Trauma is the leading cause of death in individuals younger than 45 years (1). The massive bleeding that frequently occurs with trauma causes many life-threatening sequelae. Cellular dysfunction and metabolites disarrangement occur after hemorrhagic shock in many organs and tissues (2, 3). Moreover, these patients are susceptible to sepsis after trauma injury because of a marked depression of cell-mediated immunity (4).
Resveratrol (RSV) is found in plants and red wines and possesses anti-inflammatory properties (5). Resveratrol has been shown to reduce lipid synthesis and to interfere with arachidonate metabolism (6), inhibit platelet activation and aggregation (7), and suppress the generation of reactive oxygen species (8). In our previous research, we showed that RSV protected cardiac performance during ischemic-reperfusion injury (9) and attenuated hepatic injury following trauma-hemorrhage (TH) (10). However, the difference in metabolomic profiles between animals subjected to TH with or without RSV treatment remains unclear.
Metabolomics is a novel systematic approach for analyzing metabolic profiles that can provide information on environmental effects and disease states (11). Hydrogen 1 nuclear magnetic resonance spectroscopy, gas chromatography–mass spectrometry (MS), and lipid chromatography (LC)–MS are the major tools of metabolomics. Metabolomic profiling has been used to investigate many complex traits in diseases such as cancer (12), diabetes (13), and septic shock (14). These emerging methods have identified novel biological pathways that contribute to determining the pathogenesis and prognosis of such diseases (15).
Resveratrol protects organ functions in inflammatory responses resulting from TH; however, its precise mechanism with respect to the metabolomic profiles remains to be clarified. This study was designed to analyze the plasma metabolomic profiles in rats subjected to TH and to clarify the metabolic effects of RSV in the context of TH. We investigated whether circulating metabolomes are altered after TH and, if so, whether the administration of RSV has a therapeutic effect on the altered metabolomic profile.
MATERIALS AND METHODS
Rat TH model
Adult male Sprague-Dawley rats (300- to 350-g body weight) were used in this study. All animal experiments were performed according to the guidelines of the Animal Welfare Act and The Guide for Care and Use of Laboratory Animals from the National Institutes of Health (16). All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital. The male rats were obtained from the National Science Council Experimental Animal Center. All male rats were caged individually in the animal house with controlled humidity 70% to 75%, temperature (24°C–25°C), and lighting (light-dark cycle every 12 h). The animals were provided with water and basal diet, and at least 1 week was allowed for the animals to adapt to the environment. Animals were fasted overnight but allowed free water access before the experiment. Trauma-hemorrhagic shock and resuscitation were then performed as described previously (17, 18). In brief, male rats were anesthetized with isoflurane inhalation, and soft tissue trauma was performed with midline laparotomy. The polyethylene catheters were placed in the right femoral vein and both femoral arteries. To reduce postoperative pain, wounds were bathed in 1% topical lidocaine. The lidocaine was directly dropped around 0.1 to 0.2 mL into operative wounds, including midline abdomen and both inguinal areas. The rats were allowed to awaken until slightly pulling at the restraints and placed into a Plexiglas chamber in a prone position during the wakeup period, after which they were bled within 10 min to reach the mean arterial pressure of 35 to 40 mmHg. The rats were bled spontaneously via an arterial catheter. The mean arterial pressure was maintained between 35 and 40 mmHg for 90 min. Four times the volume of the shed blood with Ringer’s lactate was then infused for 60 min with a syringe pump. Thirty minutes before the end of the resuscitation period, the rats received RSV, 3,4′,5-trihydroxy-trans-stilbene (30 mg/kg, intravenously; Sigma, St Louis, Mo) (19), or an equal volume of the vehicle (∼0.5 mL, 10% dimethyl sulfoxide; Sigma). Sham animals underwent all procedures; however, neither resuscitation nor hemorrhage was performed. Resveratrol or vehicle was administered in sham rats. The rats were humanely killed with injection of overdose of pentobarbital at 24 h after sham operation or resuscitation. The 5-mL volume of blood samples was collected from inferior vena cava using rinsed aspiration needles with heparin solution into the 10-mL centrifuge tubes, then the blood samples immediately centrifuged at 3,000 revolutions/min for 10 min. The plasma samples were collected and stored at the −80°C refrigerator. There were 10 rats in each group.
Plasma sample extraction
The extraction of plasma sample was prepared as described previously (20). Briefly, plasma sample was mixed with acetonitrile and centrifuged at 10,000g. The supernatant was collected, and the pellets were extracted with acetonitrile and methanol. Samples were then resuspended in 100 μL of 95:5 water-acetonitrile and centrifuged at 14,000g for 5 min. The clear supernatant was collected for LC-MS analysis (20).
LC/time-of-fight MS analysis
Liquid chromatographic separation was achieved on a 100 × 2.1-mm Acquity 1.7-μm C18 column (Waters Corp, Milford, Mass) using a high-performance liquid chromatography system as described previously (20). The samples were then eluted from the column. Mass spectrometry was performed on an Agilent Q-TOF (time-of-fight) MS (6510 Q-TOF MS; Agilent Technologies, Santa Clara, Calif). The data were collected using data acquisition software (Agilent MassHunter Workstation; Agilent Technologies). The metabolomics analysis was carried out at the Metabolomics Core Laboratory, Healthy Aging Research Center, Chang Gung University.
Processing of data
The information was extracted from the raw data as described previously (20). The accurate masses of the features were searched against the METLIN, HMDB, and KEGG databases. Compound prediction was performed using the Metabolite Database and Molecular Formula Generation software (Agilent Technologies). In addition, chromatographic conditions were applied for the structural identification.
Principal component analysis (PCA) was used for clustering analysis and correlation analysis. The three-dimensional partial least squares discriminant analysis (PLS-DA) was also used for classification and discrimination problems. The results are presented as the means ± SEM. One-way analysis of variance was conducted for the multiple-group comparisons, and a least significant difference post hoc test was used to evaluate the significance of the differences between the group means. P < 0.05 was considered significant.
Analysis of metabolite profiles in rat plasma after TH injury with and without RSV treatment
The results showed obvious differences between the sham group and the control group and between the control group and the treatment group (Fig. 1A). The scores of the sham groups were visibly distinguishable from those of the control and treatment groups, indicating specific differences in the metabolic profiles of the groups. In addition, typical base peak intensity chromatograms were obtained by scanning in the electrospray-positive-ion (ESI+) and electrospray-negative-ion (ESI−) modes (Fig. 1B). A series of two-dimensional score plots was constructed to highlight the ability to discriminate between the sham, control, and treatment groups based on the metabolic profiles. The data showed that the three groups were well distinguished (Fig. 1C).
Based on the PCA results showing sample grouping, a supervised PLS-DA analysis was performed to reveal specific metabolic changes in the defined groups and to improve the separation between the specimens. We constructed a PLS-DA model to optimize the separation of the data according to the sham, control, and treatment groups. The results showed distinct global shifts in metabolism between the sham and control groups and between the control and treatment groups. The distribution of the R2Y and Q2Y parameters obtained by fit to random data is useful for estimating their statistical significance. If the values are found outside such distribution, this is a sign of high validity of the model (21). In general, the R2Y shows the amount of Y variables explained by the model after cross validation and gives an overview about the fitting of the model, whereas cumulative Q2Y gives information about the predictive quality of the model. All values close to 1 resemble a good model. In Figure 1D, it showed that the value of R2Y is 0.480 and the value of Q2Y is 0.353, indicating the model had not enough good fit. Individual variation in animals, insufficient number of animals, and RSV metabolite profile change response to RSV might cause low values of R2Y and Q2Y.
Analysis of the expressed metabolites in plasma following TH with and without RSV treatment
To gain insight into the mechanisms of the protective effect of RSV after TH, we proposed that any metabolite with change after TH with and without RSV treatment would be important. In view of this, the differentially expressed metabolites frequently occurring during both conditions were particularly interesting to us. When we restricted the analysis to those features that were only significantly different between the rats in the control and treatment groups, the sets of features are illustrated in Fig. 2, A and B. The intensities of the features were significantly higher in the control group than in the sham and treatment groups. These results suggest that the levels of many metabolites were significantly increased in the plasma of the TH rats; however, RSV treatment notably reduced these metabolic derangements.
The differences in the metabolites profiles in the groups were searched against the METLIN, HMDB, and KEGG databases. The metabolites that were significantly different between the groups are listed in Table 1. Twelve metabolites were identified. The signal intensities of the targeted metabolites in the sham, control, and treatment groups are shown in Fig. 3.
Our results revealed the plasma levels of acetylcarnitine and butyrylcarnitine were lower in the treatment group than in the control group (Fig. 3, B and C). In addition, plasma ketone bodies were elevated in the control group but were significantly attenuated in the treatment group (Fig. 3, I and J). Similar changes in the plasma lysine and histidine metabolites also were observed (Fig. 3, D, E, and H). Of note, the plasma carnitine level was 44% lower in the control group than the sham group, and the plasma carnitine level of the treatment group was double the level of the control group (Fig. 3A). In addition, all animals survived during the study period. There were not any negative reactions to RSV.
According to the results, TH rats had higher plasma level of carnitine and lower levels of acetylcarnitine and butyrylcarnitine in comparison to sham rats. In contrast, RSV treatment significantly reduced carnitine and increased acetylcarnitine and butyrylcarnitine, suggesting RSV effectively ameliorated the alterations of the carnitine system associated with TH injury. There was a statistically significant increase in carnitine.
Trauma-hemorrhage shock injury leads to complex reactions in microorganism infections and in host immune, inflammatory, and coagulation responses. A series of metabolite profiles in pathophysiological alterations were reported (22–25). In view of this, these changes on energy metabolism, mitochondrial biogenesis, ketoacidosis, nitrogen excretion, and protein degradation were investigated. The results suggested that RSV significantly inhibited the metabolomics changes that resulted from TH injury.
Our results showed that TH injury decreased the plasma level of carnitine, whereas it increased the levels of acetylcarnitine and butyrylcarnitine. Carnitine is required for transporting fatty acids to the mitochondria from the cytosol during the breakdown of lipids for the generation of metabolic energy (22). Excessive acyl-CoA might overwhelm the tricarboxylic acid (TCA) cycle mechanism, leading to acyl-CoA overload in the mitochondria. Pignatelli et al. (26) demonstrated that carnitine could be helpful in modulating oxidative stress during major abdominal surgery–dependent oxidative damage. Carnitine also demonstrated a positive effect on a distally burned dorsal skin flap model (27). Recently, carnitine has been shown in humans to be a pivotal mediator of fatty acid oxidation that is the preferred metabolic pathway during sepsis and critical conditions (28). Our results suggest that the capacity for fatty acid oxidation was overloaded, and the TCA cycle activity was impaired in the control group. Resveratrol treatment effectively ameliorated the alterations of the carnitine and acylcarnitines (i.e., acetylcarnitine and butyrylcarnitine) systems associated with TH injury. The evidence strongly suggests that RSV ameliorated the overloaded and disturbed fatty acid β-oxidation pathway and improved the mitochondrial energy metabolism in rats subjected to TH shock injury.
Resveratrol has been shown to improve energy expenditure (29). Previous studies have suggested that TH shock might induce oxidative stress (19, 23). In addition, the impairment of mitochondrial function has been reported in TH (30). In the context of TH, RSV treatment has been shown to improve mitochondrial biogenesis and protect liver (23) and heart functions (31). In line with these findings, the RSV-caused alterations of plasma carnitine and acylcarnitines in our study indicate that RSV could improve fatty acid metabolism in TH injury. It is possible that the improved energy metabolism is caused by increased mitochondrial biogenesis against the TH-induced energy imbalance.
Ketoacidosis is a metabolic state associated with high concentrations of ketone bodies, formed by the breakdown of fatty acids and the deamination of amino acids. The two common ketones produced in humans are acetoacetic acid and β-hydroxybutyrate. Our data also revealed that the levels of plasma 3-ketobutyrate and 3-hydroxybutyrate in the control group were higher than those in the sham group; however, the increase was reduced in the RSV-treated group. Increases in ketone bodies might cause serious pathophysiological complications, such as ketoacidosis. Moreover, a high concentration of ketone bodies disturbs fatty acid metabolism in the mitochondria of hemorrhagic shock patients (24). Otherwise, TH is associated with increased nitrogen excretion and accelerated protein degradation. Elevated plasma levels of branched-chain amino acids and aromatic amino acids have been observed in TH (25). Consistent with these findings, we found that circulating leucine and isoleucine were significantly elevated in the control group compared with the sham and treatment groups. Moreover, RSV significantly inhibited the increases in leucine and isoleucine caused by TH, and the concentrations were almost identical to those in the sham group. To the best of our knowledge, this study is the first to suggest that RSV attenuates protein degradation in TH rats. Crystalloid in general may be very limited in current resuscitation practices. Because resuscitation strategy using plasma and red blood cells or whole blood was not applied in our study, it may have limited applicability to current.
In conclusion, our study showed RSV treatment attenuated the TH-induced disturbances of the carnitine system, TCA cycle activity, and mitochondrial biogenesis. In addition, this study demonstrated that RSV attenuated protein degradation in TH rats. We concluded that RSV exerts multiple beneficial metabolic effects during TH injury, particularly in improving energy metabolism and reducing protein degradation.
The authors thank the staff of the Metabolomics Core Laboratory, Healthy Aging Research Center, Chang Gung University, for their technical support of this study.
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