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J Appl Physiol 102: 933-941, 2007. First published November 16, 2006; doi:10.1152/japplphysiol.00919.2006
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Androstenediol inhibits the trauma-hemorrhage-induced increase in caspase-3 by downregulating the inducible nitric oxide synthase pathway

Juliann G. Kiang,1,2,3,4 Russell M. Peckham,1 Leah E. Duke,1 Tomoharu Shimizu,5 Irshad H. Chaudry,5 and George C. Tsokos1,3

1Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, Maryland; 2Armed Forces Radiobiology Research Institute and Departments of 3Medicine and 4Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; and 5Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, Alabama

Submitted 21 August 2006 ; accepted in final form 8 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Soft tissue trauma and hemorrhage (T-H) diminishes various aspects of liver function, while it increases hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 levels. Treatment with androstenediol (AED) inhibits the T-H-induced alterations of the above parameters. We sought to identify the molecular events underlying the beneficial effect of AED. Exposure of rats to T-H significantly increased the caspase-3 activity and protein, whereas treatment with AED significantly limited these increases. AED treatment also suppressed the T-H-induced increase in iNOS by effectively altering the levels of key transcription factors involved in the regulation of iNOS expression. Immunoprecipitation and immunoblotting analyses indicate that T-H increased apoptosome formation, and AED treatment significantly decreased it. Modulating the iNOS protein by transfecting cells with iNOS gene or small interfering RNA further confirmed the correlation between iNOS and caspase-3. Our data indicate that AED limits caspase-3 expression by suppressing the expression of transcription factors involved in the production of iNOS, resulting in decreased apoptosome. AED can potentially be a useful adjuvant for limiting liver apoptosis following T-H shock.

hemorrhage shock; caspase-9; cytochrome c; adiol; 5-androstene-3beta, 17beta-diol

SEVERE HEMORRHAGIC SHOCK, EVEN with resuscitation, results in depressed liver blood flow (51) that appears to contribute to liver injury and an inflammatory response. It is evident that maintenance of liver circulation after hemorrhagic shock is critical in limiting liver injury (49, 57). The maintenance of liver circulation relies partly on endothelin-1 and nitric oxide (NO) (2, 27, 37, 43, 58). However, an increased inducible NO synthase (iNOS) in hemorrhaged liver has been reported (8, 11, 23, 43, 47, 48). Treatment of animals with iNOS inhibitors and experiments in iNOS gene knockout mice have shown that iNOS derived NO participates in liver injury and in the inflammatory cascade produced by hemorrhage (15, 20, 30, 31).

Androstenediol (AED) is one of the metabolites of the most abundant steroid hormone in plasma dehydroepiandrosterone and is an intermediate in the pathway for the synthesis of testosterone and estrogen. AED has been reported to have protective effects against ionizing radiation-induced injury in mice (28, 55), lethal bacterial infections, and endotoxic shock (35). AED has also been shown to preserve cardiovascular function (42) and hepatic function (44) after trauma plus hemorrhage (T-H), to reduce injury from behavioral stress (14), to promote wound repair (14), and to attenuate inflammation caused by viral infection (5). AED treatment also inhibits hemorrhage-induced decreases in portal blood flow, bile production, and serum albumin levels, and increases portal pressure, hepatic nitrate/nitrite, iNOS, and endothelin-1 levels (43). Although it has been suggested that the decrease in endothelin-1 and the induction of endothelial NO synthase improve hepatic perfusion, downregulation of iNOS decreases liver damage. The underlying mechanism of protective effects of AED has not been elucidated, however.

It is known that many caspases are involved in the intrinsic pathway of apoptosis in which caspase-3 plays a key role in triggering the occurrence of apoptosis (17, 18, 24). Caspase-3 is an aspartate-specific cystenyl protease. It is evident that hemorrhage increases caspase-3 cellular activity (22, 23) and decreases cellular ATP levels in various tissues (6, 7, 20, 23, 38, 50). Agents that increase inducible heat shock protein 70 kDa and inhibit iNOS have been shown to ameliorate the hemorrhage-induced cell injury (19–21). In this study, we examined the mechanisms whereby AED confers liver protection in animals subjected to soft tissue trauma followed by hemorrhage (T-H). We report that AED limits the T-H-induced increases in liver caspase-3 and apoptosome formation by limiting the expression of iNOS at the transcriptional level. AED can potentially be a useful adjuvant for inhibiting liver apoptosis following T-H.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. The animal protocol was reviewed and approved by Institutional Animal Care and Use Committee at University of Alabama.

Experimental protocols.   A nonheparinized rat model of soft tissue T-H was used in this study (49). Male Sprague-Dawley rats [body weight (BW): 275–325 g; Charles River Laboratories, Wilmington, MA] were fasted overnight before the experiment, but allowed water ad libitum. The rats were anesthetized using isoflurane (Attane Minrad, Bethlehem, PA) inhalation. A 5-cm midline laparotomy was then performed to induce soft tissue trauma. Following this, the abdomen was closed in layers, and both femoral arteries and the right femoral vein were cannulated with PE-50 tubing (Becton-Dickinson, Sparks, MD). The animals were then restrained in a supine position, and the area of incision was bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ) to minimize postoperative pain. The rats were then allowed to awaken and were rapidly bled to a mean arterial pressure (MAP) of 35–40 mmHg within 10 min. The time at which the animals could no longer maintain a MAP of 35–40 mmHg without infusing some fluid was defined as maximum bleed-out volume. The rats were maintained at this MAP until 40% of the shed blood was returned in the form of Ringer lactate (LR). The animals were then resuscitated with four times the volume of shed blood with LR over 60 min. Following resuscitation, the catheters were removed, the vessels were ligated, and skin incisions were closed with sutures. Sham-operated animals underwent the same groin dissection, which included the ligation of the femoral arteries and cannulation of the femoral vein, but the animals were neither subjected to T-H nor resuscitated. The animals were returned to their cages and were allowed food and water ad libitum and were killed 24 h after the end of resuscitation. All rats subjected to this protocol survived until euthanization.

In the treatment group, AED (1 mg/kg BW; Steraloids, Newport, RI) was administered intravenously immediately at the end of the resuscitation. In the vehicle-treated group (control group), rats received the same volume of vehicle (Intralipid, 1 ml/kg BW; Sigma, St. Louis, MO). A small portion of liver was removed from the treated rats and frozen immediately at –70°C until used for immunoblotting and biochemical assays.

Tissues were first minced and then sonicated for 15 s and then centrifuged at 10,000 g for 10 min. The supernatant was saved for determining the total amount of protein in each lysate sample and performing immunoblotting analysis. The cellular caspase-3 activity was measured. Proteins and moieties were assessed by immunoblotting.

Cell culture.   Human intestinal epithelial T84 cells, Hep60 cells, FHs74 Int cells, and CRL-1550 cells (American Type Cell Culture, Rockville, MD) were grown in DMEM, hybrid-care medium, and RPMI-1640, respectively, in a humidified incubator with a 5% CO2 atmosphere. Each medium was supplemented with 2 mM glutamine, 4.5 g/l glucose, and 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, pH 7.4 (Quality Biological, Gaithersburg, MD). Cells were fed every 3–4 days.

iNOS gene construct.   iNOS gene was used in this study. cDNA of human iNOS was obtained from Dr. N. Tony Eissa (Baylor College of Medicine, Houston, TX). A 3,362-bp coding sequence with Hind III and Xho I restriction sites at each terminals of human iNOS gene was subcloned from this full length of human iNOS cDNA. With the use of PCR application, the forward and reverse primers were 5'-CT AAG CTT GTC ATG GCC TGT CCT TGG AAA TTT CTG TTC-3' and 5'-GAC TCG AGC TCA GAG CGC TGA CAT CTC CAG GCT-3', respectively. After digestion and purification of the PCR amplification product, the expression cDNA sequence was used for insertion into the vector. The vector used in this study was pcDNA3 (Invitrogen, San Diego, CA). The expression cDNA of iNOS gene was inserted between the Hind III and Xho I sites of pcDNA 3.1 vector. The expression construct then was sequenced to confirm its correct sequence and open reading frame (20).

Transient gene transfection.   Cultured cells (1.5 x 106) were grown in six-well plates. Cells in each well were added with 4-µg iNOS expression plasmid in 0.5-ml antibiotic-free growth medium using Lipofectamine 2000 transfection kit (Invitrogen). The equal amount of cells was also mixed with blank pcDNA 3.1 vector as a control group. Then 1.5-ml DMEM was added to each well, and the cells were placed back into the incubator for 24 h to allow the transient transfection.

iNOS small interfering RNA transfection.   To decrease iNOS protein, RNA interference technology was used. Two designed pairs of oligoduplexes targeted against iNOS were purchased from Qiagen (Valencia, CA). The target sequences of those oligoduplexes are NOS-S sense strand, 5'-ACAACAGGAACCUACCAGCTT-3', and NOS-AS antisense strand, 5'-GCUGGUAGGUUCCUGUUGUTT-3', respectively. A nonspecific oligoduplex (nonsilencing control, targeting on AAU UCU CCG AAC GUG UCA CGU) at the same final concentrations as the iNOS RNA duplexes was used as a negative control. To maximize small interfering RNA (siRNA) silencing potential, the siRNAs were heated for 1 min at 90°C, followed by 60 min at 37°C before the siRNA transfection. The interested cells were grown in fresh medium without antibiotics 24 h before the transfection. Then these cells were transferred to each well in 6-well plates (density: 2 x 106 cells/well). The transient transfection with siRNA duplexes at 100 nM was performed using lipofectamine reagent (Invitrogen), according to the manufacturer's protocol. Twenty-four hours after transfection, the cells were exposed to either vehicle or 10 mM NaCN for 1 h for hypoxia to occur and then recovered for another 23 h. These cells were harvested and assayed.

Immunoprecipitation.   Tissue lysates containing 300 µg protein were incubated with the specified antibody (5 µl), chilled on ice for 1 h, mixed with protein A/G agarose beads (50 µl; Santa Cruz Biotechnology, Santa Cruz, CA), and incubated overnight on a nutator at 4°C. The immunoprecipitate was collected by centrifugation at 12,500 g for 10 min and washed twice with 500 µl of stop buffer and once with 500-µl PBS wash buffer. The pellet was resuspended in the 50 µl of electrophoresis sample buffer without 2-mercaptoethanol, boiled for 5 min, then centrifuged for 30 s to remove the agarose beads. The supernatant was incubated with 5% 2-mercaptoethanol at 37°C for 1 h. Twenty-five microliters of sample were loaded onto precast 10% Tris-glycine polyacrylamide gels for Western blots.

Western blots.   Samples were resolved on SDS-polyacrylamide slab gels (precast 10% gel, Invitrogen). Protein was blotted onto a nitrocellulose membrane (type NC, 0.45 µm, Schleicher and Schuell) using a Novex blotting apparatus and the manufacturer's protocol. The nitrocellulose membrane was blocked by incubation for 90 min at room temperature in PBS containing 5% nonfat dried milk. The blot was then incubated for 60 min at room temperature with the selected antibody against actin, Kruppel-like factor (KLF)-4, KLF-6, NF{kappa}B-p65, NF{kappa}B-p50, iNOS, p53, Bcl-2 (Santa Cruz Biotechnology), inactive caspase-3, active caspase-3, caspase-9 (Epitomics, Belmont, CA), and cytochrome c (Upstate, Lake Placid, NY), at 1 µg/ml in PBS-5% BSA. The blot was washed three times (10 min each) in Tris-buffered saline-0.1% Tween 20 before incubating for 60 min at room temperature with a x1,000 dilution of species-specific IgG peroxidase conjugate (Santa Cruz Biotechnology) in PBS-1% gelatin. The blot was washed six times (5 min each) in Tris-buffered saline-0.1% Tween 20 before detection of the peroxidase activity using the Enhanced Chemiluminescence kit (Amersham Life Science Products, Arlington Heights, IL).

Measurements of caspase-3 activity.   Caspase-3 activity was determined using the CASPASE-3 Cellular Activity Assay Kit PLUS (Biomol, Plymouth Meeting, PA). Change in absorbance was measured at 405 nm with a SpectraMax 250 spectrophotometric plate reader and SOFTmax Pro 3.1.1 software (Molecular Devices, San Diego, CA). Data were normalized to total protein, and caspase-3 activity was expressed as picomoles pNitroaniline per minute per microgram protein.

Statistical analysis.   All data are expressed as means ± SE. One-way ANOVA, {chi}2 test, and Student's t-test were used for comparison of groups with 5% as a significant level.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
T-H induces dysfunction in liver.   Our laboratory has found that hemorrhage alone does not lower liver biochemical parameters, such as cellular ATP and caspase-3 enzymatic activity (20, 22). Addition of trauma to hemorrhage induces a significant ATP loss by 88 ± 2% and a marked elevation of caspase-3 by threefold (Fig. 1). Therefore, T-H becomes a useful model for studying liver under severe assaults (43). In Chaudry's laboratory, T-H has also been shown to increase portal pressure, alanine transferase, and NO production; AED treatment effectively limits these changes (43). Both groups of rats treated with T-H alone and with T-H plus AED all survived 24 h after resuscitation. Liver tissues were analyzed to investigate the effect of T-H on caspase-3 and its relationship to iNOS.


Figure 1
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  		Fig. 1. Androstenediol (AED) decreases trauma-hemorrhage (T-H)-induced increases in caspase-3. Caspase-3 activity in lysates of liver of sham-operated (sham, n = 4), T-H-treated (n = 4), AED-treated (n = 4), and T-H plus AED-treated (T-H+AED, n = 4) rats was measured. AED decreased T-H-induced increase in caspase-3 activity. *P < 0.05 vs. sham-operated group, AED group, and T-H+AED group; determined by {chi}2 test. pNA, pNitroaniline.

 
AED decreases T-H-induced increases in cellular caspase-3 enzymatic activity.   Hypoxia has been shown to alter cellular caspase-3 enzymatic activity in human cultured cells (25), rat ileum and lung (23), and mouse jejunum, lung, heart, kidney, and brain (21), and it is considered to be a reliable biomarker of apoptosis. We decided to determine whether T-H altered caspase-3 enzymatic activity in rats subjected to T-H. T-H increased cellular caspase-3 enzymatic activity in liver tissue by 332 ± 21% (P < 0.05 vs. sham controls). When rats subjected to T-H were treated with AED, the caspase-3 enzymatic activity returned to basal levels in the liver. AED alone did not alter the basal enzymatic activity of caspase-3 protein (Fig. 1). Therefore, treatment with AED eliminated the T-H-induced increase in cellular caspase-3 enzymatic activity.

AED treatment attenuates T-H-induced increases in caspase-3 expression in the absence of Akt phosphorylation.   To determine whether the T-H-induced increase in cellular caspase-3 activity was due to the increased caspase-3 protein, we measured caspase-3 levels in liver tissue lysates of animals subjected to T-H and sham-operated animals. Furthermore, to determine whether the T-H-induced increase in caspase-3 activity involved preferential increases in the inactive or active form of caspase-3, we blotted electrophoretically separated liver lysates with antibodies against the 35-kDa (inactive) and 17-kDa (active) forms of caspase-3. Both the inactive and active forms of caspase-3 were increased in the livers of T-H-treated animals (Fig. 2). The levels of inactive and active forms of caspase-3 were increased by 216 ± 5 and 246 ± 4%, respectively, in the livers of T-H-treated animals compared with sham-handled animals (P < 0.05 vs. sham controls). Treatment with AED alone reduced caspase-3 levels by only 50 ± 12 and 25 ± 3%, respectively (P < 0.05 vs. sham controls). Similarly, treatment with T-H and AED also diminished caspase-3 levels by 50 ± 2 and 34 ± 18%, respectively (P < 0.05 vs. sham controls; Fig. 2, B and C).


Figure 2
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  		Fig. 2. AED decreases T-H-induced increases in active and inactive forms of caspase-3. Using immunoblotting analysis, we measured caspase-3 levels, phosphorylation of Akt, and actin abundance in lysates of liver of sham-operated (n = 4), T-H-treated (n = 4), AED-treated (n = 4), and T-H+AED-treated (n = 4) rats. Their specific bands of proteins were quantitated densitometrically and normalized against actin. A: representative Western blots are presented. B: AED decreased T-H-induced increase in inactive caspase-3 protein. C: AED decreased T-H-induced increase in active caspase-3 protein. *P < 0.05 vs. sham-operated group, AED group, and T-H+AED group; **P < 0.05 vs. sham-operated group and T-H group, determined by {chi}2. STD, standard.

 
Because increased caspase-3 activity has been attributed to the phosphatidylinositol 3-kinase/Akt pathway (17), we determined Akt phosphorylation at Ser473 and Thr308 by immunoblotting. We failed to detect Akt phosphorylation at serine or threonine residues, which indicates that a pathway other than phosphatidylinositol 3-kinase/Akt is responsible for the increase in caspase-3 (Fig. 2A).

AED inhibits the T-H-induced increases in iNOS.   It has been established that hemorrhage upregulates the expression of iNOS in mouse jejunum, lung, heart, kidney, and brain (21), and rat ileum and lung (23). Because inhibition of iNOS results in decreases in caspase-3 activity in human intestinal cells and Jurkat T cells (25), we asked whether AED could reduce the T-H-induced altered expression of iNOS. Indeed, T-H increased iNOS in the liver (Fig. 3, lane 2), and AED treatment significantly inhibited this increase (Fig. 3, lane 4). This observation suggests that the effect of AED on the caspase-3 activity may be related to the effect of AED on the expression of iNOS.


Figure 3
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  		Fig. 3. AED inhibits T-H-induced increases in inducible nitric oxide synthase (iNOS) and Kruppel-like factor (KLF)-6 and decrease in KLF-4. Using immunoblotting analysis, proteins of iNOS, KLF-4, KLF-6, and actin were assessed in lysates of liver of sham-operated (n = 4), T-H-treated (n = 4), AED-treated (n = 4), and T-H+AED-treated (n = 4) rats. Their specific bands of proteins were quantitated densitometrically and normalized with actin protein. A: representative Western blot is presented. B: AED increased KLF-4. *P < 0.05 vs. sham, T-H, and T-H+AED; **P < 0.05 vs. sham, T-H, and AED, determined by {chi}2 test. C: AED decreased iNOS. D: AED decreased KLF-6. *P < 0.05 vs. sham group, AED group, and T-H+AED group; determined by {chi}2 test.

 
It has been known that KLF-4 and KLF-6 are transcriptional factors that control the expression of the iNOS gene. Specifically, KLF-6 upregulates, whereas KLF-4 has been shown to downregulate, iNOS gene expression at the transcriptional level (21, 23, 54). In the rat liver, T-H increased KLF-6 and decreased KLF-4. Treatment with AED decreased KLF-6 and increased KLF-4 (Fig. 3). It appears, therefore, that the effect of AED on iNOS is mediated through effects on transcription factors known to control the expression of iNOS.

AED inhibits T-H-induced increase in NF{kappa}B.   The promoter region of iNOS gene has 10 NF{kappa}B binding sites. Hemorrhage increases NF{kappa}B binding to the iNOS gene, thereby leading to an increased iNOS gene expression (40). We found that T-H increased both p65 and p50 units of NF{kappa}B in the liver tissues (Fig. 4A, lane 2). Treatment with AED vastly decreased both proteins (Fig. 4A, lane 4). Like KLF-4 and KLF-6, it appears that the effect of AED on iNOS gene expression is also mediated by preceding effects on NF{kappa}B (Fig. 4), another transcription factor known to control iNOS gene expression (56).


Figure 4
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  		Fig. 4. AED inhibits T-H-induced increases in NF{kappa}B. Using immunoblotting analysis, proteins of NF{kappa}B and actin were assessed in lysates of liver of sham-operated (n = 4), T-H-treated (n = 4), AED-treated (n = 4), and T-H+AED-treated (n = 4) rats. Their specific bands of proteins were quantitated densitometrically and normalized with actin protein. A: representative Western blot is presented. B: AED decreased NF{kappa}B-p65. C: AED decreased NF{kappa}B-p50. *P < 0.05 vs. sham group, AED group, and T-H+AED group; determined by {chi}2 test. **P < 0.05 vs. sham-operated group and T-H-treated group, determined by {chi}2 test.

 
T-H increases apoptosome formation.   Caspase-3 enzymatic activity has been shown to be activated by complexes of cytochrome c and caspase-9, termed the "apoptosome" (17, 18). Therefore, we used immunoprecipitation and immunoblotting analysis to detect and measure the apoptosome. The complexes were detected in liver lysates immunoprecipitated with anti-caspase-9 antibody first and then immunoblotted with anti-cytochrome c antibody. Lysates from T-H liver displayed an increase in cytochrome c protein (Fig. 5A). To further confirm the presence of the complex formation between caspase-9 and cytochrome c, we immunoprecipitated the lysate with anti-cytochrome c antibody and immunoblotted with anti-caspase-9 antibody. Lysates from T-H liver also displayed an increase in caspase-9 (Fig. 5B), suggesting that T-H results in an increased apoptosome formation, thereby leading to an increased caspase-3 enzymatic activity.


Figure 5
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  		Fig. 5. AED inhibits T-H-induced apoptosome formation. The cellular cytochrome c and caspase-9 were assessed in lysates of liver of sham-operated (n = 4), T-H-treated (n = 4), AED-treated (n = 4), and T-H+AED-treated (n = 4) rats. Using immunoprecipitation (IP) and immunoblotting (IB) analysis, proteins of cytochrome c and caspase-9 were detected. Their specific bands of proteins were quantitated densitometrically. A: representative Western blot of liver caspase-9 complex formation with cytochrome c. Lysates of rat liver were immunoprecipitated with anti-caspase-9 antibody, and cytochrome c was then detected with cytochrome c antibody using immunoblotting analysis. B: representative Western blot of liver cytochrome c complex formation with caspase-9. Lysates of rat liver were immunoprecipitated with anti-cytochrome c antibody, and caspase-9 was then detected with caspase-9 antibody using immunoblotting analysis. *P < 0.05 vs. sham-operated group, AED-treated group, and T-H+AED-treated group; determined by {chi}2 test.

 
AED inhibits T-H-induced increase in apoptosome formation.   Treatment with AED at the end of the resuscitation period inhibited the increase in apoptosome formation in liver of T-H-treated rats (Fig. 5). Liver lysates immunoprecipitated with anti-caspase-9 antibody and immunoblotted with anti-cytochrome c antibody exhibited a significantly low level of cytochrome c protein (Fig. 5A). Similarly, lysates immunoprecipitated with anti-cytochrome c antibody and immunoblotted with anti-caspase-9 antibody also exhibited a low level of caspase-9 protein (Fig. 5B).

AED fails to inhibit T-H-induced decreases in p53 and Bcl-2.   The p53 is a proapoptosis protein, and Bcl-2 is an antiapoptosis protein. Hemorrhage alone for 1 h has been shown to increase mRNA of p53 and Bcl-2 in rat lung and Bcl-2 in ileum (23). We wanted to determine whether p53 and Bcl-2 were altered 24 h after T-H treatment. Figure 6 shows that T-H significantly decreased p53 and Bcl-2, whereas the AED treatment failed to correct the observed decreases, suggesting that the AED inhibitory action is rather specific to the iNOS pathway.


Figure 6
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  		Fig. 6. AED fails to block T-H-induced decreases in p53 and Bcl-2. Using immunoblotting analysis, proteins of p53, Bcl-2, and actin were assessed in lysates of liver of sham-operated (n = 4), T-H-treated (n = 4), AED-treated (, n = 4), and T-H+AED-treated (n = 4) rats. Their specific bands of proteins were quantitated densitometrically and normalized with actin protein. A: representative Western blot is presented. B: AED did not block the T-H-induced decrease in p53. C: AED did not block the T-H-induced decrease in Bcl-2. *P < 0.05 vs. sham group and AED group; determined by {chi}2 test. **P < 0.05 vs. sham-operated group, T-H-treated group, and T-H+AED-treated group, determined by {chi}2 test.

 
iNOS protein regulates the level of caspase-3 activity.   To verify the correlation between the protein level of iNOS and the caspase-3 activity, cells were transiently transfected with the iNOS gene. Western blotting shows increased iNOS protein after transfection for 24 h (Fig. 7A, lane 3). No iNOS protein was detected in control cells (Fig. 7A, lane 1) or vector-transfected cells (Fig. 7A, lane 2). The caspase-3 activity in iNOS gene-transfected cells increased by twofold (P < 0.05 vs. control and vector transfected).


Figure 7
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  		Fig. 7. Correlation between the iNOS level and the caspase-3 activity. Human CRL1550 intestinal cells were transfected with iNOS gene or treated with iNOS small interfering RNA (siRNA). A: iNOS gene transfection increased iNOS protein and caspase-3 enzymatic activity (n = 3). *P < 0.05 vs. control (CON) and vector-treated groups, determined by one-way ANOVA. B: treatment with iNOS siRNA decreased iNOS and caspase-3 enzymatic activity. Cells were treated with nonsilencing oligo or iNOS siRNA for 24 h, then exposed to 10 mM NaCN for 1 h to allow hypoxia to occur, and recovered for 23 h to allow iNOS upregulation (n = 3). *P < 0.05 vs. CON group, siRNA-treated group, and siRNA+hypoxia (HX)-treated group, determined by {chi}2.

 
To further confirm this correlation, RNA interference technology was adopted. Cells were treated with iNOS siRNA 24 h before hypoxia. The hypoxic cells in the absence of iNOS siRNA displayed increases in iNOS (Fig. 7B, lane 2) and caspase-3 enzymatic activity (Fig. 7B). Treatment with iNOS siRNA before hypoxia prevented these increases (Fig. 7B), suggesting that iNOS regulates caspase-3.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLAIMER
 REFERENCES
 
Hemorrhagic shock invariably causes delayed pathology, including multiple organ dysfunction (3, 19). Pathology results from distinct molecular processes, and it is believed that reversal or prevention of the molecular abnormalities should result in tissue and organ protection. Accordingly, in this study, we sought to investigate the molecular events that underwrite the known ability of AED (a metabolite of the most abundant steroid hormone in plasma, dehydroepiandrosterone) to protect liver damage in animals subjected to T-H.

We report herein that T-H causes significant increases in cellular caspase-3 activity and protein in the liver of rats subjected to T-H. Caspase-3 is an aspartate-specific cysteinyl protease that plays a key role in cell apoptosis (17, 18). It has been shown that caspase-3 activity is increased by hypoxia (12, 25), ischemia (10, 36, 53), hemorrhage (9, 22, 26, 29, 32, 33, 60), and in other pathological conditions (1, 33, 34, 39, 46, 52). Interventions that lead to inhibition of caspase-3 activity (60), such as treadmill exercise (26), hypertonic saline resuscitation (33), or LR resuscitation (23), have been shown to reduce tissue apoptosis and brain damage in hemorrhaged animals. It is also evident that treatment of cells (25) or animals (21) with iNOS inhibitors results in decreased cellular caspase-3 activity. We found that AED treatment limits the expression of iNOS, a finding that implies that iNOS expression regulates the expression of caspase-3 through undetermined pathways. This conclusion is corroborated by previous findings (25) in human intestinal epithelial and T cells, which demonstrated that pharmaceutical manipulation of iNOS results in changes in caspase-3 expression. The fact that AED limits the increased expression caused by T-H of two iNOS transcriptional enhancers, KLF-6 and NF{kappa}B (p65 and p50 components), and prevents the decrease of the suppressor KLF-4, strongly suggests that AED controls the expression of iNOS by regulating the expression of known regulators of the iNOS gene transcription. NF{kappa}B activity has been reported increased also in septic (4) and hemorrhagic shock (40, 41). It is presumed that the affected transcription factor promoter regions define androgen response elements through which the transcription rate is affected. It should be noted that AED has been shown to downregulate the T-H-induced increase in iNOS in rat heart (45) and rat liver (44) via peroxisome proliferator-activated receptor-{gamma} activation. In addition, it has also been reported that AED promotes wound repair by inhibiting monocyte chemoattractant protein (MCP)-1 and platelet-derived growth factor (14) and that AED reduces the viral infection-induced inflammation by inhibiting MCP-1 (5). It is not clear yet whether iNOS relates to peroxisome proliferator-activated receptor-{gamma}, MCP-1, or platelet-derived growth factor. If iNOS in fact could regulate their activities, then the idea that AED inhibits them via inhibiting iNOS becomes plausible and is worthwhile to explore further.

Besides 17beta-estradiol, dehydrorpiandrosterone, and AED, flutamide (an androgen receptor antagonist) has been reported to confer cytoprotection from T-H-induced organ injury by upregulating estrogen receptors (16, 59). Therefore, the possibility of AED cytoprotecting the liver from T-H being mediated by altering estrogen receptors cannot be ruled out. It is of interest to explore the effect of AED on estrogen receptors.

Apoptosis is thought to be triggered through extrinsic pathway, intrinsic pathway, or caspase-independent-mediated pathways (17, 18, 24). The intrinsic pathway includes the formation of apoptosome (consisting of Apaf-1, cytochrome c, and caspase-9) that activates caspases-3 and -7. Then caspase-3 activates other caspases. The increased apoptosome formation after T-H was observed (Fig. 5). The increase, therefore, led to the increased caspase-3 enzymatic activity and protein. The view of iNOS protein regulating caspase-3 activity is further supported by data obtained from experiments with iNOS gene transfection and siRNA treatments (Fig. 7). Our preliminary data using confocal microscopy have also shown that iNOS overexpression by iNOS gene transfection significantly increases complex formation between caspase-9 and cytochrome c. This complex is reduced in cells treated with iNOS siRNA (Kiang JG, Bowman PD, Lu X, Li Y, Wu BW, Loh HH, Tsen KT, Tsokos GC, unpublished data). Based on our data from experiments using immunoprecipitation and immunoblotting (Fig. 5) and iNOS gene transfection/siRNA technology (Fig. 7), iNOS triggers the apoptosis cascade as well as organ injury.

The observation of the decreased p53 and Bcl-2 in liver resulted from T-H is not in agreement with that reported in jejunum and lung of rats exposed to hemorrhage (23) and cultured human cells exposed to NaCN-induced hypoxia (25). The discrepancy can be due to the differences of insults, the time to collect tissues after the insults, and types of tissues studied. Since AED failed to correct the decreased p53 and Bcl-2 in liver, it appears that the inhibitory action of AED on the iNOS pathway is rather specific.

A schematic representation of the finding of this study is shown in Fig. 8. T-H increases KLF-6 and NF{kappa}B and inhibits KLF-4, resulting in an increased expression of iNOS. This increase in iNOS leads to increase NO production (43) that reacts with O2– to form ONOO–. Peroxynitrite leads to mitochondrial swelling and release of cytochrome c that subsequently is caged by caspase-9 to form the apoptosome. The apoptosome activates caspase-3 (18), leading to apoptosis. AED treatment inhibits KLF-6 and NF{kappa}B and increases KLF-4. Subsequently, iNOS expression is reduced followed by reduction of caspase-3 activity and apoptosis.


Figure 8
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  		Fig. 8. Schematic representation depicting the T-H-induced molecular abnormalities and the proposed sites of AED action. T-H increases KLF-6 and NF{kappa}B and decreases KLF-4, resulting in an increased expression of iNOS. This increase in iNOS leads to increase NO production (35) that reacts with O2– to form ONOO–. Then peroxynitrite makes mitochondria swell up and release cytochrome c that subsequently is caged by caspase-9 to form apoptosome. The apoptosome therefore activates caspase-3 (18), leading to apoptosis. AED treatment inhibits KLF-6 and NF{kappa}B and increases KLF-4. Subsequently, iNOS expression is reduced followed by reduction in caspase-3 and apoptosis. {uparrow}, Increase; {downarrow}, decrease.

 
In summary, AED, a metabolite of the most abundant steroid hormone in plasma dehydroepiandrosterone, protects rat liver from apoptosis caused by T-H. The protective effect of AED is mediated by its ability to regulate the transcription factors of iNOS and subsequently to inhibit apoptosome formation and cellular caspase-3 activity. We conclude that the liver protective effect of AED can potentially be a useful adjuvant for inhibiting liver apoptosis after T-H shock.


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The project was supported by Department of Defense Research Area Directorate II Science and Technology Objective C.


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The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the US Department of the Army, the Uniformed Services University of The Health Sciences, US Department of Defense, or University of Alabama at Birmingham.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. G. Kiang, Armed Forces Radiobiology Research Institute, Bldg. 42, Room 2423 8901 Wisconsin Ave., Bethesda, MD 20889-5603 (e-mail: kiang{at}afrri.usuhs.mil)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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