Albumin resuscitation protects against traumatic/hemorrhagic shock-induced lung apoptosis in rats

Albumin resuscitation protects against traumatic/hemorrhagic shock-induced lung apoptosis in rats 1 first-author 1 1 2 3 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chenzuobing@hotmail.com †E-mail:chenzuobing@hotmail.com chenzuobing@hotmail.com Department of Emergency Surgery, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Emergency Surgery, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Intensive Care Unit, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Intensive Care Unit, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 To determine the effects of albumin administration on lung injury and apoptosis in traumatic/hemorrhagic shock (T/HS) rats. Methods: 2 Studies were performed on an in vivo model of spontaneously breathing rats with induced T/HS; the rats were subjected to femur fracture, ischemia for 30 min, and reperfusion for 20 min with Ringer’s lactate solution (RS) or 5% (w/v) albumin (ALB), and the left lower lobes of the lungs were resected. Results: 2 Albumin administered during reperfusion markedly attenuated injury of the lung and decreased the concentration of lactic acid and the number of in situ TdT-mediated dUTP nick-end labelling (TUNEL)-positive cells. Moreover, immunohistochemistry performed 24 h after reperfusion revealed increases in the level of nuclear factor κB (NF-κB), and phosphorylated p38 mitogen-activated protein kinase (MAPK) in the albumin-untreated group was down-regulated by albumin treatment when compared with the sham rats. Conclusion: 2 Resuscitation with albumin attenuates tissue injury and inhibits T/HS-induced apoptosis in the lung via the p38 MAPK signal transduction pathway that functions to stimulate the activation of NF-κB. Traumatic/hemorrhagic shock (T/HS) Mitogen-activated protein kinase (MAPK) Nuclear factor κB (NF-κB) Albumin Apoptosis INTRODUCTION 1 Fluid resuscitation is vital in the treatment of traumatic/hemorrhagic shock (T/HS). Shifts in paradigm have suggested that the majority of cellular injuries occur during resuscitation but not ischemic period (Waxman, 1996 1996 ). Adverse sequelae after shock treatment are the result of systemic inflammation, leading to the generation of reactive oxygen species, arachidonic acid metabolites, and inflammatory cytokines which perpetuate a pathologic state, even after the restoration of intravascular volume (Nuytinck et al., 1988 1988 ). Clinically, this can contribute to the development of adult respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS), which are important determinants of outcome in trauma (Peitzman et al., 1995 1995 ). Recent data have suggested that the type of resuscitation fluid used to treat hemorrhagic shock can affect the physiologic response (Sun et al., 1999 1999 ), and that colloid versus crystalloid resuscitation in the acute trauma setting remains a controversial subject of debate. Despite the negative perception for resuscitation, recent studies showed that human albumin, as a broadly binding protein, has been characterized as a scavenger in addition to being an anti-apoptotic agent or antioxidant (Esposito et al., 1989 1989 ; Emerson, 1989 1989 ; Zoellner et al., 1996 1996 ; Kentner et al., 2002 2002 ; Osband et al., 2004b 2004b ), and no increased mortality has been documented with albumin administration in the trauma population (Cochrane Injuries Group Albumin Reviewers, 1998 1998 ; Vincent et al., 2004 2004 ; Dubois et al., 2006 2006 ). Recently, the mechanisms involved in pulmonary apoptosis following lung ischaemia/reperfusion (I/R) have begun to be understood. Cell death that occurs following ischemic insult has long been associated with necrosis. However, recent reports have established apoptosis as a significant contributor to cell death after I/R (Genescà et al., 2002 2002 ; Wang X. et al., 2004 2004 ). A wide variety of stimuli such as oxidative stress can induce endothelial cell apoptosis and endothelial dysfunction, which may be regulated by different signaling pathways (de Nigris et al., 2003 2003 ; Jiang et al., 2006 2006 ). Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, are a family of central signaling molecules that respond to numerous stimuli and are known to participate in decisions regarding cell survival and death. ERK1/2 phosphorylation promotes cell survival, whereas p38 MAPK activation induces apoptotic responses in endothelial cells (Hoefen and Berk, 2002 2002 ). Recent studies have suggested that p38 MAPK may be an effective contributor to the development of acute organ injury, via the regulation of the nuclear factor κB (NF-κB)-activation pathway (Kim et al., 2006 2006 ; Wang H. et al., 2007 2007 ). Novel therapeutic interventions for the attenuation of T/HS-induced endothelial cell apoptosis and organ dysfunction remain a major focus area of research. All these led to our hypothesis that albumin would also protect lung epithelia from injury and apoptosis induced by T/HS. MATERIALS AND METHODS 1 Animals 2 Male Sprague-Dawley rats (8~10 weeks old, weighing 200~250 g) were purchased from the Animal Resource Center, School of Medicine, Zhejiang University, China. This study was approved by the Institutional Laboratory Review Board for the Care and Use of Animals, and based on the principles stated in the Guide for the Care and Use of Laboratory Animals by National Institutes of Health Publication in 1985. Establishment of T/HS in rats 2 The rats were anesthetized intraperitoneally with 200 mg/kg chloral hydrate, and were allowed to waken. Under aseptic conditions, catheters were inserted into the right jugular vein for fluid resuscitation, the right carotid artery for measurement of mean arterial pressure (MAP), and the left femoral artery for the induction of hemorrhage. After an open, mid-diaphyseal transverse fracture was performed in the left femur, the animals were bled through the femoral artery catheter to an MAP of (30±5) mmHg and this was maintained for 30 min. After this period, rats were resuscitated with two times the shed blood volume in the form of Ringer’s lactate solution (RS) or 5% (w/v) albumin (ALB). Finally, the catheters were removed, the vessels were ligated, and the incisions were closed. Animal grouping and sample collection 2 The rats ( n =36) were randomly divided into three groups: (1) sham group ( n =12) rats underwent the same anesthetic and surgical procedures, but neither trauma/hemorrhage nor fluid resuscitation was performed; (2) T/HS+RS group ( n =12) rats were resuscitated with RS; and (3) T/HS+ALB group ( n =12) rats with 5% (w/v) albumin. For each of the T/HS groups and the sham group, blood was collected from the rats for analysis at 1, 3, and 24 h postresuscitation, and the left lower lobes of the lungs were resected at 24 h postresuscitation. Blood analysis 2 The PaO 2 /FiO 2 ratio levels in heparinized arterial blood were measured using a Radiometer ABL 700 blood gas analyzer (Copenhagen, Denmark), and the lactate measurements were performed using a lactic acid kit (Jianchen, Nanjing, China). The plasma samples were stored at −70 °C until analysis. In situ TdT-mediated dUTP nick-end labelling (TUNEL) 2 Lung tissue samples were washed by perfusion with ice-cold phosphate buffered solution (PBS) for 1 min and then fixed using ice-cold 4% (w/v) paraformaldehyde in PBS. Following treatment with fixation solution for 24 h, tissue samples were cryosectioned at a thickness of 10 μm, and the serial sections obtained were stained with in situ TUNEL reagents (In Situ Cell Death Detection Kit, POD; Roche, Mannheim, Germany) according to the manufacturer’s instructions. The cell types were identified by hematoxylin staining (Vector Laboratories, Burlingame, CA, USA). The number of TUNEL-positive cells was counted against the total number of hematoxylin-stained cells. The nuclei were counted under a light microscope in 8~10 microscopic fields per slide; 500 cells were counted. The percentage of apoptosis (apoptotic index) was calculated by dividing the number of cells that took up the stain by the total cell counts in an open field. Immunohistochemistry 2 The paraformaldeyde-fixed tissues were cryosectioned at a thickness of 10 μm and were prepared for immunoperoxidase staining that was performed using the Envision System (Dako, CA, USA). In brief, endogenous peroxidase was quenched by treatment with 0.3% (v/v) H 2 O 2 in methanol for 30 min. The nonspecific immunoglobulin-binding sites were blocked with normal goat serum for 1 h, and the sections were then incubated with anti-phospho-p38 MAPK or anti-phospho-p65 NF-κB as the primary antibody (Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Further, the sections were incubated for 30 min with a biotinylated secondary antibody solution, followed by incubation for 30 min with the Envision System (Dako). Immunoglobulin complexes were visualized following incubation with 3,3′-diaminobenzidine (Sigma, St Louis, MO, USA); the sections were then washed, counterstained with hematoxylin, cleared, mounted, and examined by light microscopy. In total, 10 fields were observed for each slide. Tissue sections that were treated with normal serum instead of the primary antibodies were used as negative controls for the immunohistochemical staining. Routine histological examination was conducted by hematoxylin and eosin staining. The images were analyzed by using a computer-assisted image analyzer system that comprised a microscope (Olympus IPP6.0; Tokyo, Japan) equipped with a high-resolution video camera (JVC TK-890E; Japan). This analysis was performed for at least 10 fields per section and 2 sections per animal at a magnification of 400×. The staining intensity (on a scale of 0~3 for the lowest to the highest intensity) and the proportion of stained cells (on a scale of 0~4 for 0% to more than 70% cells stained) were semiquantitatively determined as described previously (Zhou et al., 2003 2003 ). A combined score of ≥6 was considered as overexpression. All the slides were scored by 2 observers who were blinded to the pathology and experiment features of T/HS in the study. Statistical analysis 2 Differences were considered significant at P < 0.05 for data comparison. The data were compared among groups by analysis of variance (ANOVA) and Tukey’s test. The data were represented as the mean± SD . RESULTS 1 Establishment of the T/HS model in rats 2 In our T/HS model, a bone fracture and continuous bleeding were induced. The MAPs of T/HS-affected rats that had been resuscitated with 5% (w/v) albumin or RS, together with the sham control rats, were longitudinally measured in Fig. 1 1 . In the sham control rats, the MAP recorded remained constant at a value of 85 mmHg, while in the experimental rat groups, it increased slightly immediately after trauma induction and then decreased rapidly to a value of about 30 mmHg after bleeding. Following a compensatory increase in the MAP during the shock phase, the MAP increased gradually toward the value recorded at baseline postresuscitation in the experimental rats, regardless of the type of fluid administered. The rapid drop in the MAP in the experimental rats following T/HS induction and the gradual recovery of MAP postresuscitation reflected the establishment of a T/HS model. Interestingly, no significant difference was observed among the rat groups with regard to the MAP recorded at the end of resuscitation, indicating that the resuscitation with albumin was not responsible for the observed MAP fluctuations in this rat model. Effects of albumin treatment on the plasma lactic acid and the PaO 2 /FiO 2 ratio in the T/HS-affected rats 2 T/HS induction significantly increased the plasma concentrations of lactic acid from the baseline values by 3~4 times immediately after the hemorrhage phase (Fig. 2a 2a ). These increases were transient, and 3 h after complete resuscitation with RS, these concentrations dropped and attained the average levels. The PaO 2 /FiO 2 ratio decreased significantly in the T/HS-affected rats 1 h after resuscitation (Fig. 2b 2b ). In contrast, the changes in lactic acid (T/HS+RS group (8.79±1.23) mmol/L vs T/HS+ALB group (4.95±1.65) mmol/L, P < 0.01) and the PaO 2 /FiO 2 ratio (T/HS+RS group (160±32) mmHg vs T/HS+ALB group (258±57) mmHg, P < 0.01) were significantly reversed 1 and 3 h after resuscitation in albumin-treated rats respectively. Thus, albumin treatment promoted the recovery of lung function following T/HS. Comparative effects of albumin treatment on T/HS-induced lung injury and apoptosis 2 The effects of albumin treatment on T/HS-induced lung injury and apoptosis were evaluated and compared. The administration of albumin during reperfusion period attenuated injury and apoptosis (Fig. 3 3 ). Injuries that developed in the T/HS rats, such as marked lung edema and inflammation, were improved in the albumin-treated group. Meantime, quantitative analysis revealed a significant decrease in the proportion of TUNEL-positive cells in the lungs of the T/HS+ALB group when compared with the T/HS+RS group (proportion of TUNEL-positive cells: (10.8±2.4)% and (19.9±6.1)% for the albumin-treated and untreated groups respectively, P < 0.05). p38 MAPK activation following T/HS induction and albumin treatment 2 To determine the role played by MAPK, if any, in the lungs following T/HS induction, we performed immunohistochemical analyses using specific phospho-antibodies against p38 MAPK. Fig. 4a 4a shows that the levels of phosphorylated p38 increased following T/HS induction and resuscitation, and that albumin treatment attenuated the activity of phosphorylated p38 MAPK in the lungs. The immunohistochemical staining results were scored to determine the inter-group differences in the distribution patterns and the intensity of p38 MAPK immunolabelling in the lung tissue. The staining intensity (scored from 0 to 3 for the lowest to highest intensity) and the proportion of stained cells (scored from 0 to 4 for 0% to more than 70% cells stained) were semiquantitatively determined as described previously. Based on the scores, the immunolabelling intensity of p38 MAPK was considered to have increased in the T/HS+RS group when compared with the sham group. Further, a significant reduction in the p38 MAPK expression was noted in the albumin-treated rats when compared with the untreated rats (T/HS+ALB group (2.5±0.8)% vs T/HS+RS group (4.3±1.2)%, P < 0.05). NF-κB p65 activation following T/HS induction and albumin treatment 2 We performed immunohistochemical staining to assess the localization of phosphorylated NF-κB p65 expression. The results were scored to determine the inter-group differences in the distribution patterns and the immunolabelling intensity of phosphorylated NF-κB p65 in the lung tissue (Fig. 4a 4a ). The staining intensity and the proportion of stained cells were scored according to the same semiquantitative system as described above. The expression patterns of phosphorylated NF-κB p65 were observed to be similar to those exhibited by phosphorylated p38 MAPK. Thus, albumin may attenuate the increased expression of phosphorylated NF-κB p65 in the lungs induced by T/HS (T/HS+ALB group (1.8±0.2)% vs T/HS+RS group (4.5±0.9)%, P < 0.01). DISCUSSION 1 T/HS patients run the risk of developing ARDS and subsequent MODS. T/HS has been shown to induce cellular apoptosis (Lu et al., 2005 2005 ), which is an important contributor to cell dysfunction, organ failure, and mortality (Deb et al., 1999 1999 ; 2000 2000 ). Blood volume therapy can help to maintain hemodynamic stability and diminish the risk of developing hypovolemia and tissue malperfusion; however, the manner in which T/HS-induced cell apoptosis can be directly attenuated remains largely unclear. Hence, the discovery that resuscitation with albumin protects lung epithelia from apoptosis in T/HS rats significantly, as well as binds fatty acid, scavenges free radicals, and improves microvascular permeability (Horstick et al., 2002 2002 ; Osband et al., 2004a 2004a ), is highly significant. We examined the effects of albumin treatment on the hemodynamics, lung functions, and cellular apoptosis in the lung tissue by using a rat model, wherein T/HS that mimicked the clinical conditions was induced and the animals were subsequently resuscitated with RS or 5% (w/v) albumin. First, we observed that immediate resuscitation following T/HS can rapidly restore hemodynamic stability, with or without albumin. However, albumin treatment increased the PaO 2 /FiO 2 ratio and decreased the plasma lactic acid concentrations when compared with those in RS resuscitated rats. These three clinically available parameters were used to test the effectiveness of volume expansion and tissue perfusion with different resuscitation fluids, and the difference in gas exchange or in lung edema formation. The results are consistent with several reports on the protective effects of albumin on I/R-induced organ dysfunction (Boura et al., 2003 2003 ; Zhang et al., 2003 2003 ; Su et al., 2007 2007 ). Second, two distinct modes of cell death, apoptosis and necrosis, have been reported to be involved in the destruction of small intestinal epithelial cells (Ikeda et al., 1998 1998 ), lung epithelial cells (Ayala et al., 2001 2001 ), and renal tubular cells (Engel et al., 2001 2001 ) during I/R; however, apoptosis is noted to be the primary mode of cell death that greatly contributes to organ dysfunction, and alveolar apoptosis after hemorrhagic shock and associated toxic byproducts have been suggested to contribute to lung injury (Buckley et al., 1998 1998 ; Compton et al., 1998 1998 ; Magnotti et al., 1998 1998 ). RS resuscitation has been demonstrated to activate neutrophils and raise apoptosis in the liver and small intestine of hemorrhaged rats (Deb et al., 1999 1999 ). On the contrary, albumin has been well characterized as a general scavenger in addition to being an anti-apoptotic agent and an antioxidant. Our laboratory has also shown that the type of fluid used affects the extent of apoptosis in rat lungs after T/HS and resuscitation. In this study, it was observed that 5% (w/v) albumin resuscitation resulted in significantly less apoptosis in the lung than RS resuscitation by the TUNEL staining. Based on this finding, we set out to determine which signaling pathways were responsible for regulating apoptosis in the lungs of T/HS rats. Considering the antiapoptotic effects of alumin in patients with I/R injury and the important role played by p38 MAPK in pulmonary injury by reperfusion (Khan et al., 2003 2003 ; 2004 2004 ; Lai et al., 2004 2004 ), we aimed to determine whether albumin-induced organ protection is mediated by regulation of the activities of MAPK family members. Our study demonstrates, for the first time, that the expression level of phosphorylated p38 in the lung is higher in animals with T/HS injury and reperfusion with RS, when compared with normal animals; this is consistent with previous findings regarding the pathologic sequelae of cardiac, hepatic, and renal reperfusion injuries (Toledo-Pereyra et al., 2004 2004 ; Matot et al., 2006 2006 ). In this study, albumin administration attenuated both p38 MAPK expression and apoptosis. Furthermore, we examined the expression of NF-κB p65 in tissue samples obtained from the T/HS-affected hosts, and found that T/HS increased this expression, while albumin treatment attenuated it. These data indicate the unfavorable role played by p38 MAPK in T/HS-induced apoptosis associated with the development of acute organ injury via its upstream action on NF-κB. In summary, our study on a rat model demonstrates that T/HS can induce cellular apoptosis in the lungs. 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Serum albumin is a specific inhibitor of apoptosis in human endothelial cells 10.1631/jzus.B0820130 Project supported by the National Natural Science Foundation of China (No. 30672071) and the Traditional Chinese Medicine Foundation of Zhejiang Province, China (No. 2004C071) 2008-04-17 2008-08-17 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 09