#These authors contributed equally to this work
Primary lung graft dysfunction could significantly attribute to ischemia-reperfusion lung injury (IRLI) during transplantation surgery. β2-adrenergic agonists were one of the bronchodilators that had been well-established in the management of asthma and chronic obstructive pulmonary disease (COPD) with anti-inflammatory potency. By applying the model of isolated rat lung, we evaluated the efficacy of short-acting β2-agonist inhalation to ameliorate ischemia-reperfusion damage. The experiment protocol was 180 min of global ischemia and then reperfusion for 60 min. In the β2-agonist inhalation group, aerosolized albuterol was administrated prior ischemia procedure. Increased weight ratios of wet to dry lung and microvascular permeability were characterized in the IRLI group. In contrast, pre-inhaled β2-agonist significantly mitigated the severity of pulmonary edema. Bronchoalveolar lavage from the β2-agonist group presented decreased leukocyte counts and cytokines production, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein 2 (MIP-2). Devastating oxidative stress was widely recognized during the ischemia-reperfusion process, while β2-agonist pretreatment revealed subsided H2O2, myeloperoxidase (MPO), and the cleavage of caspase-3. Western blotting from lung homogenates identified the blockade of NF-κB and MAPK activation in the β2-agonist inhalation group. Currently, there was no specific pharmacotherapy in IRLI management. Our results elucidated the protective effect of β2-agonist bronchodilator against ischemia-reperfusion induced oxidative stress, inflammation reaction, and pulmonary edema.
In lung transplantation, the unavoidable process of organ ischemia and subsequent reperfusion mainly contributed to acute allograft injury within the first 72 hours (
Short-acting β2-agonists as bronchodilators were extensively prescribed to relieve bronchospasm for decades in asthmatic attacks. Beyond its broncho-relaxing effect, literature had claimed the activation of β2 receptor to modulate inflammatory response (
The component of IRLI in primary graft dysfunction could be recapitulated by preclinical models, including unilateral hilar occlusion, isolated, perfused rodent lungs, and orthotopic lung transplantation (
The study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital Subcommittee for the Care of Animal Subjects. Animal care and handling practices were in accordance with the National Institutes of Health guidelines for ethical animal research. The model of the isolated perfused lung
After sternotomy, heparin (1 unit/g) was injected into the right ventricle through which the pulmonary artery was catheterized. The left atrium was also catheterized. The pulmonary venous outflow was diverted into a reservoir. To prevent backflow into the ventricles, an additional ligation was performed above the atrioventricular junction. The lungs were then perfused with 10 mL of blood mixed with 20 mL of 0.9% normal saline (Minipulse 2; Gilson Medical Electronics, Middleton, WI, USA) at a constant flow rate of 30 mL/min/g body weight. The pulmonary arterial pressure (
The
The pulmonary arterial resistance (
The pulmonary capillary filtration coefficient (
The experiment was initiated after hemodynamic stability had been attained for 15 min in the extracorporeal isolated lung circulation system. Rats were divided into three treatment groups—control, I/R, and I/R+β2 agonist—and all groups were subjected to isolated lung preparations ventilated with tidal volume settings at 5 mL/kg. I/R injuries were induced according to the following protocol: ventilation and perfusion of the isolated lung were discontinued for 180 min (ischemia) and then reinstituted (reperfusion) for 60 min at room temperature. Prior to ischemia, rats in the I/R+β2 agonist group were subjected to an inhaled β2-agonist treatment (2-puff dose using Ventolin Meter Dose Inhaler, 100 μg albuterol/inhalation; GlaxoSmithKline, Middlesex, UK) delivered by an in-line spacer adapted to the inspiratory limb of the ventilator circuit. The spacer was kept in line for 60 s after each actuation (2 actuations).
All experiments were terminated after closed extracorporeal perfusion. The lungs were removed, and the wet weights were measured. The lungs were then lavaged twice by instilling saline (2.5 mL/lavage) into the left upper lobe. Lavage samples were centrifuged at 1500×
The concentration of myeloperoxidase (MPO), an index of neutrophil sequestration, was measured in the lungs, as previously described (
The perfusate was centrifuged at 1000×
The concentrations of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein 2 (MIP-2) in BALF were measured using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Oxon, UK). For each assay, absorbance in each well was read at 450 nm (SpectraMax M5; Molecular Devices, Silicon Valley, CA, USA).
Lung tissues were homogenized in a lysis buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Roche). Total protein extracts were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and electro-transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 h. The following primary antibodies were used: phospho-p44/42 MAPK (p-ERK1/2), phospho-SAPK/JNK (p-JNK), phospho-p38 MAPK (p-P38), anti-p44/42 MAPK (ERK1/2), anti-SAPK/JNK (JNK), and anti-p38 MAP Kinase (P38) (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA); Caspase-3, P-AKT, AKT, PAI-1, and AP-1 (1:2000; Cell Signaling Technology); and GADPH (1:10000; Lab Frontier). Subsequently, an appropriate secondary antibody was used (horseradish peroxidase anti-rabbit IgG, 1:10000; Jackson Immuno Research Laboratories, West Grove, PA, USA). Labeled protein bands were visualized using enhanced chemiluminescence (Visual Protein Biotechnology Crop, Taiwan) and quantified using Kodak 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY, USA).
Lung tissues were homogenized in 5 mL of solution A (0.6% Nonidet P-40, 150 mmol/L NaCl, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.9, 1 mmol/L ethylenediaminetetraacetic acid [EDTA], and 0.5 mmol/L phenylmethylsulfonyl fluoride [PMSF]) using a Dounce tissue homogenizer. The homogenates were centrifuged for 30 s at 2000 rpm, and the supernatants were then collected and centrifuged again for 5 min at 5000 rpm. The pelleted nuclei were resuspended at 4°C in 300 µL of solution B (25% glycerol, 20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.2 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 0.5 mmol/L PMSF, 2 mmol/L benzamidine, 5 mg/mL pepstatin A, 5 mg/mL leupeptin, and 5 mg/mL aprotinin) and incubated on ice for 20 min. After the samples were centrifuged at 15000 rpm for 1 min, the total protein concentration in each extract was determined using a BCA protein assay (Pierce, Rockford, IL, USA). The membrane was blocked for 1h. Antibodies specific for NF-κB (1:1000; Cell Signaling Technology) and proliferating cell nuclear antigen (PCNA) (1:1000; Cell Signaling Technology) were diluted in TBST buffer and incubated overnight at 4°C. The appropriate secondary antibody was used (1:10000 horseradish peroxidase anti-rabbit) at room temperature for 1 h. Visualization was achieved using enhanced chemiluminescence (Visual Protein Biotechnology Corp, Taipei). Anti-PCNA antibody was used as a loading control to correct for the pixel values corresponding to NF-κB.
After the termination of each experiment, lung tissue in the right lower lobes was dissected and fixed immediately in 10% neutral buffered formalin. After fixation, the lung tissues were dehydrated through a graded series of alcohol, cleared in xylene, and embedded in paraffin. All sections were cut to 5 mm and stained with hematoxylin/eosin. The severity of perivascular, peribronchial, septal, and alveolar edema as well as perivascular, interstitial, and alveolar cell infiltration was examined by a scoring system. We used lung pathology score as previously developed (
Systat10.0 (Systat Software Inc, San Jose, CA, USA) was used for the statistical analysis. Comparisons among all groups were conducted using an analysis of variance, followed by Dunnett’s
Rats were randomly assigned as (a) control group ventilated with tidal volume (VT) of 5 mL/kg, (b) IRLI group underwent 180 min of global ischemia and then reperfusion for 60 min, and (c) β2-agonist inhalation group treated prior to I/R period. β2-agonist was prepared with aerosolized albuterol of 200 μg and administrated through an in-line spacer connected to the inspiratory limb of the ventilator circuit. After the I/R period, pulmonary hemodynamic variables were not statistically different by β2-agonist inhalation (
Group | N | |||||
---|---|---|---|---|---|---|
Before injury (baseline) | ||||||
Control (VT5) | 7 | 8.4 ± 3.6 | 4.7 ± 0.4 | 6.3 ± 1.7 | 0.04 ± 0.04 | 0.03 ± 0.03 |
I/R | 7 | 10.6 ± 2.0 | 4.3 ± 0.9 | 7.1 ± 0.7 | 0.07 ± 0.03 | 0.06 ± 0.02 |
I/R+β2 agonist | 7 | 8.8 ± 2.7 | 3.3 ± 0.3 | 5.8 ± 1.2 | 0.06 ± 0.03 | 0.05 ± 0.02 |
After injury | ||||||
Control (VT5) | 7 | 9.9 ± 3.9 | 4.6 ± 0.4 | 6.9 ± 1.9 | 0.06 ± 0.04 | 0.05 ± 0.03 |
I/R | 7 | 10.1 ± 0.2 | 4.3 ± 1.0 | 6.9 ± 0.7 | 0.07 ± 0.01 | 0.05 ± 0.01 |
I/R+β2 agonist | 7 | 9.0 ± 3.6 | 3.2 ± 0.3 | 5.7 ± 1.7 | 0.07 ± 0.04 | 0.05 ± 0.03 |
Note: Aerosolized albuterol was given prior ischemia period. Ischemia-reperfusion lung injury (IRLI) was created by the discontinuance of ventilation and perfusion for 180 min and then reperfused for 60 min. The control group underwent mechanical ventilation with a setting of 6 mL/kg. Pulmonary hemodynamic parameters were recorded at indicated time point via a pressure transducer. Abbreviations: VT5, mechanical ventilation with tidal volume at 5 ml/kg; I/R, ischemia and perfusion; N, number;
Lung wet gain (LWG) served as an indicator of pulmonary edema, as well as pulmonary capillary filtration coefficient. The IRLI group showed a significant increase in the ratio of the lung to body weight and microvascular permeability (
Moreover, the H2O2, MIP-2, IL-1β, and TNF-α concentrations in lavage fluid were higher in the I/R group than in the control group (
Next, we examined protein expression in lung homogenates that also showed decreased IL-1β and TNF-α levels (
Group | N | LWG(g) | WBC in BALF |
||
---|---|---|---|---|---|
Baseline | After injury | ||||
Control(VT5) | 7 | 0.10 ± 0.13 | 0.05 ± 0.06 | 0.18 ± 0.24 | 218.5 ± 44.15 |
I/R | 7 | 3.33 ± 1.41‡ | 0.08 ± 0.06 | 0.68 ± 0.21‡ | 429.69 ± 76.42‡ |
I/R+β2 agonist | 7 | 1.11 ± 0.72‡§ | 0.06 ± 0.07 | 0.33 ± 0.23‡§ | 267.5 ± 44.89‡§ |
Note: The weight of each rat was determined before experiments to reflect the lung weight
Oxidative stress during I/R has been observed to trigger cell apoptosis. Western blotting showed elevated cleavage caspase-3 expression in the IRLI group that was suppressed in the β2-agonist pretreated group (
At the cellular level, marked activation of MAPK signaling pathways reflected the facilitation of cell migration that has been correlated with primary lung graft dysfunction. There was elevated phosphorylation of p38, ERK, and JNK in lung tissue homogenates from the IRLI group (
In this study, we demonstrated that lung injury induced by a 3-hour period of ischemia and a 1-hour period of reperfusion led to increased pulmonary vascular permeability, inflammatory cell infiltration, pulmonary edema, cytokine responses, MAPK activation, NF-κB translocation, and apoptotic enzymes (caspase-3 and p-AKT) expression. Moreover, inhalation of β2 receptor agonist prior to ischemia inhibited MAPK activation, suppressed NF-κB activation, reduced inflammatory cytokines release and hydrogen peroxide production, and attenuated apoptotic responses (
Thus far, randomized clinical trials have failed to demonstrate the efficacy of β2 agonists in the attenuation of acute respiratory distress syndrome (ARDS). In the Albuterol Treatment for Acute Lung Injury (
Several factors might explain why β2 adrenergic aerosol therapy improved the outcomes of model animals in our study but did not yield evident benefits in clinical trials. First, our study administered aerosolized albuterol to non-edematous alveoli, in contrast to clinical trials in which β2-agonists were given after injury. The response of healthy epithelium to β2-agonist was quite different from those subjects with existing ALI/ARDS that were characterized by apoptotic and necrotic debris. A comparison of these results suggests that a relatively intact barrier of alveolar epithelium is required for pulmonary fluid clearance.
Second, the observed differences may be attributable to the
Despite the lack of support from clinical trials, significant evidence in the literature supports the findings of the present study. Several studies have suggested that β2 agonists reduced endothelial damage and enhanced repair in lung injury models (
Elevated intracellular cAMP levels, which protected lungs against injury, decreased under hypoxic conditions (
Our results also suggest that the protection provided by albuterol against IRLI was mediated partially through the anti-inflammatory effects of this agent. Specifically, we found that inhalation of a β2 agonist reduced oxidative stress, suppressed IR-related proinflammatory cytokine production, attenuated NF-κB activation, and decreased p38, ERK, and JNK MAPK phosphorylation. These findings are consistent with previous studies demonstrating that albuterol increases mitogen-activated protein kinase phosphatase 1 expression (MKP-1) and suppresses p38 MAPK phosphorylation, while adrenaline induces the expression of genes encoding anti-oxidative factors, such as nuclear factor E2 p45-related factor-2 (Nrf2), and thus guards against oxidative stress (
In conclusion, we found that the inhalation of adrenergic β2 agonist prior to ischemia reduced the injury associated with reperfusion by reducing MAPK activation and lung tissue apoptosis. Accordingly, preoperative β2 agonist therapy might help to reduce IRLI associated with lung transplantation.
ischemia-reperfusion
ischemia-reperfusion lung injury
pulmonary capillary filtration coefficient
lung weight gain
mitogen-activated protein kinase phosphatase 1
myeloperoxidase
pulmonary arterial pressure
pulmonary capillary pressure
pulmonary venous pressure
pulmonary arterial resistance
venous resistance