Trauma-hemorrhage reduces tissue perfusion and results in recruitment of neutrophils and excessive production of proinflammatory mediators that induces organ metabolism, structure, and immunology changes and dysfunction (1). Studies have shown that cardiac injury is associated with an increased neutrophil accumulation in the heart (2, 3). Neutrophils are activated following trauma-hemorrhage and can release superoxide anions and proteolytic enzymes that diffuse across the endothelium and injure parenchymal cells (2, 4). Alternatively, neutrophils can exit the microcirculation and migrate and adhere to matrix proteins or different cells (4, 5). Intercellular adhesion molecule 1 (ICAM-1) is known to play a major role in the firm adhesion of neutrophils to the vascular endothelium and is markedly upregulated following trauma-hemorrhagic shock (6, 7). Interleukin 6 (IL-6) contributes to neutrophil infiltration and plays a significant role in organ injury (8, 9). Furthermore, there is convincing evidence that IL-6 is required for the expression of adhesion molecules (10).
The PI3K/Akt pathway is involved in an endogenous negative feedback or compensatory mechanism, which affects proinflammatory cytokine production and chemotactic events in response to injury (11). Inhibition of the PI3K/Akt pathway with the PI3K inhibitor wortmannin increases serum cytokine levels and decreases the survival of rodents subjected to sepsis and hemorrhagic shock (11, 12). In addition, the PI3K/Akt pathway has a pivotal role in neutrophils migration to undergo chemotaxis (13, 14). Furthermore, the involvement of PI3K in cell migration is supported by the ability of selective PI3K inhibitors, such as wortmannin, to mitigate neutrophil chemotaxis (15). Studies have also shown that activation of the PI3K pathway protects organs or cells against hypoxia and ischemia-reperfusion injury–mediated antiapoptosis machinery (16).
It is known that 5-hydroxytryptamine subtype 3 (5-HT3) receptor antagonists have an antiemetic effect used to treat vomiting induced by surgery or chemotherapy/radiation therapies; the latter suggests the 5-HT3 receptor presence in various cell types involved in inflammation (17, 18). Our previous study has shown that 5-HT3 receptor antagonist ondansetron can attenuate hepatic injury after trauma-hemorrhage (19). Recent report shows tropisetron pretreatment possesses salutary effects in an embolic stroke rat model (20). It is suggested that tropisetron may exert anti-inflammatory and organ-protective effects in response to injury. Furthermore, previous studies have shown that an increase in Akt activity improved cardiac function following trauma-hemorrhage or ischemia injury (21). It is implied that Akt may play a role in tropisetron-mediated cardioprotection following trauma-hemorrhage. We hypothesized that the beneficial effects of tropisetron following trauma- hemorrhage are mediated by an Akt-related pathway. To test this hypothesis, animals were treated with tropisetron alone and in combination with the PI3K inhibitor wortmannin after trauma-hemorrhage. The effects of these treatments were then examined with respect to cardiac function as well as cardiac myeloperoxidase (MPO) activity, IL-6, ICAM-1, phospho(p)-Akt/Akt levels, and apoptosis after trauma-hemorrhage.
MATERIALS AND METHODS
This study was approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (no. 2008011401). 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.
Rat trauma-hemorrhage and resuscitation model
A nonheparinized rat model for trauma-hemorrhage and resuscitation was used in this study (22). Briefly, male Sprague-Dawley rats (275–325 g) obtained from the National Science Council were housed in an air-conditioned room under a reversed light-dark cycle and were allowed 1 week or more to adapt to the environment. Before the experiment, rats were fasted overnight but were allowed water ad libitum. The rats were anesthetized using isoflurane (Attane; Minrad Inc, Bethlehem, Pa) inhalation before the induction of soft tissue trauma via a 5-cm midline laparotomy. The abdomen was closed in layers, and catheters were placed in both femoral arteries and the right femoral vein (polyethylene [PE-50] tubing; Becton Dickinson & Co, Sparks, Md). The wounds were bathed with 1% lidocaine (Elkins-Sinn Inc, Cherry Hill, NJ) throughout the surgical procedure to reduce postoperative pain. Rats were then allowed to awaken, subjected to bleeding, and maintained at a mean blood pressure (MBP) of 40 mmHg. This level of hypotension was continued until the MBP could not be maintained without the use of additional Ringer’s lactate replacement fluid. This time point was defined as the maximum bleed-out time, and the amount of blood withdrawn was noted. Next, the rats were maintained at a MBP of 40 mmHg until 40% of the maximum bleed-out volume was returned in the form of Ringer’s lactate. The animals were then resuscitated with Ringer’s lactate with four times the volume of the shed blood over a period of 60 min. The time required for maximum hemorrhage was about 45 min, the volume of maximum hemorrhage was about 60% of the calculated circulating blood volume, and the total hemorrhage time was about 90 min (23). Thirty minutes before the end of the resuscitation period, the rats received tropisetron (1 mg/kg, intravenously), tropisetron plus the PI3K inhibitor wortmannin (1 mg/kg, intravenously at the beginning of resuscitation), wortmannin, or an equal volume of the vehicle (∼0.2 mL distilled water) (8). The catheters were then removed, the vessels ligated, and the skin incisions closed with sutures. Sham-operated animals underwent a surgical procedure that included a laparotomy in addition to ligation of the femoral artery and vein, but neither hemorrhage nor resuscitation was performed. The animals were then returned to their cages and were given food and water ad libitum. The animals were killed 24 h after the end of the resuscitation or sham operation. The dosage of tropisetron (1 mg/kg, intravenously) was chosen according to our previous study where 5-HT3receptor antagonist in the dosage of 1 mg/kg could attenuate hepatic injury in a rat trauma-hemorrhagic shock model (19). The combination dose of tropisetron plus the PI3K inhibitor wortmannin (1 mg/kg) is referenced by our previous studies that PI3K inhibitor wortmannin (1 mg/kg) could reverse the Akt-dependent hepatic injury following trauma-hemorrhage (6, 8). There were eight rats in each group.
Measurement of cardiac output and in vivo heart performance
Twenty-four hours after either the sham operation or trauma-hemorrhage and resuscitation, animals were reanesthetized using sodium pentobarbital; cardiac output (CO), maximal rate of left ventricular pressure increase (+dP/dtmax), maximal rate of left ventricular pressure decrease (−dP/dtmax), heart rate (HR), and MBP were measured, as described previously (24).
Measurement of MPO activity
The cardiac MPO activity in homogenates of left ventricle tissue was determined as described previously (19). Frozen tissue samples were thawed and suspended in phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, St. Louis, Mo). Samples were sonicated on ice and centrifuged at 12,000g for 15 min at 4°C, and an aliquot was transferred to phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine hydrochloride and 0.0005% hydrogen peroxide (Sigma). The change in absorbance at 460 nm was measured spectrophotometrically for 5 min. Myeloperoxidase activity was calculated using a standard curve generated using human MPO (Sigma), and values were normalized to protein concentration.
Measurement of cardiac IL-6 and ICAM-1 levels and circulating IL-6 levels
Interleukin 6 and ICAM-1 levels in the heart and plasma IL-6 levels were determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, Minn) according to the manufacturer’s instructions as described previously (19). Briefly, the samples were homogenized in phosphate-buffered saline (1:10 weight-volume, pH 7.4) containing protease inhibitors (complete protease inhibitor cocktail; Boehringer, Mannheim, Germany). Homogenates were centrifuged at 2,000g for 20 min at 4°C, and the supernatant was assayed for IL-6 and ICAM-1 levels. An aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC Protein Assay; Bio-Rad, Hercules, Calif).
Western blot assay
Rat heart tissues were homogenized in a buffer as described previously (19). The homogenates were centrifuged at 12,000g for 15 min at 4°C and analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the proteins were then transferred to nitrocellulose membranes. The membranes were incubated with antibodies for total Akt protein, p-Akt (Ser473) (Cell Signaling Technology, Beverly, Mass), or GAPDH (Abcam, Cambridge, Mass) overnight at 4°C. The membranes were incubated with horseradish peroxidase–conjugated goat anti–rabbit antibody or goat anti–mouse antibody for 1.5 h at room temperature. After the final washing, blots were probed using enhanced chemiluminescence (Amersham, Piscataway, NJ) and autoradiographed.
Analysis of DNA fragmentation by ELISA
Mononucleosomes and oligonucleosomes produced by endogenous endonucleases were detected using mouse monoclonal antibodies. The Cell Death Detection ELISA PLUS kit (Roche Diagnostics, Indianapolis, Ind) was used to assess apoptosis according to the manufacturer’s directions (25). All samples were tested in duplicate, and the absorbance values were averaged. The results were calculated after subtracting the background value of the immunoassay from the average of the absorbance values.
Results are presented as mean ± SEM (n = 8 rats/group). Data were analyzed using one-way analysis of variance and Tukey test. Differences were considered significant at a P ≤ 0.05.
Effect of tropisetron on cardiac function
Trauma-hemorrhage produced a significant decrease in CO, MBP, and ±dP/dtmax (CO: 57 ± 6 vs. 20 ± 3 mL/min; MBP 111 ± 5 vs. 57 ± 3 mmHg; +dP/dtmax: 5,635 ± 220 vs. 2,147 ± 205 mmHg/s; -dP/dtmax: 6,609 ± 854 vs. 1,850 ± 317 mmHg/s) but was not found in HR (417 ± 13 vs. 385 ± 12 beats/min) (Figs. 1 and 2). There was no significant difference in CO, HR, MBP, and ±dP/dtmax in the sham animals receiving tropisetron compared with vehicle-treated shams. Tropisetron significantly improved CO and MBP following trauma-hemorrhage (CO: 33 ± 3 vs. 20 ± 3 mL/min; MBP 77 ± 3 vs. 57 ± 3 mmHg, P < 0.05); however, the values remained lower than those of the shams (Fig. 1). Moreover, tropisetron attenuated the decrease in +dP/dtmax and -dP/dtmax (+dP/dtmax: 4,788 ± 389 vs. 2,147 ± 205 mmHg/s; -dP/dtmax: 4,810 ± 286 vs. 1,850 ± 317 mmHg/s, P < 0.05); however, these parameters still remained lower than those of the shams (Fig. 2). Administration of the PI3K inhibitor wortmannin abolished the tropisetron-induced improvement in cardiac function following trauma-hemorrhage (tropisetron + wortmannin vs. tropisetron: CO: 19 ± 3 vs. 33 ± 3 mL/min; MBP 59 ± 5 vs. 77 ± 3 mmHg; +dP/dtmax: 2,512 ± 391 vs. 4,788 ± 389 mmHg/s; -dP/dtmax: 2,119 ± 342 vs. 4,810 ± 286 mmHg/s, P < 0.05).
Alteration in cardiac MPO activity
The results revealed that there was no difference in cardiac MPO activity between vehicle- and tropisetron-treated sham groups (0.047 ± 0.005 vs. 0.051 ± 0.005 U/mg protein) (Fig. 3). After trauma-hemorrhage, MPO activity was significantly increased in vehicle-treated rats compared with sham-operated animals (0.139 ± 0.007 vs. 0.047 ± 0.005 U/mg protein, P < 0.05). Tropisetron treatment attenuated this increase in cardiac MPO activity (0.084 ± 0.004 vs. 0.139 ± 0.007 U/mg protein, P < 0.05). Administration of the PI3K inhibitor wortmannin prevented the tropisetron-mediated attenuation of cardiac MPO activity after trauma-hemorrhage (tropisetron + wortmannin vs. tropisetron: 0.143 ± 0.010 vs. 0.084 ± 0.004 U/mg protein, P < 0.05).
Alteration in cardiac IL-6 levels
There was no significant difference in cardiac IL-6 levels between vehicle- and tropisetron-treated sham groups (115 ± 9 vs. 111 ± 12 pg/mg protein) (Fig. 4). Trauma-hemorrhage significantly increased cardiac IL-6 levels in vehicle-treated rats compared with sham animals (356 ± 17 vs. 116 ± 9 pg/mg protein, P < 0.05), which was attenuated by tropisetron treatment (189 ± 11 vs. 356 ± 17 pg/mg protein, P < 0.05). Tropisetron-mediated reductions in cardiac IL-6 levels were abolished by wortmannin coadministration (tropisetron + wortmannin vs. tropisetron: 321 ± 12 vs. 189 ± 12 pg/mg protein, P < 0.05).
Alteration in cardiac ICAM-1 expression
There was no significant difference in cardiac ICAM-1 levels between vehicle- and tropisetron-treated sham groups (818 ± 82 vs. 765 ± 90 pg/mg protein, P < 0.05) (Fig. 5). Trauma-hemorrhage significantly increased ICAM-1 expression in the heart (2,302 ± 234 vs. 819 ± 82 pg/mg protein, P < 0.05), and treatment with tropisetron attenuated this increase (1,399 ± 79 vs. 2,302 ± 234 pg/mg protein, P < 0.05). Coadministration of the PI3K inhibitor wortmannin with tropisetron prevented the tropisetron-induced reduction of ICAM-1 expression. (tropisetron + wortmannin vs. tropisetron: 2,391 ± 238 vs. 1,399 ± 79 pg/mg protein, P < 0.05).
Heart Akt protein expression and activity
There was no significant difference in Akt protein expression between sham and trauma-hemorrhaged rats (Fig. 6). However, Akt activity, as determined by its phosphorylation, was significantly decreased after trauma-hemorrhage (1.0 ± 0.1 vs. 0.5 ± 0.0, P < 0.05). Administration of tropisetron after trauma-hemorrhage restored Akt activity to the levels observed in sham animals (1.0 ± 0.1 vs. 0.9 ± 0.1, P < 0.05). The increase in p-Akt induced by tropisetron was abolished by administration of wortmannin along with tropisetron (tropisetron vs. tropisetron + wortmannin: 1.0 ± 0.1 vs. 0.5 ± 0.1, P < 0.05) (Fig. 6).
Cardiac DNA fragmentation
Trauma-hemorrhage produced a significant increase in cardiac DNA fragmentation (1.4 ± 0.1 vs. 0.4 ± 0.1, P < 0.05), which was attenuated following administration of tropisetron (0.8 ± 0.1 vs. 1.4 ± 0.1, P < 0.05) (Fig. 7). To evaluate whether the antiapoptotic effect of tropisetron is mediated an Akt pathway, a group of trauma-hemorrhage rats was cotreated with tropisetron and wortmannin. Administration of wortmannin abolished the tropisetron-induced attenuation of cardiac DNA fragmentation following trauma-hemorrhage (tropisetron + wortmannin vs. tropisetron: 1.2 ± 0.1 vs. 0.7 ± 0.1, P < 0.05) (Fig. 7).
Effect of tropisetron in circulating IL-6 levels
The results revealed that plasma IL-6 levels were not detected in the vehicle- and tropisetron-treated sham groups (Fig. 8). After trauma-hemorrhage, plasma IL-6 levels were increased in vehicle-treated rats (284 ± 59 pg/mL) (Fig. 8). Tropisetron treatment attenuated this increase in plasma IL-6 levels (95 ± 21 vs. 284 ± 59 pg/mL, P < 0.05). Administration of the PI3K inhibitor wortmannin prevented the tropisetron-mediated attenuation of plasma IL-6 levels after trauma-hemorrhage (tropisetron + wortmannin vs. tropisetron: 276 ± 56 vs. 95 ± 21 pg/mL, P < 0.05).
In this study, we sought to determine whether Akt-dependent pathways play an important role in tropisetron-mediated cardioprotection following trauma-hemorrhage. The salutary effects of tropisetron at doses of 0.1, 0.3, 1, and 3 mg/kg have been evaluated in cardiac injury after trauma-hemorrhage. We found fewer beneficial effects when tropisetron was administered at a dosage of 0.1 or 0.3 mg/kg and similar results when tropisetron was administered at a dosage of 1 or 3 mg/kg (data not shown). Our results indicate that rats treated with tropisetron (1 mg/kg) displayed improvements in CO and ±dP/dtmax 24 h after trauma-hemorrhage. In addition, 24 h after trauma-hemorrhage, cardiac MPO activity, IL-6, ICAM-1 levels, and apoptosis were also markedly increased in male rats. Administration of a single dose of tropisetron (1 mg/kg) during resuscitation attenuated the increases in those parameters. Administration of tropisetron also prevented the trauma-hemorrhage–induced decrease in p-Akt expression. Furthermore, our findings indicate that administration of the PI3K inhibitor wortmannin along with tropisetron abolished the tropisetron-induced cardioprotection in rats subjected to trauma-hemorrhage. These studies collectively suggest that the salutary effects of tropisetron seem to be mediated via an Akt pathway.
Hemorrhagic shock results in excessive production of proinflammatory mediators, such as cytokines and chemokines (1). A prolonged period, despite fluid resuscitation, induces tissue and cellular damage, which plays a significant role in the development of dysfunctions in various organs (1, 19). Multiple organ failure or dysfunction secondary to a systemic inflammatory response remains the major cause of mortality and morbidity (1). Previous studies have shown that the enhanced secretion of proinflammatory cytokines by mast cells, dendritic cells, and macrophages is an important factor in the initiation and perpetuation of tissue inflammation (1). These cytokines recruit other immune cells including neutrophils, thereby increasing leukocyte trafficking and the subsequent accumulation of neutrophils in the damaged organs. Activated neutrophils seem to infiltrate the injured organs in conjunction with increased expression of adhesion molecules on endothelial cells and elevated local levels of chemokines and cytokines (26). Studies have shown that cardiac injury is associated with neutrophil accumulation (27). The infiltration of neutrophils in the heart is accompanied by increased expression of adhesion molecules and an elevation in locally produced cytokine levels (2, 27). Tropisetron is often used for prevention or treatment of postoperative or chemotherapy-induced nausea and vomiting (28, 29). 5-Hydroxytryptamine subtype 3 receptor antagonists, particularly tropisetron, possess anti-inflammatory properties (20, 30). Previous studies have shown that anti-inflammatory effects of tropisetron are related to inhibition of IL-1β, tumor necrosis factor α, and IL-6 release in lipopolysaccharide-stimulated human primary monocytes (31). However, little is known about the role of tropisetron in hemorrhagic shock and resuscitation.
The cytokine IL-6 is an important early mediator in the heart during trauma-hemorrhage and is required for the expression of adhesion molecules and chemokines (32). In this study, the ability of tropisetron to modulate expression of inflammatory cytokine (IL-6) and adhesion molecule (ICAM-1) suggests a role for tropisetron in the regulation of cardiac injury. Previous animal studies showed that tropisetron attenuated the damage of cerebral infraction (20) and reduced the severity of colonic inflammation (30) that were associated with decreased neutrophil infiltration and inflammatory cytokines. The results are consistent with our present findings.
The PI3K/Akt pathway is known to play an important role in the organ protection and cell survival through Akt phosphorylation (8, 33). Previous studies have shown that upregulation of the PI3K/Akt pathway attenuates the overproduction of cytokines and adhesion molecules and neutrophil accumulation after trauma-hemorrhage (21, 29). Reduction of IL-6, ICAM-1 levels, and MPO activity in the heart is associated with an attenuation of cardiac injury (22). Our previous study has also reported that PI3K/Akt pathway plays a critical role in cardioprotection (25). This study was performed to examine whether tropisetron can improve cardiac function through Akt-dependent attenuation of cardiac IL-6 and ICAM-1 levels, MPO activity, and apoptosis in hemorrhagic shock and resuscitation. Our finding indicated that hemorrhagic shock was accompanied by a decrease in cardiac Akt activation. The depressed Akt phosphorylation following hemorrhagic shock was restored by administration of tropisetron after trauma-hemorrhage. However, the restored Akt phosphorylation by tropisetron after hemorrhagic shock was abolished by coadministration of wortmannin. These results thus indicate that the salutary effects of tropisetron on cardiac functions after hemorrhagic-shock are in part mediated by an Akt-dependent pathway.
From this study, tropisetron activated cardiac Akt and decreased neutrophil infiltration following trauma-hemorrhage. It remains unknown whether tropisetron primarily modified neutrophil activity or modified cardiac cellular activity via an Akt-dependent pathway and somehow limited neutrophil recruitment. Further studies are still needed to investigate the precise mechanism for this effect.
In this study, the plasma IL-6 levels were significantly attenuated in the rat treated with tropisetron following trauma-hemorrhage. The result showed that tropisetron on whole-body system may also contribute to its favorable effect on the heart. However, the precise mechanisms still need to be determined. Apoptosis was evaluated in the myocardium; however, transaminase levels or other indicators of cardiac injury were not examined in the study. It remains unknown if tropisetron was able to prevent myocardial injury that did not result in apoptosis. In view of this, additional studies are needed to completely elucidate the mechanism. Wortmannin was chosen as a specific inhibitor of PI3K. However, wortmannin can have effects on other signaling pathways such as mTOR, p38 MAPK, polo-kinase, and insulin receptor activation, depending on the concentration and cell type (34, 35). Additional studies are, however, needed to precisely elucidate the mechanism by which tropisetron attenuates cardiac injury following trauma-hemorrhage.
In conclusion, administration of tropisetron ameliorates cardiac injury and production of proinflammatory mediators after trauma-hemorrhage. Blockade of Akt activation abolishes the salutary effects of tropisetron in the heart following trauma-hemorrhage. Our findings provide evidence that tropisetron-mediated cardioprotection is, in part, mediated via an Akt-dependent pathway after trauma-hemorrhage.
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