Trauma is the leading cause of death in individuals younger than 45 years in the United States (1). Improvements in trauma resuscitation have increased the survival of patients sustaining profound hemorrhagic shock (2). However, patients recovering from profound hemorrhagic shock often develop a systemic inflammatory response. Late death of these patients usually results from adult respiratory distress syndrome (ARDS) and the development of multiple organ failure (3). Splanchnic hypoperfusion and the development of gut barrier failure after hemorrhagic shock are believed to be the major pathogenic causes of systemic inflammation and its progression to ARDS and, ultimately, multiple organ failure (4, 5).
The exact mechanism of how intestinal hypoperfusion during hemorrhagic shock causes late distant organ dysfunction remains uncertain. Recent studies have shown that mesenteric lymphatic route is the primary conduit for gut-derived toxic factors (6-8). Gut ischemia invokes intestinal cells to generate immunoinflammatory factors that contribute to distant organ injury. Interrupting the flow of mesenteric lymph by lymphatic duct ligation before shock can abrogate lung damage, gut permeability, bone marrow suppression, neutrophil activation, and endothelial cell damage that are normally observed in post-hemorrhagic-shock animals (6-10).
Although mesenteric lymph may provide a mechanistic link between splanchnic hypoperfusion and lung injury after hemorrhagic shock, little is known of the harmful factors that are carried in the mesenteric lymph after hemorrhagic shock or the mechanisms by which these factors injure the distant organs. Some studies have been done to identify the toxic mediators or biomarkers released from ischemic gut to mesenteric lymph that are responsible for shock-induced distant organ injuries (11-14). These studies indicated that some proteins and/or lipid factors involved in postshock mesenteric lymph induced endothelial cell dysfunction and/or neutrophil activation. Conversely, Cheng et al. (15) proposed an alternative possibility that lung injury may be due to loss of protective mediators observable in normal mesenteric lymph. They reported that lipoproteins in normal mesenteric lymph contain anti-inflammatory properties. Decreased protective lipoproteins after hemorrhagic shock may contribute to the toxicity associated with postshock mesenteric lymph.
Previous studies have focused on examining the toxicity of mesenteric lymph and searching for the possible mediators. We hypothesized that systemic comparison of proteomic patterns between preshock and postshock mesenteric lymph samples would elucidate the possible mediators or biomarkers of remote organ injury. This study reports the results of systemic proteomic analysis of mesenteric lymph samples in response to hemorrhagic shock using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A comprehensive comparison of protein changes is made, and the mechanistic basis of these changes is discussed.
MATERIALS AND METHODS
Animal experiments used adult male Sprague-Dawley (SD) rats weighing 350 to 400 g after a minimum 7-day acclimation period. All animals were housed in a central animal care facility and received chow and water ad libitum. Approval was obtained from the Institutional Animal Care and Use Committee before the study. All animals received humane care in compliance with the principles of laboratory animal care and use.
Fourteen rats were randomly divided into two groups and subjected to trauma (laparotomy) plus hemorrhagic shock or trauma plus sham shock. The animals were anesthetized by i.p. injection with 70 to 80 mg/kg of ketamine and 5 to 7 mg/kg of xylazine. After shaving the chest and abdomen hair, the femoral artery was cannulated with a polyethylene-50 tube, and the internal jugular vein was cannulated with a silicone tube. Blood pressure was continuously monitored (Power lab; ADInstruments Inc, Colorado Springs, Colo) from the femoral artery. Midline laparotomy was then performed using aseptic technique. The main mesenteric lymphatic duct was identified and cannulated with a silicone tube (0.51/0.94 mm; HelixMark, Carpinteria, Calif) exiting the right flank. The abdomen was then closed.
Hemorrhagic shock was induced by drawing blood from the jugular vein to a MAP of 35 ± 5 mmHg and maintained for 60 min with continuous monitoring of blood pressure by withdrawing or reinfusing shed blood as needed. Shed blood was collected into a preheparinized syringe containing 1 mL heparin saline solution and kept at 37°C. The animal was kept warm with a heat blanket and/or lamp throughout the study. The resuscitation protocol was reinfusion of the shed blood followed by 2× shed blood volume in normal saline over 30 min. The sham-shock rat was anesthetized; the femoral artery and the jugular vein were cannulated, but without withdrawing blood. Maintenance normal saline was infused at 2 mL/h through the jugular vein for sham-shock rats.
Collection of mesenteric lymph
Mesenteric lymph samples were collected by direct drainage into a sterile siliconized Eppendorf tube on ice at 1-h interval before shock and for an additional 3 h after resuscitation from hemorrhagic shock. The volume of lymph was measured for each period. The lymph was centrifuged at 12,000 revolutions/min for 15 min at 4°C, and lymph specimens were aliquoted and immediately frozen at −80°C before testing.
Proteomic analysis was carried out at the Clinical Proteomics Center of Chang-Gung Memorial Hospital. All experimental procedures followed the standardized protocols established in the Core Laboratory. Protein concentrations of lymph samples were measured using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Ill).
Two-dimensional gel electrophoresis
The isoelectric focusing (IEF) was performed using Protean IEF Cell (Bio-Rad, Hercules, Calif). Ready-made IPG strips (170-mm Immobiline DryStrips, pH 4-7 [Bio-Rad]) were used for the first dimensional electrophoresis according to the manufacturer's instructions. Lymph samples were solubilized for IEF in 7 M urea, 2 M thiourea, 4% wt/vol CHAPS, 0.001% wt/vol bromophenol blue (0.1%), 0.2% wt/vol Biolyte 3/10 (Bio-Rad) (20%), and 50 mM dithiothreitol (DTT). Preliminary studies revealed that the optimal protein amount for the mesenteric lymph sample was 600 μg for the 17-cm IPG strip. This amount provided for optimal protein spot detection in SYPRO Ruby-stained (Molecular Probes, Eugene, Ore) gels without producing excessive spot overlays and blurring effects of overloading samples. Each lymph sample containing 600 μg protein was applied onto the IPG gel by in-gel rehydration using reswelling tray, and the first dimensional IEF was conducted at 60,000 Vh at 20°C. After IEF, the IPG strip was equilibrated twice for 20 min in equilibration buffer containing 6 M urea, 20% vol/vol glycerol, 2% wt/vol sodium dodecyl sulfate (SDS), and 1.5 M Tris-HCl buffer (pH 8.8) supplemented with 2.0% wt/vol DTT for the first treatment and 2.5% wt/vol iodoacetamide (IAA) for the second treatment. After rinsing with 1× running buffer, the IPG gel was then transferred to a 12.5% polyacrylamide gel, and the second dimensional SDS-polyacrylamide gel electrophoresis (PAGE) was performed. Running conditions for horizontal SDS-PAGE were 10 mA for 30 min followed by 45 mA for 5 h. Protein molecular weight (Mr) and isoelectric point (pI) were assigned after calibrating 2-D gels with broad-range Mr standard proteins and 2-D SDS-PAGE standard proteins (Bio-Rad). The 2-D SDS-PAGE was developed until the bromophenol blue dye marker reached the bottom of the gel.
Gel staining and imaging
The gels were stained with SYPRO Ruby (Molecular Probes; Invitrogen, Eugene, Ore). The gels were initially fixed in buffer containing 10% methanol and 7% acetic acid for 1 h and then stained for 6 h in a commercially available SYPRO Ruby buffer. The gels were then washed with 10% methanol in 7% acetic acid for 2 h and soaked in distilled deionized H2O overnight. Finally, the dried stained gels were scanned with a high-resolution scanner (ProXPRESS 2D proteomic imaging system; Perkin Elmer, Boston, Mass), and image analyses were performed using the Progenesis software package (Progenesis Workstation v2005; Nonlinear Dynamics, Newcastle-upon-Tyne, UK).
In-gel digestion of proteins
The protein spots of interest were manually excised from SYPRO Ruby-stained gels, washedfs and in-gel digested with trypsin following the protocol set by the Core Laboratory. Briefly, the gel slices were cut into small pieces, destained in buffer containing 50% acetonitrile (ACN) with 25 mM ammonium bicarbonate (pH 8.5), then washed with deionized water. The gel pieces were dehydrated in 100% ACN for 5 min, dried in a SpeedVac evaporator for 5 min, and then rehydrated in 2 μL of 5 ng/μL trypsin (Promega, Madison, Wis) in 25 mM ammonium bicarbonate (pH 8.5). After removing the unabsorbed solution, the gel pieces were incubated in 10 to 20 μL of 25 mM ammonium bicarbonate (pH 8.5) for 14 to 16 h at 37°C. The solution containing digested protein fragments was transferred to a new tube, and the peptide fragments remaining in the gel were extracted in 5% trifluoroacetic acid (TFA)/50% ACN for 20 min at room temperature. Crude digest mixtures were concentrated and redissolved in 10 μL of 50% ACN with 0.1% TFA. One microliter of peptide mixture was mixed with 1 μL of matrix, and then 0.5 μL of the resulting mixture was spotted onto the AnchorChip target (Bruker-Daltonics GmbH, Bremen, Germany) and allowed to air dry for approximately 5 min at room temperature.
Peptide mass fingerprint by MALDI-TOF MS
Tryptic peptide fingerprint analyses were performed by MALDI-TOF MS analysis on an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics GmbH). Searches were performed without constraint by protein Mr or pI, and the parameters allowed for carbamidomethylation of cysteine, partial oxidation of methionine residues, and 1 missed trypsin cleavage. All spectra underwent an internal two-point calibration using two peptides derived from autodigested trypsin (m/z 842.51 and 2,211.10 d). Mass lists were then used to search the NCBI database using the MASCOT software from Matrix Science (www.matrixscience.com). Proteins with at least five matching peptides and mass measurement errors of less than 100 ppm were considered a good match. Comparison of the theoretical and observed values of Mr and pI indicated that most proteins were identified at their expected position on the gels. Some different spots close to each other were identified as being different isoforms of the same protein. The MALDI-TOF MS resolution for the peptides was around 20,000, and the mass accuracy was 0.01 to 0.02 d. The MS/MS resolution was around 6,000.
Lymph containing 10 μg protein was mixed with sample buffer containing 62.5 mM Tris, pH 6.8; 2% SDS; 7.5% glycerol; 107.5 mM β-mercaptoethanol; and trace bromophenol blue; heated to 95°C for 5 min, cooled, and then subjected to electrophoresis using 4% in upper layer and 10% in bottom-layer SDS-PAGE. The protein was then transferred to a polyvinylidene fluoride membrane (Amersham Biosciences, Piscataway, NJ), blocked with 5% skim milk in Tris-buffered saline with Tween buffer followed by immunoblotting analysis. The membranes were incubated with a 1:10,000 dilution of polyclonal anti-rat β-actin, anti-rat serum albumin, anti-rat haptoglobin, and anti-rat C3 complement (all from Abcam, Cambridgeshire, UK) as primary antibodies; followed by a 1:10,000 dilution of a horseradish peroxidase-conjugated anti-chicken IgY for complement C3 and haptoglobin; and anti-mouse IgG for albumin and actin as secondary antibodies; and were visualized by Coomassie blue.
The Progenesis software package was used to identify significantly upregulated or downregulated spots by comparing preshock with 3-h postshock (or sham shock) gels of the same animal. The levels of expression of specific proteins measured by abundance were obtained. Data were analyzed by the Student t tests using SPSS statistical software program (version 10.1 for Windows; SPSS, an IBM company, Chicago, Ill). P < 0.05 was considered statistically significant. Only protein spots that have 2-fold or more differences in abundance were selected for identification by MS.
The lymph flow rates and protein concentrations in the mesenteric lymph samples are shown in Table 1. The production of lymph decreased during hemorrhagic shock and increased significantly at each postshock period, whereas lymph flow rates of sham-shock rats decreased slightly as time passed. Protein concentrations in the mesenteric lymph samples were stationary as time elapsed in sham-shock animals, whereas they changed dramatically in hemorrhagic-shock animals, which may be due to a marked increase in lymph production in postshock periods. Although protein concentrations in the mesenteric lymph after resuscitation from hemorrhagic shock were significantly lower (P < 0.001 at each time interval) when compared with those of sham-shock lymph samples, total protein amount in each period lymph sample remained similar.
Two-dimensional gel electrophoresis of normal mesenteric lymph
Comparing 2-D gel maps of preshock mesenteric lymph using a pH 3 to 10 and a pH 4 to 7 nonlinear IPG for the first, and the 12.5% polyacrylamide gel for the second dimension, gel electrophoresis showed some protein spots grouped into clusters or charge trains within a pI range of 3 to 10 and an Mr range of 15 to 100 kd. Better resolution of acidic proteins was obtained using a pH 4 to 7 nonlinear IPG and a 12.5% polyacrylamide gel for the second dimension. Data accumulated from separations conducted on pH 4 to 7 gradients indicated that preshock (normal) mesenteric lymph expressed approximately 401 protein spots (369-438 protein spots on the individual gel). High reproducibility was observed for all gels from preshock, postshock, and sham-shock mesenteric lymph samples. The numbers of protein spots of the postshock mesenteric lymph samples increased significantly when compared with those of preshock lymph samples (483 ± 62 vs. 401 ± 23; P = 0.002).
Comparison of protein profiles of preshock and 3-h postshock mesenteric lymph
To identify deregulated proteins in the postshock mesenteric lymph, 2-DE was used to compare protein profiles between preshock and 3-h postshock mesenteric lymph samples of the same animal using pH 4 to 7 nonlinear IPG and 12.5% polyacrylamide gels for the second dimension from seven SD rats. Each sample was conducted at least twice to confirm the reproducibility. Figure 1 shows the representative 2-DE images. To compare between different gels, the volume (density multiplied by area) of each spot was calculated, and the background was subtracted. The relative volume of each spot was normalized by dividing the volume of each spot by the sum volume of all spots present in the gel and used for comparison. The protein spots exhibiting more than a 2-fold difference in abundance were regarded as real variation. With a 2-fold-change threshold, 21 protein spots revealed increased abundance, and 10 protein spots showed decreased abundance in the 3-h postshock mesenteric lymph. These spots were selected for further study. Figure 2 shows the focal 2-D gel images of six (A-F) consistently deregulated proteins (with a P < 0.05). For protein spots showing increased abundance, the volume changes ranged from 35.6 folds for spot 13 (rat 5, group D, albumin precursor) to 2.1 folds for spot 9 (rat 6, group C, actin cytoplasmic 1). In contrast, for protein spots showing decreased abundance, the volume changes ranged from 10.8 folds for spot 6 (rat 5, group B, unknown protein) to 2.1 folds for spot 4 (rat 2, group A, haptoglobin). The peak-volume ratio (amount of protein volume change) was quantified with n = 7.
Among these 31 differentially expressed protein spots, 12 distinct proteins were identified by a combination of peptide mass fingerprinting and MS/MS peptide sequencing. These included nine upregulated and three downregulated proteins in the 3-h postshock mesenteric lymph samples. As shown in Figure 1, spots 1 to 4, 7 to 10, 11 to 17, 22 to 24, and 30 and 31 presented as apparent "clusters" or "charge trains" of spots, each of which gave peptide mixtures similar to those of other spots within the group. The proteins were identified as rat haptoglobin precursor (spots 1-4), two isoforms (β- and γ-) of actin (spots 7-10), serum albumin precursor (spots 11-17), α1-macroglobulin precursor (spots 22-24), and fibrinogen γ-chain precursor (spots 30 and 31).
Table 2 shows the details of protein identification and results of protein quantification. In the downregulated protein spots, haptoglobin precursor, α1-macroglobulin precursor, and apolipoprotein E precursor were identified. Two protein spots could not be identified based on peptide mass fingerprinting and MS/MS peptide sequencing. Among the four downregulated proteins, only two (haptoglobin precursor and the unidentified protein) were consistently downregulated (P < 0.05) in all seven animals. Nine distinct proteins were upregulated. These included cytoplasmic 1 and 2 actin (β- and γ-actin), serum albumin precursor, major urinary protein (MUP) precursor, complement C3 precursor, creatine kinase (CK) B type, transthyretin precursor, C-reactive protein precursor, fibrinogen γ-chain precursor, and fetuin B precursor. Of the upregulated proteins, four were consistently upregulated (P < 0.05) in all seven animals: rat β- and γ-actin, serum albumin precursor, MUP precursor, and complement C3 precursor.
Comparison of protein profiles of 3-h postshock and 3-h sham-shock lymph samples
Figure 3 shows protein patterns of initial and 3-h post-trauma/sham-shock mesenteric lymph samples using a pH 4 to 7 nonlinear IPG and a 12.5% polyacrylamide gel for the second dimension. The protein images of 3 h after sham-shock mesenteric lymph revealed a pattern very similar to that of preshock lymph, indicating that most differences in the 3-h postshock mesenteric lymph proteomes were due to hemorrhagic shock itself. However, three exceptions were noted. First, a consistently decreased abundance of haptoglobin with similar peak-volume ratio to that of 3-h postshock mesenteric lymph was noted in the 3-h sham-shock mesenteric lymph samples. Another exception is increased abundance of MUP precursor in some (3/7) 3-h sham-shock mesenteric lymph samples with similar peak-volume ratio to that of 3-h postshock samples. The other exception was increased abundance of actin in some (4/7) 3-h sham-shock lymph samples. However, the mean peak-volume ratio of actin in the 3-h sham-shock mesenteric lymph samples was much lower than that of 3-h postshock mesenteric lymph (2.79 ± 0.83 vs. 4.17 ± 1.06; P = 0.019)
The consistently deregulated proteins identified by 2-DE and MS in the mesenteric lymph were further confirmed using Western blotting analysis for β-actin, serum albumin, C3 complement, and haptoglobin. Compared with that derived from preshock lymph, upregulated β-actin as well as C3 complement and downregulated haptoglobin in the 3-h postshock mesenteric lymph samples were verified (Fig. 4). However, increased expression of C3 complement and decreased expression of haptoglobin were also noted in 3-h sham-shock lymph samples.
Peltz et al. (16) reported the first proteomic exploration of postshock mesenteric lymph in 2009. No proteomic pattern of sham-shock mesenteric lymph was performed in their article. In this study, using a greater change in abundance (≧2-fold) threshold, the authors included sham control samples to show the complete picture of mesenteric lymph proteome changes modulated by hemorrhagic shock from a larger sample size. At 3 h after resuscitation from hemorrhagic shock, the abundance of 12 distinct proteins (31 spots) in the mesenteric lymph samples was significantly altered. The consistently upregulated proteins were two isoforms of actin (β- and γ-actin), serum albumin precursor, complement C3 precursor, and MUP precursor, whereas the consistently downregulated proteins were haptoglobin precursor and an unidentified protein. The protein images of 3 h after sham-shock mesenteric lymph revealed a pattern very similar to that of pre-sham-shock lymph, suggesting that most of the differences in 3-h postshock mesenteric lymph proteomes were due to hemorrhagic shock itself. The exception of decreased abundance of haptoglobin precursor in 3-h post-sham-shock mesenteric lymph samples indicated that the differential expression of haptoglobin might not result from the effects of hemorrhagic shock.
Most deregulated proteins are canonical acute-phase proteins. Although many acute-phase proteins are predominantly derived from the liver, some evidences suggest that acute-phase response can also be elicited from extrahepatic tissues and cell types (17-19). Intestinal epithelial cells are now known to participate in the acute-phase response that may affect not only the mucosa itself but also the function and integrity of remote organs (17-22). Significant differences in the amounts of acute-phase proteins in the mesenteric lymph implicate active innate immune responses to gut ischemia and suggest that inflammatory response may play an important role in the development of remote organ (lung) injury. For many acute-phase proteins, specific biological functions have not yet been identified. Some of them exhibit various immunomodulatory effects (23, 24). Thus, the effects of these acute-phase proteins in the mesenteric lymph after hemorrhagic shock deserve further study.
Tissue injury is responsible for the increased abundance of some proteomes in postshock mesenteric lymph. In our study, serum albumin precursor is the most increased in abundance protein in postshock mesenteric lymph (about 8.5-fold). Serum albumin precursor was reported to be upregulated in body fluids in some inflammatory situations such as mycotic keratitis (25) and passive Heymann nephritis (26). Creatine kinase B type is a component of cytoskeletal network, and CK system is postulated to play an important role in muscular energy metabolism. Elevated CK in the circulation is generally regarded to be a passive or active release of CK from muscle damage. Actin typically constitutes the framework of cytoskeletal machinery. Previous studies showed that hemorrhagic shock resulted in selective intestinal actin cytoskeleton disruption (27). All these three proteins may serve as a novel marker of injury severity after hemorrhagic shock.
Recently, several studies have reported the cooperation of so-called structural proteins with well-characterized signaling proteins. Structural components may also play functional roles in signal transduction. All three previously mentioned proteins were reported to be structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after gut ischemia (28-31). Serum albumin precursor and α-actin had been suggested to be present in the signaling complex as accessory factors of the redox sensing machinery serving multiple functions (28). Disruption of the actin cytoskeleton was reported to result in nuclear factor κB activation and inflammatory mediator production (29). Increased actin level in the postshock mesenteric lymph was suggested to contribute to the pathogenesis of acute lung injury after hemorrhagic shock (30). Actin monomer may polymerize into filaments in extracellular environment, and previous study by Haddad et al. (30) suggested that increased intravascular abundance of filamentous actin contributes to the development of ARDS if these actin filaments are not removed by actin scavengers and the reticuloendothelial system. Creatine kinase contains highly reactive cysteines in their active sites and is sensitive to oxidative stress. Zhang et al. (31) reported that other than playing a key role in highly energy-demanding process, CK B type also plays a critical role in modulating immune response such as proliferation, activation, and cytokine secretion of T cells. Whether the upregulation of these three proteins in the postshock mesenteric lymph has biological effects on remote lung injury deserves further study.
The observation of consistently upregulated complement C3 precursor in the 3-h postshock mesenteric lymph is theoretically important. It has been reported that endotoxemia stimulates production of complement C3 in the mucosa of the small intestine (20). Complement also reportedly has an important role in local and remote tissue injury associated with gastrointestinal I/R injury (32).
Major urinary protein is a pheromone-carrying protein of the lipocalin family. The lipocalin superfamily encompasses a large set of distantly related proteins that act as carriers for small lipophilic molecules. Studies have shown that lipocalin is able to bind N-fMLP (N-formyl-MET-Leu-Phe), a neutrophil chemoattractant produced by Escherichia coli, suggesting a function of lipocalin in initiating inflammatory response (33). Moreover, lipocalin is also reported to participate in the systemic response to I/R injury (34). Fibrinogen binds through its γ-chains to cell surface receptors, growth factors, and coagulation factors. These binding interactions reportedly contribute to pathophysiological processes, including inflammation and thrombosis (35). Fibrinogen γ-chain region Gly190-Val202 functions as a recognition sequence for the leukocyte integrin CD11b/CD18 that promotes leukocyte adhesion (36). Further studies to address whether these proteins act as mediators of remote lung injury after hemorrhagic shock are warranted.
Reduced protective proteins in mesenteric lymph have been proposed to contribute to remote organ injury after hemorrhagic shock (15). Theoretically, decreased protective proteins in the postshock mesenteric lymph are less likely to be a key factor in initiating remote lung injury because previous studies showed that diversion of the mesenteric lymph from the thoracic duct before subjecting rats to hemorrhagic shock reduced the severity of acute lung injury (7-10). In this study, we observed a consistently decreased abundance of haptoglobin precursor in 3-h postshock mesenteric lymph samples. Haptoglobin is reportedly a potent anti-inflammatory agent (37, 38) and known to inhibit the respiratory burst and rise in intracellular calcium of neutrophils after stimulation with N-formylmethionyl-leucyl phenylalanine (39). Whether haptoglobin plays any role in acute lung injury after hemorrhagic shock cannot be ascertained from this study because a similar change pattern of haptoglobin precursor was found in 3-h sham-shock mesenteric lymph samples, indicating that the downregulation of haptoglobin precursor may not be resulted simply from the effect of hemorrhagic shock.
The anti-inflammatory action of (apo)lipoproteins has been extensively discussed. Cheng et al. (15) first reported that decreased protective lipoproteins in the mesenteric lymph after hemorrhagic shock contributed to the toxicity of postshock mesenteric lymph. The current study did not reveal consistently downregulated apolipoprotein in postshock mesenteric lymph samples. However, we did observe downregulated apolipoprotein E in some postshock mesenteric lymph. In this study, we also observed a depletion of the protease inhibitors α1-macroglobulin in some postshock mesenteric lymph. This finding may implicate the loss of protective protease inhibitor in the development of postshock inflammation.
The mesenteric lymph is a complex biological fluid. We can hardly expect that a single mediator in the postshock mesenteric lymph triggers the development of remote lung injury. An intriguing pathomechanism involving various insults may synergize to produce acute lung injury. The initiation of inflammatory response caused by increased complement C3 may be synergistic with the effects of reduced haptoglobin, α1-macroglobulin, and apolipoprotein E in losing inhibition of respiratory burst. Major urinary protein precursors acting as carriers for small lipophilic molecules, increased fibrinogen γ-chain promoting leukocyte adhesion, and the addition of other acute-phase proteins as accessory cofactors may further exaggerate the lung injury after hemorrhagic shock. Further studies are warranted to identify and determine exact abundance of each protein in the mesenteric lymph samples at various time intervals during and after hemorrhagic shock. Nevertheless, the present data provide basic information to identify the critical junctures in the signaling networks involved in the molecular pathology of remote organ (lung) injury after resuscitation from hemorrhagic shock.
One limitation of this study is that only proteome was analyzed in the mesenteric lymph. It is likely that other nonprotein components in the mesenteric lymph may have bioactivities. Although the expression of 12 distinct proteins was found to be influenced by hemorrhagic shock, some proteins might be missed because of technical limitations. Additional proteins might have been missed because of the limited sensitivity of 2-DE techniques. Some classes of proteins such as very basic proteins, very small proteins, and hydrophobic proteins were also difficult to be resolved.
The authors thank Dr Jen-Kun Chen from Clinical Proteomics Center of Chang-Gung Memorial Hospital for technical assistance and valuable discussion. Ted Knoy is appreciated for his editorial assistance.
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