Inflammation ( # 2014) DOI: 10.1007/s10753-014-9893-2
Remifentanil Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Downregulating the NF-κB Signaling Pathway Ying Zhang,1 Zhaohui Du,1 Qing Zhou,1 Yanlin Wang,1 and Jianguo Li1,2
Abstract—Remifentanil significantly represses cell immune responses and influences neutrophil migration through endothelial cell monolayers. The present study determines the beneficial effects of remifentanil and the mechanisms by which it attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI). Rats were intratracheally instilled with 2 mg/kg LPS to induce ALI. Results showed that remifentanil could resolve lung injury, as evidenced by remarkable decreases in lung edema (wet-to-dry weight ratio), neutrophil infiltration (myeloperoxidase activity), and pulmonary permeability [total number of cells and protein concentrations in bronchoalveolar lavage fluid (BALF)]. Remifentanil also attenuated the concentrations of proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β, and interleukin-6 in BALF, as well as effectively repressed the activation of nuclear factor-kappaB (NF-κB), which has been associated with the inhibition of IκBα degradation.These results suggest that remifentanil may be a suitable treatment for LPS-induced ALI. Remifentanil exerts beneficial effects on the inhibition of proinflammatory cytokine production by downregulating the NF-κB pathway. KEY WORDS: remifentanil; lipopolysaccharide; acute lung injury; cytokines; nuclear factor-kappaB.
INTRODUCTION Acute respiratory distress syndrome (ARDS) is the most common complications of sepsis. Despite extensive investigations on new strategies for treatment, the morbidity and mortality of sepsis-induced ARDS in critically ill patients remain unacceptably high [1]. Lipopolysaccharide (LPS), the major constituent of the outer cell wall of Gram-negative bacteria, is known to induce the production of several inflammatory cytokines, which are recognized as principal components in the causation of sepsis-induced acute lung injury (ALI) [2]. Intratracheal instillation of LPS has been widely accepted as an experimental model of ALI [3]. LPS exerts its toxic effects on the lungs through direct injury to endothelial cells and indirect activation of neutrophils and macrophages, thereby releasing proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1β 1
Department of Anesthesia, Critical Care Medicine & Emergency Medic i n e C e n t e r , Z h o n g n a n H o s p i t a l , Wu h a n U n i v e r s i t y, Wuhan, 430071Hubei Province, People’s Republic of China 2 To whom correspondence should be addressed at Department of Anesthesia, Critical Care Medicine & Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan, 430071Hubei Province, People’s Republic of China. E-mail: [email protected]
(IL-1β), and interleukin-6 (IL-6). The complex network of these proinflammatory cytokines initiates, amplifies, and perpetuates the inflammatory response, severely altering gas exchange and inducing refractory hypoxemia. Therefore, an anti-inflammatory therapeutic strategy may be crucial in the management of LPS-induced ALI. Li et al. [4] reported that nuclear factor-kappaB (NF-κB) plays an important role in the pathogenesis of LPS-induced ALI. Increased activation of NF-κB has been found in peripheral blood neutrophils and alveolar macrophages in patients with ALI after LPS exposure [5]. Therefore, inhibitors of NF-κB function may be useful as anti-inflammatory agents for the treatment of ALI [6]. Remifentanil, an opioid agonist, interacts primarily with the mu-opioid receptor and is metabolized by esterases that are widely found throughout the plasma, red blood cells, and interstitial tissues [7]. It has been reported to possess inhibitory effects on IL-6 production and induces nitric oxide synthase in septic mice [8]. In addition, remifentanil significantly represses cell immune responses in animals [9]. Treatment with remifentanil could influence neutrophil migration through endothelial cell monolayers, as well as adhesion molecule expression [10]. However, the protective potential of remifentanil for ALI has yet to be reported. We hypothesize that remifentanil suppresses LPS-induced ALI.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Du, Zhou, Wang, and Li MATERIALS AND METHODS
and LPS groups rats received the same volume of saline. The samples were collected after 8 h of LPS treatment.
Experimental Animals Male Sprague–Dawley rats (Wuhan Universtity Experiment Animals Center) weighing 200 to 250 g were used for the experiment. All animals were maintained in a sterile, standard laboratory supplied with food and water as needed. The rats were placed in individual ventilated cages under specific pathogen-free conditions in the animal facility of Wuhan University. All protocols were approved by the Wuhan University of Science and Technology Animal Care and Use Committee. In addition, the animals received humane care in compliance with the Principles of Laboratory Animal Care.
Histopathological Evaluation of Lung Lungs were harvested immediately after the rats (n=8, respectively) were sacrificed at 8 h after the LPS challenge, and specimens were embedded in paraffin. The lung tissues were deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E), and observed by light microscopy. The tissues were evaluated blindly without prior knowledge of the animal treatment group. Ten randomly selected fields from each specimen were observed by light microscopy. Lung Wet-to-Dry Weight Ratio
Reagents Remifentanil was purchased from Yichang Renfu Co., Ltd. (Yichang, China, batch no. H200301970). LPS from Escherichia coli serotype 055:B7 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rat TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D systems (Minneapolis, MN, USA). Antibodies to NF-κB p65 were purchased from Invitrogen (San Francisco, CA, USA). Kits used to determine myeloperoxidase (MPO) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Experimental Protocols The LPS-induced ALI model was prepared by following a previous protocol [4]. Rats were anesthetized with sodium pentobarbital (40 mg/kg) and fixed on a board at an angle of 45° in a supine position. After sterilization, a midline incision was made to the neck to isolate the trachea. Rats were intratracheally instilled with LPS (2 mg/kg) dissolved in 1 mL of sterile saline using a 20-gauge needle syringe. The rats were divided randomly into four groups, namely, the control, LPS, remifentanil control (REM group), and remifentanil treatment (LPS+REM group) groups, with eight rats in each group. In the LPS and LPS+REM groups, rats were instilled endotracheally with 2 mg/kg LPS dissolved in 1 mL of sterile saline. In the control and REM groups, rats were treated in the same manner as rats in the LPS group but received saline instead of LPS. After 6 h of LPS treatment, the rats of the REM and LPS+REM groups were continuously administered remifentanil (0.04 mg/kg) intravenously for 10 min. The control
The lung wet-to-dry (W/D) ratio was used as a parameter of lung edema induced by LPS and calculated at 8 h in the rats that were not subjected to histological examination (n=8, respectively). After the rats were sacrificed, their lungs were removed, weighed, and then dried in an oven at 80 °C for 48 h. The dried lungs were weighed again to calculate pulmonary W/D ratios. Determination of Bronchoalveolar Lavage Protein, Cell Counts, and Proinflammatory Cytokines The animals (n=8, respectively) were sacrificed at 8 h after the LPS challenge. The lung was lavaged three times through a tracheal cannula with 5 mL of autoclaved phosphate-buffered saline (PBS) instilled up to a total volume of 13.5 mL. Bronchoalveolar lavage fluid (BALF) samples were centrifuged at 3,000 rpm for 10 min at 4 °C. The sediment cells were washed and resuspended in PBS, after which the total number of cells in BALF was counted double-blindly using a hemocytometer. The concentrations of cytokine TNF-α, IL-1β, and IL-6 in the supernatant were measured by ELISA according to the manufacturer’s instructions, and the concentrations of protein in the supernatants of the BALF were quantified according to the method of Hartree [11]. MPO Activity in Lung Tissue MPO activity was assessed as an index of neutrophil infiltration in lung tissues and measured at 8 h in the rats that were subjected to W/D ratio measurement (n=8, respectively). Lung tissues were homogenized in 0.5 % hexadecyltrimethylammonium bromide solution using a homogenizer. The homogenate was then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant obtained
Remifentanil Attenuates LPS-Induced Acute Lung Injury was used for assays of MPO activity according to the kit instructions. The absorbance was measured at 460 nm with a spectrometer. MPO acitvity was expressed in units per gram of tissue. Tissue Nuclear Protein Extraction The nuclear extracts were prepared following a previous protocol [12]. The homogenates were centrifuged at 14,000 rpm for 5 min, and the supernatant nuclear extracts were then harvested and stored at −70 °C. The extracted proteins were quantified by the Lowry-Kalckar assays [13]. Western Blot Analysis of Lung Tissue Western blot analysis was carried out to measure the protein expressions of NF-κB and I-κBα. Equal amounts of each extract were electrophoresed in 6 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad), and then incubated with the primary antibody overnight at 4 °C and with the secondary antibodies for 90 min at room temperature. The bands were detected by enhanced chemiluminescence, and the band intensities were quantified by densitometry and expressed as mean area density using the accompanied software. Mean area density was expressed for all protein blots relative to β-actin expression. Statistical Analysis Data were presented as mean±SEM and compared through one-way analysis of variance (ANOVA). The Student-Newman-Keuls q test was used for statistical analysis to compare the data among all groups. A significant difference was presumed when P0.05), indicating that remifentanil has little effect on lung water content and neutrophil infiltration in normal rats. In the LPS group, the W/D ratio was markedly increased compared with the control group (*P
Remifentanil Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Downregulating the NF-κB Signaling Pathway Ying Zhang,1 Zhaohui Du,1 Qing Zhou,1 Yanlin Wang,1 and Jianguo Li1,2
Abstract—Remifentanil significantly represses cell immune responses and influences neutrophil migration through endothelial cell monolayers. The present study determines the beneficial effects of remifentanil and the mechanisms by which it attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI). Rats were intratracheally instilled with 2 mg/kg LPS to induce ALI. Results showed that remifentanil could resolve lung injury, as evidenced by remarkable decreases in lung edema (wet-to-dry weight ratio), neutrophil infiltration (myeloperoxidase activity), and pulmonary permeability [total number of cells and protein concentrations in bronchoalveolar lavage fluid (BALF)]. Remifentanil also attenuated the concentrations of proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β, and interleukin-6 in BALF, as well as effectively repressed the activation of nuclear factor-kappaB (NF-κB), which has been associated with the inhibition of IκBα degradation.These results suggest that remifentanil may be a suitable treatment for LPS-induced ALI. Remifentanil exerts beneficial effects on the inhibition of proinflammatory cytokine production by downregulating the NF-κB pathway. KEY WORDS: remifentanil; lipopolysaccharide; acute lung injury; cytokines; nuclear factor-kappaB.
INTRODUCTION Acute respiratory distress syndrome (ARDS) is the most common complications of sepsis. Despite extensive investigations on new strategies for treatment, the morbidity and mortality of sepsis-induced ARDS in critically ill patients remain unacceptably high [1]. Lipopolysaccharide (LPS), the major constituent of the outer cell wall of Gram-negative bacteria, is known to induce the production of several inflammatory cytokines, which are recognized as principal components in the causation of sepsis-induced acute lung injury (ALI) [2]. Intratracheal instillation of LPS has been widely accepted as an experimental model of ALI [3]. LPS exerts its toxic effects on the lungs through direct injury to endothelial cells and indirect activation of neutrophils and macrophages, thereby releasing proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1β 1
Department of Anesthesia, Critical Care Medicine & Emergency Medic i n e C e n t e r , Z h o n g n a n H o s p i t a l , Wu h a n U n i v e r s i t y, Wuhan, 430071Hubei Province, People’s Republic of China 2 To whom correspondence should be addressed at Department of Anesthesia, Critical Care Medicine & Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan, 430071Hubei Province, People’s Republic of China. E-mail: [email protected]
(IL-1β), and interleukin-6 (IL-6). The complex network of these proinflammatory cytokines initiates, amplifies, and perpetuates the inflammatory response, severely altering gas exchange and inducing refractory hypoxemia. Therefore, an anti-inflammatory therapeutic strategy may be crucial in the management of LPS-induced ALI. Li et al. [4] reported that nuclear factor-kappaB (NF-κB) plays an important role in the pathogenesis of LPS-induced ALI. Increased activation of NF-κB has been found in peripheral blood neutrophils and alveolar macrophages in patients with ALI after LPS exposure [5]. Therefore, inhibitors of NF-κB function may be useful as anti-inflammatory agents for the treatment of ALI [6]. Remifentanil, an opioid agonist, interacts primarily with the mu-opioid receptor and is metabolized by esterases that are widely found throughout the plasma, red blood cells, and interstitial tissues [7]. It has been reported to possess inhibitory effects on IL-6 production and induces nitric oxide synthase in septic mice [8]. In addition, remifentanil significantly represses cell immune responses in animals [9]. Treatment with remifentanil could influence neutrophil migration through endothelial cell monolayers, as well as adhesion molecule expression [10]. However, the protective potential of remifentanil for ALI has yet to be reported. We hypothesize that remifentanil suppresses LPS-induced ALI.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Du, Zhou, Wang, and Li MATERIALS AND METHODS
and LPS groups rats received the same volume of saline. The samples were collected after 8 h of LPS treatment.
Experimental Animals Male Sprague–Dawley rats (Wuhan Universtity Experiment Animals Center) weighing 200 to 250 g were used for the experiment. All animals were maintained in a sterile, standard laboratory supplied with food and water as needed. The rats were placed in individual ventilated cages under specific pathogen-free conditions in the animal facility of Wuhan University. All protocols were approved by the Wuhan University of Science and Technology Animal Care and Use Committee. In addition, the animals received humane care in compliance with the Principles of Laboratory Animal Care.
Histopathological Evaluation of Lung Lungs were harvested immediately after the rats (n=8, respectively) were sacrificed at 8 h after the LPS challenge, and specimens were embedded in paraffin. The lung tissues were deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E), and observed by light microscopy. The tissues were evaluated blindly without prior knowledge of the animal treatment group. Ten randomly selected fields from each specimen were observed by light microscopy. Lung Wet-to-Dry Weight Ratio
Reagents Remifentanil was purchased from Yichang Renfu Co., Ltd. (Yichang, China, batch no. H200301970). LPS from Escherichia coli serotype 055:B7 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rat TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D systems (Minneapolis, MN, USA). Antibodies to NF-κB p65 were purchased from Invitrogen (San Francisco, CA, USA). Kits used to determine myeloperoxidase (MPO) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Experimental Protocols The LPS-induced ALI model was prepared by following a previous protocol [4]. Rats were anesthetized with sodium pentobarbital (40 mg/kg) and fixed on a board at an angle of 45° in a supine position. After sterilization, a midline incision was made to the neck to isolate the trachea. Rats were intratracheally instilled with LPS (2 mg/kg) dissolved in 1 mL of sterile saline using a 20-gauge needle syringe. The rats were divided randomly into four groups, namely, the control, LPS, remifentanil control (REM group), and remifentanil treatment (LPS+REM group) groups, with eight rats in each group. In the LPS and LPS+REM groups, rats were instilled endotracheally with 2 mg/kg LPS dissolved in 1 mL of sterile saline. In the control and REM groups, rats were treated in the same manner as rats in the LPS group but received saline instead of LPS. After 6 h of LPS treatment, the rats of the REM and LPS+REM groups were continuously administered remifentanil (0.04 mg/kg) intravenously for 10 min. The control
The lung wet-to-dry (W/D) ratio was used as a parameter of lung edema induced by LPS and calculated at 8 h in the rats that were not subjected to histological examination (n=8, respectively). After the rats were sacrificed, their lungs were removed, weighed, and then dried in an oven at 80 °C for 48 h. The dried lungs were weighed again to calculate pulmonary W/D ratios. Determination of Bronchoalveolar Lavage Protein, Cell Counts, and Proinflammatory Cytokines The animals (n=8, respectively) were sacrificed at 8 h after the LPS challenge. The lung was lavaged three times through a tracheal cannula with 5 mL of autoclaved phosphate-buffered saline (PBS) instilled up to a total volume of 13.5 mL. Bronchoalveolar lavage fluid (BALF) samples were centrifuged at 3,000 rpm for 10 min at 4 °C. The sediment cells were washed and resuspended in PBS, after which the total number of cells in BALF was counted double-blindly using a hemocytometer. The concentrations of cytokine TNF-α, IL-1β, and IL-6 in the supernatant were measured by ELISA according to the manufacturer’s instructions, and the concentrations of protein in the supernatants of the BALF were quantified according to the method of Hartree [11]. MPO Activity in Lung Tissue MPO activity was assessed as an index of neutrophil infiltration in lung tissues and measured at 8 h in the rats that were subjected to W/D ratio measurement (n=8, respectively). Lung tissues were homogenized in 0.5 % hexadecyltrimethylammonium bromide solution using a homogenizer. The homogenate was then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant obtained
Remifentanil Attenuates LPS-Induced Acute Lung Injury was used for assays of MPO activity according to the kit instructions. The absorbance was measured at 460 nm with a spectrometer. MPO acitvity was expressed in units per gram of tissue. Tissue Nuclear Protein Extraction The nuclear extracts were prepared following a previous protocol [12]. The homogenates were centrifuged at 14,000 rpm for 5 min, and the supernatant nuclear extracts were then harvested and stored at −70 °C. The extracted proteins were quantified by the Lowry-Kalckar assays [13]. Western Blot Analysis of Lung Tissue Western blot analysis was carried out to measure the protein expressions of NF-κB and I-κBα. Equal amounts of each extract were electrophoresed in 6 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad), and then incubated with the primary antibody overnight at 4 °C and with the secondary antibodies for 90 min at room temperature. The bands were detected by enhanced chemiluminescence, and the band intensities were quantified by densitometry and expressed as mean area density using the accompanied software. Mean area density was expressed for all protein blots relative to β-actin expression. Statistical Analysis Data were presented as mean±SEM and compared through one-way analysis of variance (ANOVA). The Student-Newman-Keuls q test was used for statistical analysis to compare the data among all groups. A significant difference was presumed when P0.05), indicating that remifentanil has little effect on lung water content and neutrophil infiltration in normal rats. In the LPS group, the W/D ratio was markedly increased compared with the control group (*P
