Mechanism of estrogen-mediated attenuation of hepatic ... - CiteSeerX

Uncorrected Version. Published on July 26, 2007 as DOI:10.1189/jlb.0607355

Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation Jun-Te Hsu,1 Wen-Hong Kan, Chi-Hsun Hsieh,2 Mashkoor A. Choudhry, Martin G. Schwacha, Kirby I. Bland, and Irshad H. Chaudry3 Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA

Abstract: Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)-1. Up-regulation of HO-1 mediates potent, anti-inflammatory effects and attenuates organs injury. Although studies have shown that 17␤-estradiol (E2) prevents organ damage following traumahemorrhage, it remains unknown whether Akt/ HO-1 plays any role in E2-mediated attenuation of hepatic injury following trauma-hemorrhage. To study this, male rats underwent trauma-hemorrhage (mean blood pressure, ⬃40 mmHg for 90 min), followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body weight), E2 plus the PI-3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma-hemorrhage, plasma ␣-GST and hepatic tissue myeloperoxidase (MPO) activity, IL-6, TNF-␣, ICAM-1, cytokine-induced neutrophil chemoattractant-1, and MIP-2 levels were measured. Hepatic Akt and HO-1 protein levels were also determined. Trauma-hemorrhage increased hepatic injury markers (␣-GST and MPO activity), cytokines, ICAM-1, and chemokine levels. These parameters were markedly improved in the E2-treated rats following trauma-hemorrhage. E2 treatment also increased hepatic Akt activation and HO-1 expression compared with vehicletreated, trauma-hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following traumahemorrhage are in part mediated via an ER-related, Akt-dependent up-regulation of HO-1. J. Leukoc. Biol. 82: 000 – 000; 2007.

ries and severe blood loss [1]. Multiple organ failure or dysfunction, secondary to a systemic inflammatory response, remains the major cause of mortality and morbidity following trauma [2– 8]. The PI-K3/protein kinase B (PKB; Akt) is known to be an endogenous negative-feedback or compensatory mechanism, which serves to limit proinflammatory and chemotactic events in response to injury [9, 10]. Inhibition of the PI-3K/Akt pathway with a PI-3K inhibitor Wortmannin increases serum cytokine levels and decreases the survival of mice subjected to sepsis [10, 11]. Neutrophils are part of an innate immune response to infection [12], and thus, these cells have a protective effect. However, under conditions such as trauma-hemorrhage, the tissue infiltration of neutrophils is increased [13]. Neutrophils also release superoxide anions and proteolytic enzymes, which diffuse across the endothelium and cause tissue damage [14, 15]. It has also been reported that adhesion molecules and chemotactic factors mediate neutrophil movement and migration [16]. Among adhesion molecules, ICAM-1, which is constitutively present on the surface of endothelial cells, plays a critical role in firm adhesion of neutrophil to the vascular endothelium and is markedly up-regulated following hemorrhagic shock [15, 17, 18]. The recruitment of neutrophil to sites of inflammation is also driven by locally produced cytokines and chemokines [19]. Studies have shown that IL-6 and TNF-␣ play important roles in the pathophysiology of hepatic ischemia-reperfusion [3, 20] and are required for expression of adhesion molecules and production of chemokines [21]. The CXC chemokines, such as cytokine-induced neutrophil chemoattractant (CINC)-1 and MIP-2, are potent chemoattractants for neutrophil in rats [16, 22]. Moreover, it has been shown that treatment of rats with antibodies to neutralize the release of CINC and MIP-2 in rat inflammation models decreases myeloperoxidase (MPO) buildup and neutrophil levels [23, 24].

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Key Words: protein kinase B 䡠 CINC-1 䡠 MIP-2 䡠 MPO

INTRODUCTION The liver has been shown to play a critical role in the development of delayed organ dysfunction following traumatic inju0741-5400/07/0082-0001 © Society for Leukocyte Biology

Current address: Department of Surgery, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan. 2 Current address: Department of Surgery, China Medical University Hospital, China Medical University, Taichung, Taiwan. 3 Correspondence: Center for Surgical Research, University of Alabama at Birmingham, 1670 University Blvd., Volker Hall, Room G094, Birmingham, AL 35294-0019, USA. E-mail: [email protected] Received June 7, 2007; revised July 3, 2007; accepted July 3, 2007. doi: 10.1189/jlb.0607355

Journal of Leukocyte Biology Volume 82, October 2007

Copyright 2007 by The Society for Leukocyte Biology.

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A growing body of evidence indicates that Akt activation induces heme oxygenase (HO)-1 [25–27], which is to known to play a protective role in many organs under various deleterious conditions including trauma-hemorrhage [15, 16, 28, 29]. Upregulation of HO-1 causes a reduction of adhesion molecules, chemokines, and neutrophil and ameliorates hepatic injury following hepatic ischemia-reperfusion [3, 30]. Studies have also shown that administration of 17␤-estradiol (E2) following trauma-hemorrhage increased HO-1 expression, which prevents the organs from dysfunction and injury [28, 29]. Nonetheless, it remains unknown whether Akt has any role in the E2-mediated HO-1 up-regulation. We hypothesized that the beneficial effects of E2 following trauma-hemorrhage are mediated via an Akt-dependent up-regulation of HO-1. To test this hypothesis, we treated animals with E2 alone and in combination with the PI-3K inhibitor Wortmannin following trauma-hemorrhage. The effects of these treatments were then examined on plasma ␣-GST levels as well as hepatic tissue MPO activity, Akt/HO-1, cytokines (IL-6 and TNF-␣), ICAM-1, and chemokine (CINC-1 and MIP-2) levels following trauma-hemorrhage.

10 min), immediately frozen, and stored at – 80°C. Hepatic injury was determined by measuring plasma levels of ␣-GST using a commercially available enzyme immunosorbent immunoassay kit according to the manufacturer’s instructions (Biotrin International Ltd., Dublin, Ireland).

Measurement of MPO activity MPO activity in homogenates of whole liver was determined as described previously [15, 18]. All reagents were purchased from Sigma Chemical Co. Briefly, equal weights (100 mg wet weight) of liver from various groups were suspended in 1 ml buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer, pH 6.0) and sonicated at 30 cycles, twice, for 30 s on ice. Homogenates were cleared by centrifuging at 17,000 g at 4°C for 10 min, and the supernatants were stored at – 80°C. Protein content in the samples was determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The samples were incubated with a substrate o-dianisidine hydrochloride. This reaction was carried out in a 96-well plate by adding 290 ␮l 50 mM phosphate buffer, 3 ␮l substrate solution (containing 20 mg/ml o-dianisidine hydrochloride), and 3 ␮l H2O2 (20 mM). Sample (10 ␮l) was added to each well to start the reaction. Standard MPO (Sigma Chemical Co.) was used in parallel to determine MPO activity in the sample. The reaction was stopped by adding 3 ␮l sodium azide (30%). Light absorbance at 460 nm was read. MPO activity was determined by using the curve obtained from the standard MPO.

Western blot analysis MATERIALS AND METHODS Rat trauma-hemorrhagic shock model Male (275–325 g) Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were fasted overnight before the experiment but were allowed water ad libitum. All experiments were performed in adherence with National Institutes of Health (NIH; Bethesda, MD, USA) guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham (Birmingham, AL, USA). A nonheparinized model of trauma-hemorrhage was used as described previously [31]. Briefly, rats were anesthetized by isoflurane inhalation prior to the induction of soft tissue trauma via a 5-cm midline laparotomy. The abdomen was closed in layers, and polyethylene catheters (PE-50, Becton Dickinson, Sparks, MD, USA) were placed in femoral arteries and the right femoral vein. The wounds were bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ, USA) throughout the surgical procedure to reduce postoperative pain. Rats were then placed into a Plexiglas box in a prone position and allowed to awaken, after which time, they were bled rapidly to a mean arterial blood pressure (BP) of 35– 40 mmHg within 10 min. This level of hypotension was maintained until the animals could no longer keep a mean BP of 35 mmHg, unless additional fluid in the form of Ringer’s lactate (RL) was administered. This time was defined as maximum bleed-out, and the amount of withdrawn blood was recorded. Following this, the rats were maintained at mean BP of 35– 40 mmHg until 40% of the maximum bleed-out volume was returned in the form of RL (⬃90 min from the onset of bleeding). The animals were then resuscitated with four times the volume of the shed blood with RL over 60 min. Sham-operated animals underwent the same groin dissection, which included the ligation of the femoral artery and vein, but neither hemorrhage nor resuscitation was carried out. Animals subjected to trauma-hemorrhage were allocated randomly into four groups receiving vehicle (cyclodextrin, i.v., Sigma Chemical Co., St. Louis, MO, USA), E2 [1 mg/kg body weight (BW), i.v., Sigma Chemical Co.], E2 plus the PI-3K inhibitor Wortmannin (1 mg/kg BW, i.p., Sigma Chemical Co.), or coadministration of E2 with a high-affinity estrogen receptor (ER) antagonist ICI 182,780 (3 mg/kg BW, i.p., Tocris Cookson Ltd., Ballwin, MO, USA) at the beginning of the resuscitation. Following resuscitation, the catheters were removed, the vessels were ligated, and the skin incisions were closed with sutures. The animals were killed at 2 h after the end of resuscitation or sham operation.

Measurement of hepatic injury At 2 h following trauma-hemorrhage or sham operation, heparinized blood samples were obtained, and plasma was separated by centrifugation (2000 g,

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Approximately 0.1 g freshly collected liver tissue from each rat was homogenized in 1 ml lysis buffer containing 50 mM HEPES, 10 mM sodium pyrophosphate, 1.5 mM MgCl2, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.15 M NaCl, 0.1 M NaF, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail (Sigma Chemical Co.). Tissue lysates were centrifuged at 17,000 g for 20 min at 4°C, and an aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC protein assay). The lysates (50 ␮g per lane) were then mixed with 4⫻ SDS sample buffer and were electrophoresed on 4 –12% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and transferred electrophoretically onto nitrocellulose membranes (Invitrogen). The membranes were immunoblotted with the following primary antibodies against Akt, phospho-Akt (Cell Signaling Technology, Beverley, MA, USA), HO-1 (Stressgen Bioreagents, Ann Arbor, MI, USA), or ␤-actin (Abcam, Cambridge, MA, USA). The membranes were then washed and incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody for detection of bound antibodies by ECL (Amersham, Piscataway, NJ, USA). Rabbit polyclonal ␤-actin antibody was used to determine ␤-actin as the loading control. Signals were quantified using ChemiImager 5500 imaging software (Alpha Innotech Corp., San Leandro, CA, USA).

Determination of IL-6, TNF-␣, ICAM-1, CINC-1, and MIP-2 levels in the liver IL-6, TNF-␣, ICAM-1, CINC-1, and MIP-2 levels in the liver were determined using ELISA kits (R&D, Minneapolis, MN, USA, and Biosource, Camarillo, CA, USA), according to the manufacturers’ instructions. Briefly, the samples were homogenized in 0.5 ml lysis buffer containing 50 mM HEPES, 10 mM sodium pyrophosphate, 1.5 mM MgCl2, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.15 M NaCl, 0.1 M NaF, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail (Sigma Chemical Co.). The homogenates were centrifuged at 17,000 g for 20 min at 4°C, and the supernatant was assayed for IL-6, TNF-␣, ICAM-1, CINC-1, and MIP-2 levels. An aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC protein assay), which for these proinflammatory mediators, is expressed as pg/mg protein in each sample.

Statistical analysis Results are presented as mean ⫾ SEM (n⫽6 rats/group). One-way ANOVA and Tukey’s test were used for the comparison among groups, and differences were considered significant at P ⬍ 0.05.

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RESULTS Effects of E2 on plasma levels of ␣-GST As shown in Figure 1A, trauma-hemorrhage increased plasma ␣-GST levels significantly in vehicle-treated animals. E2 treatment attenuated the trauma-hemorrhage-induced increase in plasma ␣-GST levels. To determine whether E2 reduces hepatic injury following trauma-hemorrhage via an ER-mediated pathway, a group of animals was treated with ER antagonist ICI 182,780 plus E2. The results indicate that administration of ICI 182,780 following trauma-hemorrhage prevented the E2induced decrease in ␣-GST levels. No significant difference in plasma ␣-GST levels was observed between vehicle- and E2treated sham groups.

Effects of E2 on hepatic MPO activity Trauma-hemorrhage produced a significant increase in hepatic MPO activity in vehicle-treated animals (Fig. 1B), which was attenuated by E2 treatment. However, coadministration of ER antagonist ICI 182,780 abolished E2-induced decrease in hepatic MPO activity (Fig. 1B). E2 did not alter hepatic MPO activity in sham-operated animals (Fig. 1B).

Activation of Akt in the liver Trauma-hemorrhage induced a marked decrease in Akt phosphorylation compared with shams (Fig. 2). Administration of

Fig. 2. Expression of total and phosphorylated (activated) PKB (p-Akt) in the liver at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin (W) or ICI 182,780 before resuscitation. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values were pooled from six animals in each group and are shown as means ⫾ SEM. *, P ⬍ 0.05, veruss sham or traumahemorrhage-E2.

E2 prevented the trauma-hemorrhage-induced decrease in Akt phosphorylation, and the values were similar to shams. The increase in Akt phosphorylation following trauma-hemorrhage by E2 was abolished by coadministration of Wortmannin (Fig. 2). Furthermore, coadministration of E2 along with ICI 182,780 prevented the E2-induced increase in hepatic Akt phosphorylation (Fig. 2). There was no difference in Akt phosphorylation in sham animals treated with vehicle or E2 (Fig. 2). No change in the total Akt protein expression was observed in the trauma-hemorrhage-E2-treated rats compared with shams (Fig. 2).

Expression of HO-1 in the liver Hepatic HO-1 expression was increased significantly in the vehicle-treated, trauma-hemorrhage group compared with shams (Fig. 3). Administration of E2 following trauma-hemorrhage induced a further, marked increase in hepatic HO-1 protein expression (Fig. 3). The up-regulation of HO-1 expression induced by E2 was abolished by coadministration of Wortmannin or ICI 182,780 (Fig. 3). HO-1 expression was not altered in sham animals treated with vehicle or E2 (Fig. 3).

Effects of Wortmannin on plasma levels of ␣-GST Fig. 1. Effects of E2 and ICI 182,780 (ICI) on plasma ␣-GST levels (A) and liver tissue MPO activity (B) at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus ICI before resuscitation. Data are shown as means ⫾ SEM of six animals in each group. *, P ⬍ 0.05, versus sham or trauma-hemorrhage-E2.

To determine whether E2 reduces hepatic injury following trauma-hemorrhage via an Akt-mediated pathway, a group of animals was treated with PI-3K inhibitor Wortmannin along with E2. The results indicate that administration of Wortman-

Hsu et al. Estrogen effect on Akt/HO-1 after trauma-hemorrhage

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rats. E2 treatment decreased the trauma-hemorrhage-induced increase of hepatic CINC-1 and MIP-2 levels, and these were also abolished by coadministration of Wortmannin or ICI 182,780 (Fig. 7). No change was found in hepatic ICAM-1, CINC-1, and MIP-2 levels in sham animals treated with vehicle or E2 (Figs. 6 and 7).

DISCUSSION

Fig. 3. HO-1 protein expression in the liver at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin or ICI 182,780 before resuscitation. Blots obtained from several experiments were analyzed using densitometry, and the densitometric values were pooled from six animals in each group and are shown as means ⫾ SEM. *, P ⬍ 0.05, versus sham; #, P ⬍ 0.05, versus trauma-hemorrhage-vehicle, E2 plus Wortmannin, or E2 plus ICI.

Our studies collectively suggest that the E2-mediated hepatic protection following trauma-hemorrhage is in part mediated via Akt-dependent up-regulation of HO-1. Induction of HO-1 plays an important role in organ protection against the deleterious pathophysiological conditions such as trauma-hemorrhage, ischemia, oxidative stress, and endotoxemia [18, 28, 32, 33]. Additional findings also demonstrate that inhibition of HO-1 abolished E2-induced improvement in organ function following trauma-hemorrhage [29]. Consistent with these findings, our results indicate that E2 treatment following traumahemorrhage led to a further increase in HO-1 expression and attenuated the trauma-hemorrhage-induced increase in the levels of plasma ␣-GST and hepatic MPO activity. However, our results further show that up-regulation of HO-1 is not entirely

nin following trauma-hemorrhage prevented the E2-induced decrease in ␣-GST levels (Fig. 4A).

Effects of Wortmannin on hepatic MPO activity To determine the role of Akt in an E2-induced decrease in hepatic MPO activity following trauma-hemorrhage, rats were treated with Wortmannin along with E2. The results show that the E2-induced decrease in hepatic MPO activity following trauma-hemorrhage was inhibited by coadministration of Wortmannin (Fig. 4B).

Levels of hepatic IL-6 and TNF-␣ Trauma-hemorrhage led to a significant increase in hepatic IL-6 and TNF-␣ levels compared with shams (Fig. 5). E2 administration following trauma-hemorrhage attenuated the increase in hepatic IL-6 and TNF-␣ levels (Fig. 5), which was abolished by coadministration of Wortmannin or ICI 182,780 (Fig. 5). E2 did not affect the levels of hepatic IL-6 and TNF-␣ in sham-operated animals (Fig. 5).

Levels of hepatic ICAM-1, CINC-1, and MIP-2 Trauma-hemorrhage increased hepatic ICAM-1 expression markedly, which was normalized by the administration of E2 following trauma-hemorrhage (Fig. 6). Coadministration of Wortmannin or ICI 182,780 abolished the E2-mediated reduction in ICAM-1 expression in the trauma-hemorrhage group (Fig. 6). In addition, hepatic CINC-1 and MIP-2 levels were increased significantly in vehicle-treated, trauma-hemorrhage 4


Fig. 4. Effects of Wortmannin on plasma ␣-GST levels (A) and liver tissue MPO activity (B) at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin before resuscitation. Data are shown as means ⫾ SEM of six animals in each group. *, P ⬍ 0.05, versus sham or trauma-hemorrhage-E2.


Fig. 5. Levels of IL-6 (A) and TNF-␣ (B) in the liver at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin or ICI 182,780 before resuscitation. Data are shown as means ⫾ SEM of six animals in each group. *, P ⬍ 0.05, versus sham or trauma-hemorrhage-E2.

dependent on the Akt pathway, as we observed that HO-1 is increased following trauma-hemorrhage. This initial increase in HO-1 is likely independent of Akt, as there was a concomitant, significant decrease in Akt phosphorylation in vehicletreated, trauma-hemorrhage rats compared with shams. However, E2-mediated increases in HO-1 are found to be Aktdependent, as coadministration of Wortmannin with E2 abolished the increase in HO-1 and the salutary effects of E2 on the liver. Furthermore, E2-induced increase in hepatic Akt activation and the protective effects on the liver following trauma-hemorrhage were prevented by coadministration of ER antagonist ICI 182,780. HO-1, the inducible member of the heme oxygenase enzyme family, is responsible for heme elimination. The accumulation of free heme under hypoxic conditions entails the development of its toxic effects. Therefore, its elimination from the cellular milieu is important. Carbon monoxide (CO), a principle byproduct of heme catabolism by HO-1, activates soluble guanylate cyclase and induces vasodilatation via cyclic GMP [34]. It has also shown that HO-1-mediated tissue protection may be a result of the CO-induced activation of the Ca2⫹-dependent potassium channels, which results in hyperpolarization of the smooth muscle cells, leading to decreased vascular contractility [35]. In addition, overexpression of HO-1 reduces the

expression of adhesion molecules and cytokine/chemokine levels and therefore prevents subsequent leukocyte-endothelial cell interactions [18, 36]. It has been reported that NO increases HO-1 transcription and stabilization of HO-1 mRNA [37]. The other product of HO-1 enzyme activity, bilirubin, can be regenerated by biliverdin reductase, forming a potent redox cycle [38], and reduces ICAM-1 expression in the ischemic intestine [39]. Additional findings also indicate that up-regulation of HO-1 protects mitochondrial function and prevents ATP depletion after oxidative stress [40]. Thus, multiple mechanisms are involved in the protection of HO-1 against various injury insults. Activation of PI-3K/Akt signaling cascade by E2 has been observed in different cells/tissues [41– 44]. For example, E2 induced the activation of Akt in mouse cardiomyocytes [41] and Chinese hamster ovary cells [42]. Studies have also shown that the neuroprotective effects of E2 on glutamate-induced neurotoxicity in rat neurons are through the phosphorylation of Akt [43]. Furthermore, it has been reported that human endothelial cells stimulated with E2 increase PI-3K activity, leading to the activation of Akt [44]. In addition, the action of E2 on Akt phosphorylation is observed to be mediated by ERdependent and -independent mechanisms [45]. In this study, we found that E2-induced attenuation of hepatic injury is partly via increases in Akt activation, which were blocked by ER antagonist ICI 182,780. These findings thus indicate that E2 binds to its receptor and in turn mediates the activation of Akt. It has been reported that CXC chemokines play a critical role in trauma-hemorrhage-induced extravasation of neutrophils in the liver [15, 46]. Studies have also demonstrated that hepatic injury is associated with an increased neutrophil accumulation [15, 19, 46]. The infiltration of neutrophils in the liver is also accompanied with increased expression of adhesion molecules and elevation of locally produced cytokine/ chemokine levels [19, 46]. In the present study, our results indicate that trauma-hemorrhage resulted in a significant in-

Fig. 6. Levels of ICAM-1 in the liver at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin or ICI 182,780 before resuscitation. Data are shown as means ⫾ SEM of six animals in each group. *, P ⬍ 0.05, versus sham or trauma-hemorrhage-E2.


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Fig. 7. Levels of CINC-1 (A) and MIP-2 (B) in the liver at 2 h after sham operation or trauma-hemorrhage. Animals were treated with vehicle, E2, or E2 plus Wortmannin or ICI 182,780 before resuscitation. Data are shown as means ⫾ SEM of six animals in each group. *, P ⬍ 0.05, versus sham or trauma-hemorrhage-E2.

crease in hepatic proinflammatory cytokine/chemokine levels and ICAM-1 expression, which was accompanied with increased hepatic MPO activity, an indicator for neutrophil infiltration. However, E2 administration following trauma-hemorrhage attenuated the above-mentioned proinflammatory mediators and prevented hepatic injury under those conditions. Several potential mechanisms have been proposed for the salutary effects of E2 in ameliorating hepatic injury following trauma-hemorrhage [15, 28, 29, 47]. Szalay et al. [28, 29] found that E2 administration following trauma-hemorrhage increased hepatic heat shock protein 60 and 70 mRNA expression and HO-1 mRNA expression, HO-1 protein levels, and HO-1 enzymatic activity, leading to improvement of hepatic function. Hsieh et al. [47] suggested that the beneficial effects of E2 on hepatic injury following trauma-hemorrhage are in part via the activation of the PKA pathway, which protects cells from apoptosis through Bcl-2. The present study demonstrates that E2 treatment following trauma-hemorrhage normalized the Akt phosphorylation and produced a further increase in HO-1 protein expression. Thus, these findings indicate that the protective effects of E2 on hepatic integrity after trauma-hemorrhage are mediated via Akt-dependent HO-1 up-regulation. Studies have shown that E2 treatment following traumahemorrhage improves cardiac and hepatic functions [28, 29, 6


31] and prevents hepatic neutrophil infiltration in male rats [15]. It is therefore possible that the protective effects of E2 on hepatic injury following trauma-hemorrhage may also be a result of an E2-mediated improvement on cardiac function. Nonetheless, as E2 administration following trauma-hemorrhage increased hepatic Akt activity and subsequent overexpression of HO-1, it is also possible that E2 may have a direct effect on the liver. Additional studies are, however, needed to elucidate precisely the mechanism by which E2 ameliorates hepatic injury following trauma-hemorrhage. It can also be argued that Wortmannin or ICI 182,780 should have been administered alone in sham or traumahemorrhage rats to determine if either of those per se has any adverse effect. However, as our recent studies have shown that administration of Wortmannin or ICI 182,780 alone in sham or trauma-hemorrhage animals did not produce any deleterious effects on cardiac function [48, 49], administration of Wortmannin or ICI 182,780 alone was not carried out in this study. It can also be argued that the present study did not provide direct evidence that HO-1 directly prevents hepatic injury following trauma-hemorrhage. Although this may be the case, our previous studies have shown that administration of HO enzyme inhibitor (chromium-mesoporphyrin) abolished the salutary effects of E2 on hepatic function following trauma-hemorrhage [28]. In view of this information, we did not treat rats with a HO enzyme inhibitor in this study, as that has already been shown to abolish the effects of E2. In summary, our results indicate that the E2 up-regulated Akt-dependent HO-1 expression and attenuated hepatic injury following trauma-hemorrhage. Blockade of Akt activation or ER and the associated deterioration of the examined parameters suggest that the reduction of neutrophil accumulation in the liver is in part mediated via an ER-related, Akt-dependent HO-1 pathway. Although the precise mechanism of the salutary effects of E2 in attenuating hepatic injury and the contribution of Akt/HO-1 in protecting against hepatic damage following trauma-hemorrhage remain unclear, our study provides evidence that Akt-dependent up-regulation of HO-1 may be critical in reducing hepatic injury following trauma-hemorrhage.

ACKNOWLEDGMENT This work was supported by NIH Grant R37 GM39519.

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