Published online before print June 22, 2007
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Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA
2 Correspondence: Center for Surgical Research, University of Alabama at Birmingham, 1670 University Boulevard, Volker Hall, Room G094, Birmingham, AL 35294-0019, USA. E-mail: irshad.chaudry{at}ccc.uab.edu
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300 g body weight) underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min), followed by resuscitation. E2 conjugated to BSA (E2-BSA; 1 mg/Kg E2), with or without an estrogen receptor antagonist (ICI 182,780), a PI-3K inhibitor (Wortmannin), or vehicle, was injected i.v. during resuscitation. At 2 h after trauma-hemorrhage or sham operation, intestinal myeloperoxidase (MPO) activity, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and IL-6 levels were measured (n=6 rats/group). Intestinal PI-3K, phosphorylation of Akt (p-Akt), and Akt protein expressions were also determined. One-way ANOVA and Tukey’s test were used for statistical analysis. The results indicated that trauma-hemorrhage increased intestinal MPO activity and ICAM-1, CINC-1, CINC-3, and IL-6 levels. These parameters were improved significantly in the E2- or E2-BSA-treated rats subjected to trauma-hemorrhage. Although trauma-hemorrhage decreased intestinal PI-3K and p-Akt protein expressions, E2 or E2-BSA treatment following trauma-hemorrhage prevented such decreases in intestinal PI-3K and p-Akt protein expressions. Coadministration of ICI 182,780 or Wortmannin abolished the beneficial effects of E2-BSA on attenuation of intestinal injury following trauma-hemorrhage. Thus, the PI-3K/Akt pathway plays a critical role in mediating the nongenomic, salutary effects of E2 on attenuation of shock-induced intestinal tissue damage.
Key Words: shock • hormones • receptors • ICAM-1 • CINC-1 • CINC-3
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Estrogen has been shown to prevent neutrophil accumulation in the gut in a model of inflammatory bowel disease, and this effect is mediated via the estrogen receptor (ER) [8 ]. The classic ER functions as a ligand-dependent transcription factor. However, new information indicates signal transduction via ERs, which does not involve transcriptional gene regulation [9 ]. The nongenomic effects of estrogen are believed to be mediated by receptors located in or close to the plasma membrane [10 , 11 ]. To dissociate the influence of genomic events mediated by the nuclear ERs, investigators often use membrane-impermeable conjugates of 17ß-estradiol (E2), such as E2 conjugated to BSA (E2-BSA) [12 ].
Genomic action of estrogen is mediated by nuclear ERs and involves transcription; however, nongenomic action of estrogen is mediated by membrane ERs and does not require transcription [13 , 14 ]. Nongenomic activation of the PI-3K/Akt signaling cascade by E2 has been observed in multiple cell types [15 ]. PI-3Ks constitute a family of evolutionarily conserved lipid kinases, which regulate a vast array of fundamental cellular responses, including proliferation, protection from apoptosis, superoxide production, cell migration, and adhesion [16 17 18 19 ]. These responses result from the activation of membrane-trafficking protein such as the serine/threonine (kinase) Akt [20 ]. We therefore hypothesized that the PI-3K/Akt pathway is involved in mediating the nongenomic, protective effects of E2 on intestine following trauma-hemorrhage. The aim of our study, therefore, was to determine whether the nongenomic actions of E2 have any beneficial effect on small intestine following trauma-hemorrhage and whether those effects are mediated via the PI-3K/Akt pathway.
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Preparation of intestinal samples
Immediately after anesthetizing the rats, the intestine was exposed. After approximately the first 15-cm-long proximal segment of intestine, a 3-cm-long piece of jejunum was removed and flushed gently with saline and snap-frozen in liquid nitrogen.
Measurement of myeloperoxidase (MPO) activity
MPO activity in homogenates of whole intestine was determined as described previously [1
, 2
]. All reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Briefly, equal weights (100 mg wet weight) of intestine 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 2000 g at 4°C, and the supernatants were stored at –80°C. Protein content in the samples was determined using the Bio-Rad (Hercules, CA, USA) assay kit. The samples were incubated with a substrate o-dianisidine hydrochloride. This reaction was carried out in a 96-well plate by adding 290 µl 50 mM phosphate buffer, 3 µl substrate solution (containing 20 mg/ml o-dianisidine hydrochloride), and 3 µl H2O2 (20 mM). Sample (10 µl) 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 µl sodium azide (30%). The plates were read for light absorbance at 460 nm. MPO activity was determined by using the curve obtained from the standard MPO.
Measurement of IL-6 levels
Intestinal IL-6 levels were measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
Determination of CINC-1, CINC-3, and ICAM-1 levels
Intestinal CINC-1, CINC-3, and ICAM-1 levels were determined using ELISA kits (R&D Systems), according to the manufacturer’s instructions. Briefly, the samples were homogenized in PBS (1:10 weight:vol; pH 7.4) containing protease inhibitors (Complete Protease Inhibitor Cocktail, Boehringer Mannheim, Germany). The homogenates were centrifuged at 2000 g for 20 min at 4°C, and the supernatant was assayed for CINC-1, CINC-3, and ICAM-1 levels. An aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC protein assay, Bio-Rad).
Western blot assay
Rat intestine tissues were homogenized in a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.5 mM PMSF, 1 mM sodium vanadate, 1% Triton X-100, 0.5% Nonidet P-40, and 1 µg/mL aprotinin. The homogenates were centrifuged at 2000 g for 15 min at 4°C. An aliquot of the supernatant was used to determine protein concentration (Bio-Rad DC protein assay). Protein aliquots were mixed with 4x sample buffer and were electrophoresed on 4–12% SDS-polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and transferred electrophoretically onto nitrocellulose transfer membranes (Invitrogen), which were then incubated with antibodies to total Akt protein, phospho (p)-Akt (Ser473; Cell Signaling Technology, Inc., Beverly, MA, USA), or antibodies to total PI-3K protein (p85; Upstate Biotechnology, Inc., Lake Placid, NY, USA) overnight at 4°C. The membranes were later incubated with HRP-conjugated goat anti-rabbit antibody or goat anti-mouse antibody for 1.5 h at room temperature. After washing, blots were probed using ECL (Amersham, Piscataway, NJ, USA) and then exposed to film. Signals were quantified using Cheminogen 5500 imaging software (Alpha Innotech Corp., San Leandro, CA, USA).
Statistical analysis
Results are presented as mean ± SEM (n=6 rats/group). The data were analyzed using one-way ANOVA and Tukey’s test, and differences were considered significant at a P value of 
0.05.
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Figure 1. MPO activity in intestinal tissue in rats after sham operation (Sham) or trauma-hemorrhage and resuscitation and treated with BSA (B), E2-BSA (E2B), E2, or a combination of E2-BSA and ICI 182,780 (E2B+I). Data are mean ± SEM of six rats in each group. *, P < 0.05, versus Sham + BSA; #, P < 0.05, versus trauma-hemorrhage + BSA and trauma-hemorrhage + E2-BSA + ICI 182,780.
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Figure 2. Intestinal tissue PI-3K protein expression (A), p-Akt, and protein expressions (Akt) (B, C) from shams receiving BSA (Sham+B) or E2-BSA (Sham+E2B) and trauma-hemorrhage receiving BSA (trauma-hemorrhage+B), E2-BSA (trauma-hemorrhage+E2B), E2-BSA and ICI 182,780 (trauma-hemorrhage+E2B+I), E2 (trauma-hemorrhage+E2), E2-BSA and Wortmannin (trauma-hemorrhage+E2B+W), or Wortmannin (trauma-hemorrhage+W). For equal protein loading, membranes were reprobed for ß-actin using mouse anti-ß-actin mAb. The bands were analyzed using densitometry, and the values are presented as mean ± SEM from five rats in each group. *, P < 0.05, versus all other groups.
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As E2-BSA up-regulated intestinal PI-3K following trauma-hemorrhage (Fig. 2A) , we examined if administration of the PI-3K inhibitor Wortmannin had any effect on Akt activity. The results of these experiments indicate that coadministration of Wortmannin prevented the E2-BSA-induced restoration of intestinal p-Akt following trauma-hemorrhage (Fig. 2C) .
Effect of Wortmannin on small intestinal MPO activity
To determine the role of the PI-3K/Akt pathway in a E2-BSA-induced decrease in intestinal MPO activity following trauma-hemorrhage, rats were treated with the PI-3K inhibitor Wortmannin along with E2-BSA. There was no significant difference in intestinal MPO activity in shams receiving Wortmannin compared with vehicle-treated shams (data not shown). Furthermore, the results indicated that coadministration of Wortmannin with E2-BSA prevented the E2-BSA-induced attenuation of intestinal MPO activity in trauma-hemorrhage rats (Fig. 3
).
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Figure 3. MPO levels in intestinal tissue in rats after sham operation (Sham) or trauma-hemorrhage and resuscitation receiving BSA, E2-BSA, E2-BSA and Wortmannin, or Wortmannin. Data are mean ± SEM of six rats in each group. *, P < 0.05, versus Sham + BSA; #, P < 0.05, versus trauma-hemorrhage + BSA, trauma-hemorrhage + E2-BSA + Wortmannin, and trauma-hemorrhage + Wortmannin.
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Figure 4. Intestinal IL-6 levels in animals after sham operation or trauma-hemorrhage and resuscitation and receiving BSA, E2-BSA, E2-BSA and Wortmannin, or Wortmannin. Data are mean ± SEM of six rats in each group. *, P < 0.05, versus Sham + BSA; #, P < 0.05, versus trauma-hemorrhage, trauma-hemorrhage + BSA, trauma-hemorrhage + E2-BSA + Wortmannin, and trauma-hemorrhage + Wortmannin.
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Figure 5. Intestinal CINC-1 (A), CINC-3 (B), and ICAM-1 (C) levels in rats after sham operation or trauma-hemorrhage and resuscitation and receiving BSA (B), E2-BSA, E2-BSA and Wortmannin, or Wortmannin. Data are mean ± SEM of six rats in each group. *, P < 0.05, versus Sham + BSA; #, P < 0.05, versus trauma-hemorrhage + BSA, trauma-hemorrhage + E2-BSA + Wortmannin, and trauma-hemorrhage + Wortmannin.
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The gut is considered a critical organ in the development of the delayed organ dysfunction in patients suffering from traumatic injuries 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 [22
]. Neutrophils are the principal cells involved in host defense against acute bacterial and fungal infections [23
], and thus, these cells have a protective effect. However, under conditions such as those described in this study, the infiltration of these cells can cause tissue damage [24
]. The cytokines IL-1, IL-6, and TNF-
are important early mediators in the intestine [24
, 25
] and are required for expression of adhesion molecules and chemokines [26
]. Furthermore, neutrophil movement and migration are mediated by multiple adhesion molecules on the neutrophils and endothelial cell surfaces and chemotactic factors. Initially, neutrophils interact with endothelial selectins, resulting in neutrophils rolling along the endothelial surface. This rolling process appears to allow neutrophils to become activated ("primed") by chemokines and other mediators secreted by the endothelium, resulting in their firm adhesion to endothelial adhesion molecules [27
]. Among adhesion molecules, ICAM-1 is an important mediator in the firm adhesion of neutrophils to the vascular endothelium and is strongly up-regulated following trauma-hemorrhagic shock [2
]. With regard to chemokines, rat CINC-1 and CINC-3 are members of the IL-8 family and are potent, chemotactic factors for neutrophils [7
]. Chemotaxis of neutrophils is an important functional response to chemokines and is a key event in the recruitment of neutrophils at the site of inflammation. Our previous study has indicated that following trauma-hemorrhage, CINC-1 and CINC-3 levels correlated with tissue MPO activity, a marker of neutrophil content [2
]. The ability of E2-BSA to mediate expression of inflammatory cytokines as well as adhesion molecules and chemokines suggests a role for nongenomic effects of estrogen in the regulation of intestinal inflammation. Although we did not perform histology in this study, our recent findings suggest that MPO activity and histology were altered at 4 h following trauma-hemorrhage [28
]. However, it remains unknown whether the changes in intestinal histology persist at 24 h after trauma-hemorrhage and resuscitation.
The PI-3K/Akt pathway is known to play a pivotal role in the ability of neutrophils to undergo chemotaxis [16 , 17 ]. Furthermore, the involvement of PI-3K in cell migration is supported by the ability of selective PI-3K inhibitors, such as Wortmannin and LY294002, to mitigate neutrophil chemotaxis [18 , 19 ]. There is now considerable evidence demonstrating a role for the PI-3K/Akt pathway in mediating the production of cytokines [29 ], which are important for the expression of chemokines and adhesion molecules [26 ]. Collectively together, PI-3K may reduce neutrophil infiltration through down-regulation of cytokine production. As various cells are known to release IL-6, it is possible that the observed IL-6 release is a result of release by cells other than the inflammatory cells [30 , 31 ]. Mucosal scraping studies or immunohistochemistry was, however, not performed, and this particular aspect, therefore, remains unknown. Additional studies are therefore needed to elucidate more comprehensively the mechanism by which nongenomic effects of estrogen attenuate trauma-hemorrhage-induced, intestinal injury. In the present study, E2 administration per se restored PI-3K activity following trauma-hemorrhage. As E2-BSA also restored PI-3K activity through the nongenomic pathway, it is possible that E2 prevented a trauma-hemorrhage-induced decrease in PI-3K activity through the nongenomic pathway. Additional studies using an inhibitor for the genomic effects of E2 are therefore needed to determine the relative contribution of the genomic and nongenomic effects of E2 on PI-3K activity.
We used a cell-impermeable E2-BSA preparation in this study to limit the actions of estrogen to the cell membrane. It should be noted that although E2 can dissociate from E2-BSA over time, the rate is very low (0.00063%/ml/h) [32
]. Based on this dissociation rate, the amount of free or unbound E2 in our model would be expected to be too low to stimulate even an extremely responsive E2 cell line, i.e., MCF-7 cell line [13
]. Several lines of evidence indicate that E2-BSA acts only on membrane ERs. First, by using a fluorescent form, E2-BSA-FITC (E2-BSA conjugated to FITC), E2-BSA has been shown with confocal [33
] and regular microscopy [34
] to bind only to the plasma membrane, even after a prolonged (40 min) incubation, which far exceeded the exposure time needed for achieving rapid E2 effects [13
]. Second, this membrane binding is specifically a result of E2, as BSA-FITC, devoid of E2, does not bind to the plasma membrane [33
]. Third, E2 binds to membrane ERs, as no binding was observed in cells devoid of ERs; and ER antagonist ICI 182,780 blocked the binding of E2-BSA-FITC to the plasma membrane [34
]. Other studies have also shown that E2-BSA does not activate ER element-dependent transcription, again indicating that the compound remains extracellular [35
]. Thus, it is safe to state that the actions of E2-BSA are mediated on the cell membrane. As such effects occur in the whole animal, it indicates that this type of hormonal potentiation is physiologically meaningful.
Besides the small intestine, the reduction in p-Akt, relative to total Akt in whole heart tissue and cardiomyocytes (data not shown), was also observed. It is therefore tempting to postulate that administration of Wortmannin resulted in in vivo inhibition of PI-3K. Previous studies have shown that Wortmannin is a highly specific inhibitor of PI-3K [36 ]. In addition to Wortmannin, LY294002 was used as a PI-3K inhibitor in previous studies [21 ]. Stimulation of PI-3K requires activation of the 85-kD regulatory subunit, which relies on tyrosine phosphorylation, one consequence of which is activation of the 110-kD catalytic subunit. Wortmannin, as well as LY294002, can block PI-3K activity through direct interaction with the regulatory subunit (85 kD) and the catalytic subunit (110 kD) of PI-3K [36 ]. As Wortmannin has similar effects as LY294002, we used this agent to evaluate the role of PI-3K in this study. Administration of Wortmannin alone following trauma-hemorrhage, however, reduced intestinal p-Akt expression, which suggests the possibility that the effects of coadministration of E2-BSA and Wortmannin may also be a result of the inhibition of one of these parallel pathways. Additional studies are therefore needed to elucidate precisely the mechanism by which E2-BSA attenuates intestinal injury following trauma-hemorrhage.
Understanding the exact mechanism by which E2-BSA induced Akt activation is essential for understanding the nongenomic, protective effects of E2-BSA following trauma-hemorrhage. We believe there might be two possible mechanisms for the activation of Akt by E2-BSA. First, studies have demonstrated that activation of growth factors play an important role in the PI-3K pathway, which in turn, activates Akt. It is therefore possible that E2-BSA treatment could activate certain growth factors [12
]. Second, the E2 receptor has been shown to activate PI-3K activity by association with the p85 regulatory subunit of PI-3K in a ligand-dependent manner [37
]. Despite this information, additional studies are needed to clarify the exact mechanism by which E2-BSA produces its effects on PI-3K/Akt pathway.
It could be argued that the present study used measurements at 2 h after treatment, and thus, it remains unclear whether the salutary effects of E2 or E2-BSA are sustained for longer periods of time. However, our previous studies have shown that if improvement in organ functions by any pharmacological agent is evident at 2 h after treatment, those salutary effects are sustained for prolonged intervals, and those agents also improve the survival of animals [38 ]. Thus, although a time-point other than 2 h was not examined, based on our previous studies, it would appear that the salutary effects of E2 or E2-BSA on the measured parameters in different organs would be evident, even if those effects were measured at another time-point following trauma-hemorrhage and resuscitation. It can also be argued that Wortmannin should have been administered alone in sham groups to determine if each of those per se has any adverse effects. However, as our recent study has shown that administration of Wortmannin alone in sham-operated groups did not produce any deleterious effects (unpublished data), administration of Wortmannin alone was not carried out in this study.
In summary, our results indicate that nongenomic actions of E2 also attenuate intestinal injury following trauma-hemorrhage. Furthermore, we found that up-regulation of the PI-3K/Akt pathway likely plays a significant role in the E2-BSA-mediated attenuation of intestinal injury following trauma-hemorrhage.
Received March 22, 2007; revised May 19, 2007; accepted May 31, 2007.
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and CINC-2ß, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization Biochem. J. 301,545-550[Medline]
in promoting neutrophil trafficking into inflamed tissue Blood 103,3448-3456 and ERß expressed in Chinese hamster ovary cells Mol. Endocrinol. 13,307-319 are substrates, in vitro, for a constitutively associated protein tyrosine kinase in platelets Blood 91,930-939This article has been cited by other articles:
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