation and oxidative stress via the activation of SIRT1 and their downstream targets, including PGC-1α in COPD rats. In COPD, more studies are still needed to confirm whether resveratrol has a lung protective effect due to the improvement of mitochondrial function through its activation of SIRT1/PGC-lα and then the reduction of oxidative stress. We will attempt to determine the exact mechanism in future work.
4. Materials and Methods
4.1. Animals and Cigarette Smoke Exposure
This research was conducted according to internationally recognized guidelines on animal welfare and conducted in accordance with the guidelines of the Chinese Council on Animal Care and the experimental protocol was approved by Medical Ethics Committee of Jishou university (No. 2015021). Thirty Wistar male rats (250–300 g) (12–13 weeks) were obtained from the experimental animal center of technology services in Changsha, China. The Wistar rats were housed in temperature controlled room (25 ± 2 °C) with relative humidity (55 ± 10%) on a 12 h light/dark cycle during the study. After one weeks’ acclimatization to laboratory conditions, the Wistar rats were randomly divided into three groups as follows: (1) control group (n = 10) (2) COPD group (n = 10) (3) resveratrol intervention group (n = 10). Rats in the resveratrol intervention group were fed resveratrol by gavage device before smoking every day (50 mg/kg [28
]) for 20 days. Rats in the COPD and control group were fed 0.5% w
sodium Carboxy Methyl Cellulose(CMC) for 20 days. The COPD group and resveratrol intervention group were challenged with passive cigarette smoking and repeatedly instilled with 200 µg of LPS (Sigma-Alderich, St. Louis, MO, USA) intratracheally on the first day and on the 14th day, while the control rats were injected with 200 µL saline as previously described [29
]. On days 2–13 days and 15–30 days of model establishment, the rats were placed in a self-produced fumigating box for smoking. The rats were exposed to the smoke of 15 cigarettes (Hongqi Canal®
Filter tip cigarette, Henan Tobacco Industry, Zhengzhou, China) for 20 min, twice daily, at an interval of 4 h except for one and 14 days [30
] (Figure 8
). The tar content was 17 mg per cigarette and the concentration of smog was about 18% (v
) within the box with five cigarettes burning concurrently. The control group was not given fumigation.
Resveratrol was purchased from Sigma (Sigma, St. Louis, MO, USA) and suspended in 0.5% w/v sodium CMC, and administered 1 h before the cigarette smoke exposure or LPS instillation.
4.2. Lung Histological Examination
The inferior lobes of the right lung tissues were fixed with 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin embedded and sliced into 4 µm sections and stained with hematoxylin and eosin. The slides were observed under a Leica photograph microscope (Leica Microscope Ltd., Wetzlar, Germany) at 200× magnification to evaluate the morphological changes in the lungs.
4.3. Serum Collection
Thirty days after exposure to cigarette smoke, the rats were anesthetized by pentobarbital sodium. After anesthesia, blood samples were collected from the common carotid artery intubation by tube, and then centrifuged at 3500 r/min for 10 min. The serum supernatant fluids were taken into tubes and taken into a −80 °C low temperature refrigerator.
4.4. Preparation of BALF and Tissue Processing
Thirty days after the exposure to cigarette smoke, the day after collecting the blood samples, the right side of the main bronchus was tied. The left lungs were lavaged three times with a 3 mL saline solution warmed at 37 °C via the tracheal cannula, and the BALF was collected. All BALF samples were immediately centrifuged at 1200 rpm for 10 min at 4 °C. The supernatants were obtained and stored at −80 °C for further analysis. The cells were resuspended in a phosphate-buffered saline (PBS) solution (300 μL) and counted via a hemocytometer. The cell differential was determined from an aliquot of the cell suspension (100 μL) by centrifugation on a slide and Wright-Giemsa stain. A total of 200 leukocytes were counted in each BALF sample, and the percentage of neutrophils was calculated based on morphological criteria.
The right middle lobes of the lung tissues were homogenized in potassium phosphate buffer (pH 7.4) and centrifuged at 4800 rpm for 30 min at 4 °C; the supernatants were employed for the analysis of SOD and MDA. The inferior lobes of right lung tissues were for the histopathological examination and immunohistochemical assay. The upper lobes of the right lung tissues were perfused with ice-cold heparinized saline, isolated and stored at −80 °C until analysis. The upper lobes of the right lung tissues were used for western blotting and real-time PCR.
4.5. Determination of Levels of Inflammation
The levels of IL-6 and IL-8 in the serum were measured with the ELISA method using commercially available kits (R&D Systems, Minneapolis, MN, USA). Mouse IL-8 and IL-6 assay kits were purchased from Wuhan Boster Bio-engineering limited company. The procurement operation was performed according to the manufacturer’s instructions. The serum samples were added to 96-well microtiter plates and incubated for 30 min at 37 °C with anti-rat IL-6 and IL-8 in a coating buffer at a dilution of 1:100. Then, the wells were washed five times with PBS before adding peroxidase-labeled biotinylated secondary antibodies. After 30 min of induction at 37 °C, the plates were treated with a tetramethylbenzidine (TMB) substrate solution for 10 min, and the reaction was stopped by the addition of a TMB stop solution. Finally, the optical density (OD) was measured at 450 nm by a microplate reader (1510, Thermo Fisher Scientific, Vantaa, Finland). All specimens were tested at the same time.
4.6. Determination of Levels of Oxidative Stress
In order to determine the antioxidant defenses, we measured the enzymatic defense activities in the BALF and lung tissues [31
]. The SOD and MDA in both BALF and pulmonary tissue homogenate were measured in this research. The levels of MDA and SOD in BALF and tissue homogenates were determined using the MDA and SOD kits (Nanjing Jiancheng Bioengineering Institute, China), with xanthinoxidase (SOD) and thiobarbituric acid (TBA) (MDA) chromometry, with enzyme-labelled meters at the 550 nm (SOD) and 532 nm (MDA) wave lengths, according to the manufacturer’s instructions. The homogenate supernatant and the BALF were used on the same day for the assay of the oxidative biomarkers MDA and SOD.
The immunohistochemical study of SIRT1 and PGC-1α were performed on formalin-fixed, paraffin-embedded tissue sections obtained from the rats with the inferior lobes of right lung tissues. Paraffin-embedded 4 μm thick serial sections were subjected to paraffin removal and rehydrated through graded alcohol. To block the endogenous peroxidase activity, the slides were pretreated with 3% H2O2. Tissue sections were boiled in a 0.01 M sodium citrate buffer (pH 6.0) in a 1000-watt microwave oven for 10 min to retrieve cell antigens. Primary antibodies were diluted to 1:100 for rabbit anti-SIRT1 and rabbit anti-PGC-1α. The sections were incubated with the primary antibody at 4 °C overnight. Subsequently, the slides were incubated with goat anti-rabbit biotinylated secondary antibody at a concentration of 1:100 for 30 min at 37 °C and then reacted with streptavidin-peroxidase conjugate for 30 min at 37 °C. After several further washes with phosphate buffer, slides were treated with diaminobenzidine (DAB) and counterstained with hematoxylin. The sections were dehydrated, mounted and observed under a light microscope. Omitting the primary antibody for each protein was used as the negative control, and the sections did not show any background staining.
4.8. Real-Time PCR
For total RNA extraction, the upper lobes of the right lung tissues (100 mg) were dissolved in Trizol (1 mL) (Life Technologies, Grand Island, NY, USA). RNA was reverse transcribed by use of Moloney Murine Leukemia Virus Reverse (M-MLV) reverse transcriptase (Fermentas, Glen Burnie, MD, USA). An ABI 7500 real-time thermocycler (Applied Biosystems, Foster City, CA, USA) was used to monitor the amplification reactions in real time. The initial activation was at 95 °C for 10 s, 60 °C for 30 s, and 40 cycles; and 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 30 s (ABI, Foster City, CA, USA). Real-time PCR was performed using theLightCycler1.5 (Applied Biosystems, Carlsbad, SC, USA) and the SYBRGreenqRCR Mix (Takara, Otsu, Japan).The mouse SIRT1 and PGC-1α primers used for the PCR were as follows: 5′-TGTGGTGAAGATCTATGGAGGC-3′ (forward) and 5′-TGTACTTGCTGCAGACGTGGTA-3′ (reverse) for SIRT1 and 5′-TATGGAGT GACATAGAGTGT GCT-3′ (forward) and 5′-GTCGCTACACCACTTCAATCC (reverse) for PGC-1α and 5′-CCAAGGCCAACCGCGAGAAGATGAC (forward) and 5′-AGGGTACATGGTGGTGCCGC CAGAC (reverse) for β-action. The values of the target genes were normalized using the value of the housekeeping gene β-action. All samples were run in triplicate and the average values were calculated. Omitting cDNA was used as the negative control.
4.9. Western Blotting of SIRT1 and PGC-1α
The upper lobes of the right lung tissues (100 mg) were homogenized inRadio Immunoprecipitation Assay (RIPA ) lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 2% Triton-X100, 1 mM PMSF, 2 µg/mL leupeptin) supplemented with appropriate protease inhibitors (Auragene, Changsha, China). Soluble proteins were recovered after centrifugation at 13,000 rpm for 20 min at 4 °C. Subsequently, the protein concentration was measured with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) spectrophotometrically at 570 nm. The sample was incubated at 100 °C for 20 min. An 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel was run at 200 V for 45 min. The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and were blocked with 5% nonfat dry milk in 0.05% TBS-Tween-20 for 1 h at room temperature. Then blots were incubated with rabbit anti-SIRT1 (110 kDa) (1:1000 dilution, Proteintech, Wuhan, China) and rabbit anti-PGC-1α (91 kDa) (1:1000 dilution, Proteintech, Wuhan, China) and β-actin (1:2000 dilution, Proteintech, Wuhan, China) antibodies in blocking buffer overnight at 4 °C. After washing with 0.5% TBS-Tween three times, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:18,000 dilutions, Auragene, Changsha, China) for 1 h at room temperature. Proteins were detected with enhanced luminol-based chemiluminescent (ECL) reagents (Amersham Pharmacia Biotech, Basel, Switzerland). Densitometry evaluation was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
4.10. Statistical Analysis
Data is presented as mean ± SEM (n = 10). For the purpose of multiple comparison, one way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test were applied using the biostatistics software SPSS15.0. (International Business Machines Corporation, Palo Alto, SC, USA) Significance was assigned at p < 0.05.