Introduction
Sepsis is a disseminated inflammatory response elicited by microbial infection (1) and is the major cause of mortality in critically ill patients (2–4). Acute lung injury (ALI) is a clinical syndrome associated with respiratory dysfunction and is often a complication of sepsis. ALI has a mortality rate of ~50% (5). Since the most common cause of ALI in humans is sepsis, the administration of gram-negative bacterial endotoxin, lipopolysaccharide (LPS), has been used as an animal model of sepsis-related lung injury in a number of species (6–13). Previously, Rojas et al(14) reported that intraperitoneal administration of LPS to mice leads to a transient systemic inflammatory response and transient lung injury and dysfunction.
Sirtuin 1 (Sirt1), a mammalian homolog of Sir2, is a NAD+-dependent class III histone deacetylase. Sirt1 has been demonstrated to be involved in a number of pathophysiological processes, including anti-inflammation (15–17), by the regulation of specific proinflammatory mediators. Knockdown of the Sirt1 gene leads to increased cytokine release, whereas Sirt1 activation inhibits the production of tumor necrosis factor-α, monocyte chemoattractant protein 1 and interleukin (IL)-8 (18–21). Resveratrol (trans-3,5,4′-trihydroxystilbene), a polyphenolic phytoalexin, is a potent activator of Sirt1 (22). A number of studies have demonstrated that resveratrol exerts anti-inflammatory properties (23–25). Resveratrol exhibits a chondroprotective function by the suppression of IL-1β production and reactive oxygen species (26). In human primary airway epithelial cells, resveratrol inhibits cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitrite production (27). Resveratrol also protects cartilage against the development of experimentally induced inflammatory arthritis (28).
Sirt1 may represent a promising target for anti-inflammatory therapy (29). In the present study, the role of Sirt1 in LPS-induced ALI was investigated in mice by the activation of Sirt1 with resveratrol. In addition, the inhibitory role of Sirt1 on LPS-induced inflammation in TC-1 cells was determined by the activation of Sirt1 with resveratrol or the downregulation of Sirt1 by RNA interference. The results of the study indicate that resveratrol inhibits inflammation and ALI.
Materials and methods
Cell culture and treatment
Mice pulmonary alveolar epithelial cells, TC-1 (ScienCell Research Laboratories, Carlsbad, CA, USA), were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 10% fetal bovine serum, at 37°C in a humidified incubator with 5% CO2. LPS (E. coli serotype, O111:B4) and resveratrol (both Sigma-Aldrich, St. Louis, MO, USA) were used in this study.
Resveratrol was added 1 h prior to LPS treatment. The cells were treated with 15 or 30 μM resveratrol for 1 h followed by administration of 100 ng/ml LPS.
RNA interference
Independent siRNA sequences were used to silence SIRT1 expression. The sequences used were as follows: sense, 5′-ACUUUGCUGUAACCCUGUA(dTdT)-3′ and antisense, 5′-UACAGGGUUACAGCAAAGU(dTdT)-3′ (4). The siRNA concentration was 0.58 μg/1.5×105 cells (17,30).
Animal preparation and experimental protocol
This study was approved by the Ethics Committee of the Beijing Anzhen Hospital and Beijing Ditan Hospital, Capital Medical University (Beijing, China).
Male mice (8–10 weeks old) were used in all experiments. All adult male Wistar rats (270–300 g) were kept under specific pathogen-free conditions in the animal care facility at the Beijing Institute of Cardiopulmonary Vascular Disease, Beijing Anzhen Hospital (Beijing, China).
Mice were administered with LPS intraperitoneally (10 mg/kg body weight) and sacrificed at 18 h. To study recovery from endotoxemic ALI, a subset of mice were intraperitoneally injected with 15 or 30 mg/kg resveratrol at 6 and 12 h following LPS administration, and then sacrificed 18 h following initial LPS injection. The mice were used to evaluate the lung wet-to-dry (W/D) ratio, and the histology and molecular biology were analyzed.
Lung W/D ratio
The W/D ratio was determined in the right lung as described previously (31). Briefly, the right lung was separated, weighed (wet weight) and then dried in a microwave at low power (200 W) for 5 min.
Respiratory parameters
Airflow, airway and esophageal pressures were measured (32,33). Changes in esophageal pressure, which reflect chest wall pressure, were measured with a water-filled catheter (PE205) with side holes at the tip connected to a SCIREQ differential pressure transducer (SC-24; SCIREQ, Montreal, QC, Canada) (34,35). Transpulmonary pressure was calculated by the difference between airway and esophageal pressures (32). All signals were filtered (100 Hz), amplified in a four-channel conditioner, sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A; Data Translation, Marlborough, MA, USA) and continuously recorded throughout the experiment using a personal computer. All data were analyzed using ANADAT data analysis software (RHT-InfoData, Inc., Montreal, QC, Canada).
Immunohistochemistry for Sirt1
The right lungs were removed, fixed in 3% buffered formaldehyde and embedded in paraffin. Sections (4 μm thick) were cut and stained with hematoxylin and eosin (H&E). Formalin-fixed paraffin-embedded lung biopsies of mice were deparaffinized with xylane and rehydrated in ethanol. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide solution for 15 min. Next, the sections were blocked with l% BSA for l h and subsequently incubated with 0.25 mg/ml anti-Sirt1 monoclonal antibody overnight at 4°C. Following extensive washing, the sections were treated with a secondary antibody for 20 min (36).
Matrix metalloproteinase-9 (MMP-9), iNOS, IL-1β, IL-6 and Sirt1 mRNA expression
Quantitative real-time RT-PCR was performed to measure the expression of the MMP-9, iNOS, IL-1β, IL-6 and SIRT1 genes. PCR primers for target genes were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA; Table I).
Gelatin zymography
Gelatin zymography was performed as described previously (37,38). MMP-9 expression and proteolytic activities were presented as marked bands against the background of stained gelatin.
Western blot analysis
The cells were lysed in RIPA buffer and the protein concentration was detected using the DC™ protein assay (Bio-Rad, Hercules, CA, USA). Protein expression levels were determined by general methods using 30 mg protein with primary antibodies against MMP-9 (1:500, Cell Signaling Technology, Inc., Danvers, MA, USA) and iNOS, IL-1β, IL-6 and Sirt1 (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were used for ECL-plus (GE Healthcare, Waukesha, WI, USA) detection. The results were normalized against β-actin (1:5,000, Abcam, Cambridge, UK).
Statistical analysis
Both conditions were satisfied, one-way analysis of variance (ANOVA) for repeated measures was used to compare the time course of the mean airway pressure (MAP), inferior vena cava (IVC) and right atrium (RA) dimensions. The W/D ratio was analyzed using two-way ANOVA followed by Tukey’s test. To compare non-parametric data, two-way ANOVA on ranks followed by the Dunn’s post-hoc test were selected. All statistical analysis was performed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Resveratrol decreases pulmonary edema induced by LPS
Pulmonary edema is a hallmark of ALI and the gold standard for the measurement of edema is the amount of water in the lungs.
Fig. 1 presents the wet-dry weight ratio (uncorrected for residual blood) for the control, LPS- and resveratrol-treated animals at various concentrations following LPS administration. Edema was marked following LPS administration and then gradually decreased following treatment with resveratrol, particularly in 30 μM resveratrol-treated cells (P<0.01).
Resveratrol improves the lung function of mice treated with LPS
As demonstrated in Table II, changes in lung functions were observed following the administration of LPS and a significant difference was demonstrated between LPS-treated and control animals in Penh (a dimensionless number hypothesized to be associated with airway resistance), relaxation time, minute ventilation, tidal volume, end inspiratory pause and respiratory frequency. All changes in function were restored following treatment with resveratrol in a dose-dependent manner. These variables, identified to be significantly different between LPS-treated and control animals, are presented at 18 h following LPS.
Resveratrol reduces pathological changes in the lungs of mice treated with LPS via Sirt1
Photomicrographs of H&E-stained sections of lung tissues from the control (Fig. 2A), LPS-untreated (Fig. 2B), 15 (Fig. 2C) and 30 μM resveratrol-treated mice (Fig. 2D) are presented. Following LPS administration, congestion and infiltration of inflammatory cells, which appeared to be predominantly neutrophils, were identified. Further inflammation and septal thickening were observed. The changes were restored by resveratrol, particularly at 30 μM, although increased numbers of neutrophils were still present. The results revealed that resveratrol blocked ALI of mice induced by LPS in a dose-dependent manner.
The expression of Sirt1 in mouse lungs treated with LPS (Fig. 2E) was analyzed by immunohistochemistry and was demonstrated to be significantly decreased compared with the control (Fig. 2F). Sirt1 expression was induced by resveratrol in a dose-dependent manner. Sirt1 expression was higher in samples treated with 15 μM resveratrol (Fig. 2G) compared with 30 μM (Fig. 2H). These observations indicate that resveratrol-induced inhibition of ALI induced by LPS correlates with Sirt1 expression in mice.
Resveratrol blocks LPS-induced overexpression of MMP-9 and other inflammatory factors in mice and TC-1 cells
As demonstrated in Fig. 3A, LPS markedly induced the upregulation of MMP-9 in the lungs of LPS-treated mice. mRNA expression levels of MMP-9, iNOS, IL-1β and IL-6 (Fig. 3B) were shown to be significantly induced by LPS in cells, however, the levels of Sirt1 were markedly decreased (P<0.01; Fig. 3C).
Release of MMP-9 in the culture medium was observed in LPS-treated cells and was inhibited by resveratrol in a dose-dependent manner (Fig. 3A). In addition, resveratrol inhibited a number of proinflammatory factors in a dose-dependent manner (Fig. 3B). Western blot analysis demonstrated that the upregulation of MMP-9, iNOS, IL-1β and IL-6 (Fig. 3D) were attenuated by resveratrol-treatment prior to the administration of LPS.
Resveratrol inhibits inflammation via Sirt1
RNA interference was used to knockdown Sirt1 expression in TC-1 cells. Expression of Sirt1 was reduced 48 h following treatment with siRNA targeting the Sirt1 gene (Fig. 4A). The inhibitory effect of resveratrol on the upregulation of MMP-9, iNOS, IL-1β and IL-6 was attenuated in cells in which Sirt1 expression was knocked down (Fig. 4B and C).
Discussion
Sepsis is the most common clinical setting in which ALI develops. Bacterial endotoxin, also known as LPS, is well-known to induce lung injury and a number of studies have used LPS-treated mice as a model of lung inflammation and injury (39–41). Compared with other species (e.g. swine and sheep), mice are highly resistant to LPS, therefore, large doses of the toxin are required to cause a response in the lungs (42).
In humans, a clinical constellation of findings called the systemic inflammatory response syndrome (SIRS) defines a population at risk for ALI (43); SIRS is caused by the systemic release of an array of proinflammatory cytokines (15). The intraperitoneal administration of endotoxin to mice causes transient SIRS and transient lung injury and dysfunction. The response is characterized by successive waves of cytokine release into the circulation, early evidence of lung fibrogenesis, and prolonged increases in growth factors that may participate in lung repair. The response of mice to LPS includes lethargy, weight loss and an acutely increased release of a host of inflammatory cytokines into the circulation. This systemic inflammatory response is hypothesized to reflect that of humans with sepsis. At present, effective treatment methods and therapeutics have not been developed against sepsis and ALI induced by sepsis. Resveratrol is a potent activator of Sirt1 and is known to exhibit a number of effects on metabolism, as well as anticancer, anti-ageing and anti-inflammatory properties (23,44,45,22).
In the present study, resveratrol was shown to decrease pulmonary edema induced by LPS. Rojas et al found that edema was identified 2 h following LPS, peaked at 6 h and then gradually decreased. Even 48 h following LPS administration, lung water was still ~10% higher compared with control animals (14). In the present study, Fig. 1 demonstrates that edema in the sepsis mouse model was marked following LPS administration and then decreased following treatment with resveratrol, particularly at a concentration of 30 μM. The results indicate that resveratrol may decrease pulmonary edema induced by LPS in a dose-dependent manner.
Whole body plethysmograph was used to measure a number of variables associated with breathing patterns and respiratory functions, a number of which were affected by LPS administration. Changes of lung function were identified to be significantly different between LPS-treated and control animals. All changes in function were restored following treatment with resveratrol in a dose-dependent manner. These alterations in breathing pattern are likely to be associated with the systemic inflammatory response and local inflammation and pulmonary edema. The 6 variables identified to be significantly different between LPS-treated and control animals are presented at 18 h following LPS. The results indicated that resveratrol improved lung function in mice treated with LPS.
Following LPS administration, the congestion and infiltration of inflammatory cells was observed, which appeared to be predominantly neutrophils (Fig. 2B) but not in the control (Fig. 2A). There was further inflammation and septal thickening (Fig. 2B). The changes were restored by resveratrol (Fig. 2C and D), particularly at the concentration of 30 μM (Fig. 2D), although increased numbers of neutrophils were still present. The expression of Sirt1 in the lungs of mice treated with LPS (Fig. 2F) significantly decreased compared with the control (Fig. 2E). Sirt1 expression was induced by resveratrol in a dose-dependent manner (Fig. 2G and H), particularly in the group treated with 30 μM (Fig. 2H). These results demonstrated that resveratrol blocked ALI of mice induced by LPS in a dose dependent manner and correlated with Sirt1.
LPS is commonly used to stimulate lung injury in mice and induces inflammation in various cell types (14). Mice and TC-1 cells were treated with LPS followed by resveratrol. The release of MMP-9 in the culture medium was observed in LPS-treated cells and was markedly inhibited by resveratrol in a dose-dependent manner (Fig. 3A). Resveratrol inhibited the expression of MMP-9, iNOS, IL-1β and IL-6 in TC-1 cells in a dose-dependent manner (Fig. 3). The results in the mouse model were consistent with those of TC-1 cells (data not shown). Resveratrol is a potent Sirt1 agonist and increases Sirt1 activity (46). Resveratrol was used to investigate the anti-inflammatory function of Sirt1 in LPS-induced ALI in mice. The results in mice indicated that resveratrol blocked LPS-induced inflammation of the lung, consistent with results observed in TC-1 cells (data not shown). These observations indicated that resveratrol blocked LPS-induced overexpression of MMP-9 and other inflammatory factors in mice and TC-1 cells.
Since resveratrol is a pharmacological activator of Sirt1 and may have off-target effects, the importance of Sirt1 in the anti-inflammatory activity of resveratrol was analyzed. Sirt1 expression is induced by LPS or inflammation (47,48). The inhibitory effect of resveratrol on the upregulation of MMP-9, iNOS, IL-1β and IL-6 was attenuated in TC-1 cells in which Sirt1 expression was knocked down (Fig. 4). These results indicate that the anti-inflammatory effects of resveratrol are largely dependent on the ability of Sirt1 to negatively regulate inflammation.
In conclusion, the upregulation of MMP-9, IL-1β, IL-6 and iNOS was induced in LPS-induced sepsis mouse models and the TC-1 cell line and resveratrol suppressed the overexpression of these pro-inflammatory molecules in a dose-dependent manner. Resveratrol decreased pulmonary edema in the sepsis mouse model. In addition, resveratrol improved lung function and prevented pathological alterations. Knockdown of Sirt1 by RNA interference rendered TC-1 cells more susceptible to LPS stimulation and diminished the anti-inflammatory effect of resveratrol. Resveratrol inhibited LPS-induced ALI and inflammation via Sirt1, indicating that Sirt1 is an efficient target for the regulation of LPS-induced ALI and inflammation. The results of the present study provide insights into the treatment of ALI during sepsis.