Research Of α1-antitrypsin In Attenuating Acute Lung Injury Following Cardiopulmonary Bypass

Background:Despite the improvement in the cardiopulmonary bypass (CPB) techniques as well as the postoperative intensive care, impaired pulmonary function is a well-documented complication of CPB, resulting in increased morbidity and mortality. Although the technical refinement of CPB as progressively improved, but it inevitably leads to systemic inflammatory response syndrome (SIRS). The SIRS is the main cause of post-CPB lung injury. The disturbance may be manifested as conditions ranging from subclinical functional changes in most patients to full-blown ARDS in 2% of cases after CPB. If acute lung injury (ALI) evolved into acute respiratory distress syndrome (ARDS), the mortality of ALI may be as high as 40-70%. ALI complication could prolong the length of intensive care unit (ICU) and hospital stay, and raise treatment expenses. Despite years of research into this phenomenon, the understanding behind the complex pathophysiology of CPB induced lung injury remains incomplete. Predicting and reducing ALI/ARDS would be extremely valuable for making clinical decisions and guiding subsequent research.α1-Antitrypsin(α1-AT), a 52-kDa glycoprotein, is a member of the serine protease inhibitor superfamily. al-AT has demonstrated anti-inflammatory, anti-neutrophil influx and activation, and anti-apoptotic effects. In addition，α1-AT has been shown to be effective in preventing renal, hepatic, and myocardial IR-induced organ injury and improved lung function and oxygenation in a rabbit lipopolysaccharide challenge model. Despite the above facts, little is known about the mechanism of al-AT in lung tissues after CPB. We hypothesized that α1-AT effectively prevents CPB-induced ALI. Therefore, the aim of this study was to determine the potential effect of al-AT on cell apoptosis and the expression of inflammatory cytokines and oxidative stress markers. This study was first done with a beagle dog CPB-induced ALI model, followed by a cell culture model that simulates the oxidative stress process of CPB.Part Ⅰ:Plasma level of advanced oxidation protein products as a novel biomarker of acute lung injury following cardiac surgeryObjective:The aim of this prospective study was to examine the plasma levels of AOPPs in patients with ALI following cardiac surgery and explore whether these molecules serve as useful markers in predicting the incidence of ALI in these patients.Methods and materialsThis prospective study was performed in Nanfang Hospital affiliated to Southern Medical University, China. Inclusion criteria should be met if seventy patients aged>18 years undergoing cardiac surgery with CPB at our department between January and May 2014 were included in this clinical trial. The study protocols were approved by the Ethics Committee of Nanfang Hospital of Southern Medical University. Patients were categorized into the ALI (n=18) and non-ALI groups (n=52) according to American-European Consensus Conference (AECC) definition (Bernard et al.1994). For each patient, serial plasma samples were obtained at the baseline level,1 h,12 h,24 h, and 48 h after operation and stored at-80 ℃. Plasma AOPPs were determined using the semi-automated method. Laboratory operators were blinded to the ALI patients, and relevant investigators involved in the interpretation of ALI were blinded to AOPPs levels. All statistical analyses were performed with SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA). Continuous data were analyzed for normal distribution with one-sample Kolmogorov-Smirnov test. Normally-distributed data were further analyzed using unpaired student’s t-test. Abnormally-distributed data were analyzed using Mann-Whitney U test. A multivariate analysis of variance procedure (MANOVA) was used to assess whether a significant difference existed in the assessment of AOPPs levels between two groups. Receiver operating characteristic curve (ROC) was computed, and area under the curve (AUC) was calculated to evaluate the significance of these biomarkers in diagnosing ALI. Risk factors related to the development of ALI, such as age, sex, body mass index (BMI), CPB time or clamp time, were introduced into the univariate analysis. Multivariate stepwise forward logistic regression analysis was then performed for statistically significant univariate predictors to determine the independent risk factor for ALI after CPB. P<0.05 was accepted as statistically significant.Results:A total of 70 patients fulfilled the inclusion criteria. Among them,18 (25.7%) developed ALI. The incidence of COPD, hypertension and the proportion of male were significantly increased in patients with ALI compared with non-ALI counterparts. In addition, the duration of SICU stay and hospital stay were significantly prolonged in the ALI group compared with those in the non-ALI group. For all patients, postoperative plasma levels of AOPPs were increased compared to the baseline level. The levels of plasma AOPPs were first significantly increased at 1 h after operation and declined at postoperative 12 h in all patients. The levels of plasma AOPPs in the ALI group were significantly higher than those in the non-ALI group at 1 h,12 h,24 h and 48 h after operation. Plasma level of AOPPs in non-ALI patients decreased to the baseline level, whereas those in ALI counterparts remained higher than the baseline level at 24 h after operation. In the ALI group, the level of AOPPs peaked at postoperative 1h, dramatically decreased at 12 h after operation, and began to increase at postoperative 24 h after operation. After adjustment for age, diabetes mellitus and hypertension by multivariable logistic analysis, plasma level of AOPPs at 1 h after operation (OR=1.1674; 95% CI,1.068-1.269; P=0.001) and COPD (OR=28.706,95% CI 1.770-465.640, P=0.018) were highly associated with increased risk of ALI. The area under the ROC curve of plasma level of AOPPs at postoperative 1 h was calculated as 0.875, indicating the clinical significance of plasma level of AOPPs at 1 h after operation in the diagnosis of ALI. Plasma level of AOPPs at 1 h after operation yielded the highest sensitivity and specificity at the cutoff value of 81.89μmol/L.Conclusion:In conclusion, this is the first study identifying the potential role of AOPPs involved with the incidence of ALI following open cardiac surgery with the technique of CPB. We found that plasma levels of AOPPs could be used as an early biomarker of the occurrence of ALI after cardiac surgery with CPB in adult patients. The plasma levels of AOPPs are associated with lung function and clinical outcome after CPB. The precise role of AOPPs in the clinical presentations and progression of ALI following cardiac surgery remain to be elucidated.Part Ⅱ:The study on the protective effect of al-antitrypsin in beagle dogs with acute lung injury following cardiopulmonary bypassObjective:1. Use α1-antitrypsin (α1-AT) to attenuate CPB-induced acute lung injury in beagle dogs and explain the protective effect on the lung in the view of oxidation-antioxidation.2. To investigate whether pretreatment with α1-AT could ameliorate lung inflammatory response in a beagle dog CPB model.Methods and materials:1. Establishment of acute lung injury model of beagle dogs. A total of 30 healthy beagle dogs (14.5± 0.63 kg) were randomly divided into three groups:blank control group (group I), acute lung injury group (group Ⅱ), acute lung injury+ prertreatment with al-AT group (group Ⅲ). Sodium pentobarbital in 2% concertration was used for intravenous anesthesia in dosage of 30mg/kg. During the experiment, sodium pentobarbital was continuous intravenous infused in dosage of 10 mg/kg/h with a infusion pump. Beagle dogs were fixed in supine position, then orotracheal intubation and mechanical ventilation were given routinely. Rrespiratory rate was kept on 18 beats/min while tidal volume was kept in 10 ml/kg. Right internal jugular vein puncture and the right femoral artery puncture were performed in beagle dogs, catheters were placed in appropriate site. The sheath side of the catheter was connected to the PiCCO temperature probe, and the right femoral artery catheter was connected to the PICCO monitor. Sternotomy was performed in group I without CPB, while the other two groups were performed with left pulmonary artery clamped and single-lung ventilation in CPB duration. Arterial blood gas was monitored during experiment.2. The drug delivery methods and indicators for monitoring. Group Ⅲ was pretreated with al-AT in dosage of 50 mg/kg before CPB.We recorded the vital signs of beagle dogs of each group at the time before CPB, and at the time during CPB (30min,2h), at 6h,12h post-CPB. At the same time points, arterial blood samples were collected for blood gas analysis and determination of PaO2 for calculation for oxygenation index. PiCCO monitoring technology was utilized for extravascular lung water index (EVLWI) and pulmonary vascular permeability index (PVPI) at each time point. Beagle dogs were killed at 12h after experiment, meanwhile lung tissues and bronchoalveolar lavage fluid were harvested. Blood samples were drawn from the radial artery at several time points respectively.The serum levels of tumor necrosis factor-α (INF-α), interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), advanced oxidation protein products (AOPPs) were measured. MPO activity and MDA levels of the lung tissue were determined postoperatively. Part of the lung tissue was used to measured the W/D ratio, another part of the lung tissue was used for HE staining and observed the histopathology through light microscopy.Results:1. The results of blood gas analysis and hemodynamic status:Acute lung injury group (group Ⅱ) had significantly lower ratio of PaO2/FiO2 than the blank control group (P<0.001). al-antitrypsin pretreated group (group Ⅲ) had significantly higher ratio of PaO2/FiO2 than group Ⅱ(P<0.001). α1-AT could increase partial pressure of oxygen obviously, and the oxygenation index had been further improved. PiCCO monitoring results also showed that the extravascular lung water index (EVLWI) and the pulmonary vascular permeability index (PVPI) could be lowered significantly.2. Compared with the blank control group, the levels of interleukin-8 (IL-8), tumor necrosis factor-α, (TNF-α), and matrix metalloproteinase-9 (MMP-9) in serum plasma were up-regulated in all time points post-CPB, whereas pretreatment with al-AT markedly reduced these inflammatory markers in serum post-CPB (P<0.001 versus group Ⅱ).3. Oxidative stress injury is serious during the process of acute lung injury after CPB.The serum levels of AOPPs increased after CPB in both group Ⅱ and group Ⅲ (P<0.001, P<0.001). Pretreatment with α1-AT could significantly decrease the serum levels of AOPPs and the expression of MPO and MDA in lung tissue (P<0.001).4. The results of W/D ratio and HE staining of lung tissue showed that α1-AT reduce the degree of pulmonary edema, alveolar congestion, hemorrhage and leukocytes accumulation in lung tissue of beagle dogs.Conclusion:1. α1-AT can mitigate the damage of lung tissue, improve its function and tissue oxygenation, reduce extravascular lung water and capillary permeability index.2. Our findings showed that al-AT could protect CPB-induced lung injury by suppressing the expression of inflammatory cytokines.3. The protective effect of al-AT in acute lung injury is partly related to inhibition of oxidative stress and production of ROS.Part Ⅲ:Protective effect of α1-antitrypsin in oxidative damage on human alveolar epithelial cellObjective:To study the effect and mechanism of oxidative stress on apoptosis of human alveolar epithelial cell (HPAEpiCs) and explore the protective effects of α1-antitrypsin (α1-AT).Methods and materialsThere are three groups in the experiment:blank control group, H2O2 group and α1-AT intervention group (2mg/mL). HPAEpiC cells were soaked with 500μmol/L concentration of H2O2 for 24 h. The morphological changes of HPAEpiCs were observed with inverted phase contrast microscope. The apoptosis rate of HPAEpiCs was measured by using flow cytometry. The protein expression of FasL, Fas, caspase-8, caspase-3, caspase-9, and UCP-2 were analyzed by Western blot assay.Results:There was a remarkable increase in apoptosis of HPAEpiCs induced by H2O2 compared with that of blank control group (P<0.05). Meanwhile, the protein expression of FasL, Fas, caspase-8, caspase-3, caspase-9 were obviously up-regulated in HPAEpiCs (P<0.05) while the expression of UCP-2 was obviously down-regulated in HPAEpiCs (P<0.05). Intrestingly, compared to control group, pretreatment with al-AT could reduce apoptosis in HPAEpiCs (P<0.05) and downregulate the expression of FasL, Fas, caspase-8, caspase-3, caspase-9 in HPAEpiCs and up-regulate UCP-2 in HPAEpiCs (P<0.05).Conclusion:These results indicated oxidative stress could lead to apoptosis of HPAEpiCs. Its mechanism of inducing apoptosis is related to Fas/FasL-mediated death signaling pathway and mitochondrion-mediated death signaling pathway. α1-AT could inhibit apoptosis of HPAEpiCs induced by oxidative stress through these ways.