Am J Physiol Lung Cell Mol Physiol 290: L1004 –L1009, 2006.
First published December 16, 2005; doi:10.1152/ajplung.00504.2005.
Salutary effects of estrogen receptor-␤ agonist on lung injury
after trauma-hemorrhage
Huang-Ping Yu,1,2 Ya-Ching Hsieh,1 Takao Suzuki,1 Tomoharu Shimizu,1
Mashkoor A. Choudhry,1 Martin G. Schwacha,1 and Irshad H. Chaudry1
1
Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama;
and 2Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan
Submitted 2 December 2005; accepted in final form 15 December 2005
shock; propyl pyrazole triol; diarylpropiolnitrile
HEMORRHAGIC SHOCK RESULTS in the development of acute respiratory distress syndrome and multiple organ dysfunction syndrome in patients sustaining major mechanical trauma (8, 20).
Patients who survive the initial traumatic insult remain susceptible to sepsis, multiple organ failure, septic shock, and death
(4, 26, 36). Cellular dysfunction occurs in many organs, including heart, lung, liver, and gut after hemorrhagic shock, and
these alterations persist for a prolonged period of time despite
fluid resuscitation (12, 28, 32, 33).
Sex hormones are known to modulate immune functions in
animals and in humans under normal and stress conditions
Address for reprint requests and other correspondence: I. H. Chaudry,
Center for Surgical Research, Univ. of Alabama at Birmingham, 1670 University Blvd., Volker Hall, Rm. G094, Birmingham, AL 35294-0019 (e-mail:
[email protected]).
L1004
(11). Studies have shown that proestrus female mice show
normal immune responses; however, male mice have markedly
altered immune responses after trauma-hemorrhage (35). Studies have also demonstrated that male sex steroids appear to be
responsible for producing depression in cell and organ functions after trauma-hemorrhage (34). Additional support for this
notion comes from studies that showed castration of male
animals 14 days before trauma-hemorrhage attenuated the lung
injury observed in noncastrated animals under those conditions
(2). Furthermore, administration of flutamide, a testosterone
receptor antagonist, after trauma-hemorrhage improved the
depressed organ function in male animals (24). These studies
suggest that male and female sex steroids, such as 5␣-dihydrotestosterone and 17␤-estradiol (E2), have an opposite effect
on cell and organ function after injury.
E2 is the predominant circulating sex hormone in females
and has been shown to protect lung after adverse circulatory
conditions such as trauma-hemorrhage (2). Studies have also
shown that these salutary effects of E2 are mediated via estrogen
receptors (ER), which are expressed in various organs (15).
Nitric oxide (NO) is produced as part of the inflammatory
response (27). NO is generated by inducible nitric oxide
synthase (iNOS), which is expressed in many cell types including alveolar macrophages (1, 18). A variety of inflammatory cytokines, including IL-6, induce the expression of iNOS
(10). Furthermore, iNOS expression has been reported in the
lung (31) after several inflammatory conditions such as sepsis
(18) and hemorrhagic shock (30). Thus the above-mentioned
pathophysiological conditions are known to be associated with
an elevated NO production (18, 27, 30). Previous studies have
shown that E2 can decrease iNOS expression and NO production in the lung and protect against lung injury (6, 7). In
addition, it appears that the salutary effects of E2 on attenuation of iNOS expression and NO production in the lung are
receptor dependent. Support for this suggestion comes from a
study that showed administration of E2 with the ER antagonist
ICI 182,780 abolished the salutary effects of E2 in the lung (7).
Although two primary subtypes of ERs (ER-␣ and ER-␤) are
known to exist, it remains unknown which of the two subtypes
is responsible for producing the salutary effects of E2 after
trauma-hemorrhage. Because our recent studies have shown
that the salutary effects of E2 on the myocardium after traumahemorrhage are mediated via the ER-␤ (37), we hypothesized
that ER-␤ is also responsible for mediating the salutary effects
of E2 in the lung after trauma-hemorrhage. The aim of our
The costs of publication of this article were defrayed in part by the payment
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1040-0605/06 $8.00 Copyright © 2006 the American Physiological Society
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Yu, Huang-Ping, Ya-Ching Hsieh, Takao Suzuki, Tomoharu
Shimizu, Mashkoor A. Choudhry, Martin G. Schwacha, and
Irshad H. Chaudry. Salutary effects of estrogen receptor-␤ agonist
on lung injury after trauma-hemorrhage. Am J Physiol Lung Cell Mol
Physiol 290: L1004 –L1009, 2006. First published December 16,
2005; doi:10.1152/ajplung.00504.2005.—Although 17␤-estradiol
(E2) administration after trauma-hemorrhage attenuates lung injury in
male rodents, it is not known whether the salutary effects are mediated
via estrogen receptor (ER)-␣ or ER-␤. We hypothesized that the
salutary effects of E2 lung are mediated via ER-␤. Male SpragueDawley rats underwent trauma-hemorrhage (mean blood pressure 40
mmHg for 90 min, then resuscitation). E2 (50 ␮g/kg), ER-␣ agonist
propyl pyrazole triol (PPT; 5 ␮g/kg), ER-␤ agonist diarylpropiolnitrile (DPN; 5 ␮g/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after trauma-hemorrhage or
sham operation, bronchoalveolar fluid (BALF) was collected for
protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels. Moreover, lung tissue was used for inducible nitric oxide synthase
(iNOS) mRNA/protein expression, nitrate/nitrite and IL-6 levels, and
wet/dry weight ratio (n ⫽ 6 rats/group). One-way ANOVA and
Tukey’s test were used for statistical analysis. The results indicated
that E2 downregulated lung iNOS expression after trauma-hemorrhage. Protein concentration, LDH activity, and nitrate/nitrite and IL-6
levels in BALF and nitrate/nitrite and IL-6 levels in the lung increased
significantly after trauma-hemorrhage; however, administration of
DPN but not PPT significantly improved all parameters. Moreover,
DPN treatment attenuated trauma-hemorrhage-mediated increase in
iNOS mRNA/protein expression in the lung. In contrast, no significant
change in the above parameters was observed with PPT. Thus the
salutary effects of E2 on attenuation of lung injury are mediated via
ER-␤, and ER-␤-induced downregulation of iNOS likely plays a
significant role in the DPN-mediated lung protection after traumahemorrhage.
ESTROGEN RECEPTOR AND LUNG INJURY AFTER TRAUMA-HEMORRHAGE
study, therefore, was to determine which ER is predominantly
responsible for the salutary effects of E2 in attenuation of lung
injury after trauma-hemorrhage and whether these effects are
mediated via modulation of iNOS under those conditions.
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
GACACCCTTCACCACAAG-3⬘ (forward) and 5⬘-TTGAGGCAGAAGCTCCTCCA-3⬘ (reverse). 18S was used for endogenous control. All the samples were amplified for 1 cycle at 50°C for 2 min and
at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 60°C
for 1 min.
Western blot assay. The lungs 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 phenylmethylsulfonyl fluoride, 1 mM
sodium vanadate, 1% Triton X-100, 0.5% Nonidet P-40, and 1 ␮g/ml
aprotinin. The homogenates were centrifuged at 12,000 g for 15 min
at 4°C, and protein aliquots were mixed with 4⫻ sample buffer,
subjected to electrophoresis, and transferred to nitrocellulose membranes. The membranes were incubated with anti-iNOS overnight at
4°C. The membranes were later incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody for 1.5 h at room temperature. The blots were immersed for 5 min in Super Signal West Pico
detection reagent and then exposed to film. Signals were quantified
using Cheminogen 5500 imaging software (Alpha Innotech).
Water content assay. In a separate cohort, left upper lobe of the
lung was weighed and dried for 24 h at 80°C. Water content assay of
lung tissue was calculated as wet/dry weight ratio.
Nitrate/nitrite assay. Levels of nitrite/nitrate in cell-free BALF and
lung tissue was measured using a commercially available colorimetric
assay kit (Cayman Chemical, Ann Arbor, MI).
IL-6 assay. Concentration of IL-6 in cell-free BALF and lung tissue
was determined using ELISA kits (Pharmingen, San Diego, CA)
according to the manufacturer’s instructions.
Protein assay in lung lavage. Cell-free BALF was evaluated for
total protein content (DC Protein Assay, Bio-Rad).
Lactate dehydrogenase activity assay. To evaluate lactate dehydrogenase (LDH) activity in cell-free BALF, a commercially available kit
was used (LDH Optimiert; Roche Diagnostics, Mannheim, Germany).
Statistical analysis. Results are presented as means ⫾ SE. The data
were analyzed using one-way analysis of variance and Tukey’s test,
and differences were considered significant at a P value of ⱕ05.
RESULTS
Effect of ER-␣ agonist PPT, ER-␤ agonist DPN, and E2 on
lung iNOS mRNA and protein expression. Trauma-hemorrhage
induced a significant increase in lung iNOS mRNA expression
compared with shams (Fig. 1A). E2 treatment prevented
trauma-hemorrhage-mediated increase in iNOS mRNA expression in the lung; however, it remained higher than shams.
Furthermore, DPN administration also attenuated the increase
in iNOS mRNA in the lung after trauma-hemorrhage. In
contrast, PPT did not prevent the increase in iNOS mRNA
expression after trauma-hemorrhage. In addition to mRNA
expression, we also examined the effect of PPT, DPN, and E2
on the iNOS protein level. Consistent with mRNA expression,
the results as shown in Fig. 1B indicate that the traumahemorrhage-induced increase in iNOS expression was attenuated in rats treated with E2 and DPN. Administration of PPT,
on the other hand, did not influence iNOS protein expression in
the lung after trauma-hemorrhage.
Effect of ER-␣ agonist PPT, ER-␤ agonist DPN, and E2 on
lung tissue edema. There was a significant increase in wet/dry
weight ratio in the lung tissue obtained from trauma-hemorrhage rats compared with sham-operated rats (Fig. 2), suggesting that trauma-hemorrhage increases water content in the lung
tissue. Administration of E2 and ER-␤ agonist DPN significantly prevented the increase in wet/dry weight ratio after
trauma-hemorrhage. In contrast, treatment of animals with PPT
did not influence this ratio after trauma-hemorrhage.
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Trauma-hemorrhage procedure. Our previously described nonheparinized rat model of trauma-hemorrhage was used in this study
(38). Briefly, male Sprague-Dawley rats (275–325 g; Charles River,
Wilmington, MA) were fasted overnight before the experiment but
were allowed water ad libitum. The rats were anesthetized by isoflurane (Attane; Minrad, 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 (PE-50 tubing; Becton Dickinson,
Sparks, MD). The wounds were bathed with 1% lidocaine (ElkinsSinn, Cherry Hill, NJ) throughout the surgical procedure to reduce
postoperative pain. Rats were then allowed to awaken and bled to a
mean arterial pressure (MAP) of 40 mmHg. This level of hypotension
was continued until the animals were no longer able to maintain MAP
of 40 mmHg unless additional fluid in the form of Ringer lactate (RL)
was administered. This interval was defined as maximum bleed-out
time, and the amount of withdrawn blood was noted. After this, the
rats were maintained at a MAP of 40 mmHg until 40% of the
maximum bleed-out volume was returned in the form of RL. The
animals were then resuscitated with four times the volume of the shed
blood over 60 min with RL. Thirty minutes before the end of the
resuscitation period, the rats received E2 (50 ␮g/kg subcutaneously),
ER-␣ agonist propyl pyrazole triol (PPT; 5 ␮g/kg subcutaneously),
ER-␤ agonist diarylpropiolnitrile (DPN; 5 ␮g/kg subcutaneously), or
an equal volume of the vehicle (⬃0.2 ml, 10% DMSO, Sigma). The
doses of E2, PPT, and DPN used in this study were the same as those
used in our recent study that examined the effect of these agents on
cardioprotection after trauma-hemorrhage and resuscitation (37). The
catheters were then removed, the vessels ligated, and the skin incisions closed with sutures. 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. The
animals were then returned to their cages and were allowed food and
water ad libitum. 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.
This project was approved by the Institutional Animal Care and Use
Committee of the University of Alabama at Birmingham.
Preparation of lung tissue and collection of bronchoalveolar fluid.
At 24 h after the completion of fluid resuscitation or sham operation,
the animals were anesthetized with isoflurane and then killed. The
chest was opened, and the left side of the lung was obtained after
clamping the hilum. Excess blood was blotted, and the left upper lobe
of the lung was measured for tissue water content. Other tissue section
was stored at ⫺80°C until analyzed. The trachea was then cannulated,
and bronchoalveolar fluid (BALF) was carried out with 5 ml of
phosphate-buffered saline (37°C). The BALF was centrifuged at
1,200 rpm at 4°C for 7 min. The supernatant was collected and stored
at ⫺80°C until analyzed.
Isolation of lung RNA. Lung RNA was isolated immediately after
harvesting the lung using a Nucleospin RNA purification kit (BA
Bioscience, Palo Alto, CA) according to the manufacturer’s instructions. The total RNA content in samples was determined by a
spectrophotometer (Smart TM 300, Bio-Rad), and the isolated RNA
was then stored at ⫺80°C until analyzed.
mRNA expression assay. Lung iNOS gene expression was determined by real-time quantitative RT-PCR as described previously (38).
iNOS primer was purchased from ABI (Foster City, CA). Amplification of cDNA was performed on an ABI PRISM 7900HT Sequence
Detection System. Oligonucleotide primers for iNOS were 5⬘-CAC-
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L1006
ESTROGEN RECEPTOR AND LUNG INJURY AFTER TRAUMA-HEMORRHAGE
Fig. 1. Inducible nitric oxide synthase (iNOS) mRNA (A) and protein (B)
expressions in the lung from sham rats with vehicle (sham), trauma-hemorrhage with vehicle (T-H⫹Veh), trauma-hemorrhage with propyl pyrazole triol
(T-H⫹PPT), trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and
trauma-hemorrhage with 17␤-estradiol (T-H⫹E2). For equal protein loading,
membranes were reprobed for ␤-actin using mouse monoclonal antibody. The
intensity of the bands was analyzed using densitometry and plotted as histograms in B. In each experiment, the densitometric values obtained from sham
operation with vehicle are normalized to 1, and then the fold increases in other
groups over sham values are calculated. Data are shown as means ⫾ SE of 5
animals in each group. *P ⬍ 0.05 compared with sham; #P ⬎ 0.05 compared
with T-H⫹Veh. RQ, relative quantification.
Effect of ER-␣ agonist PPT, ER-␤ agonist DPN, and E2 on
lung and BALF nitrate/nitrite levels. Trauma-hemorrhage significantly increased nitrate/nitrite levels in the lung and BALF
(Fig. 3, A and B). However, treatment with DPN or E2, but not
PPT, prevented the increase in lung and BALF nitrate/nitrite
levels.
Effect of ER-␣ agonist PPT, ER-␤ agonist DPN, and E2 on
lung and BALF IL-6 levels. There was a significant increase in
IL-6 levels in the lung and BALF (Fig. 4, A and B) after
trauma-hemorrhage. Administration of DPN or E2 after
trauma-hemorrhage prevented the trauma-hemorrhage-induced
AJP-Lung Cell Mol Physiol • VOL
Fig. 3. Nitrate/nitrite levels in the lung (A) and bronchoalveolar fluid (BALF)
(B) from sham rats with vehicle (sham), trauma-hemorrhage with vehicle
(T-H⫹Veh), trauma-hemorrhage with propyl pyrazole triol (T-H⫹PPT),
trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and trauma-hemorrhage with 17␤-estradiol (T-H⫹E2). Data are shown as means ⫾ SE of 6 rats
in each group. *P ⬍ 0.05 compared with sham, T-H⫹DPN, and T-H⫹E2;
#P ⬎ 0.05 compared with T-H⫹Veh.
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Fig. 2. Lung tissue wet/dry weight ratio from sham rats receiving vehicle
(sham), trauma-hemorrhage with vehicle (T-H⫹Veh), trauma-hemorrhage
with propyl pyrazole triol (T-H⫹PPT), trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and trauma-hemorrhage with 17␤-estradiol (T-H⫹E2).
Data are shown as means ⫾ SE of 6 rats in each group. *P ⬍ 0.05 compared
with sham, T-H⫹DPN, and T-H⫹E2; #P ⬎ 0.05 compared with T-H⫹Veh.
ESTROGEN RECEPTOR AND LUNG INJURY AFTER TRAUMA-HEMORRHAGE
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Fig. 4. IL-6 levels in the lung (A) and BALF (B) from sham operation with
vehicle (sham), trauma-hemorrhage with vehicle (T-H⫹Veh), trauma-hemorrhage with propyl pyrazole triol (T-H⫹PPT), trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and trauma-hemorrhage with 17␤-estradiol (TH⫹E2). Data are shown as means ⫾ SE of 6 rats in each group. *P ⬍ 0.05
compared with sham, T-H⫹DPN, and T-H⫹E2; #P ⬎ 0.05 compared with
T-H⫹Veh.
increase in lung and BALF IL-6 levels. However, treatment of
animals with PPT did not influence lung and BALF IL-6
expressions.
Effect of ER-␣ agonist PPT, ER-␤ agonist DPN, and E2 on
BALF total protein content and LDH activity. Trauma-hemorrhage significantly increased total protein content and LDH
activity in BALF (Figs. 5 and 6). However, treatment with
DPN or E2 prevented the trauma-hemorrhage-induced increase
in BALF total protein content and LDH activity. In contrast, no
significant change in the above parameters was observed in
trauma-hemorrhage rats treated with PPT.
There are two major subtypes of ERs, ER-␣ and ER-␤ (15,
25). In this study, we attempted to determine which of the
estrogen receptors plays a predominant role in lung protection
after trauma-hemorrhage. Our results indicate that iNOS expression and water content in lung tissue, nitrate/nitrite and
IL-6 levels, total protein content, and LDH activity in the
BALF were significantly increased after trauma-hemorrhage.
However, treatment of rats with ER-␤ agonist DPN after
trauma-hemorrhage demonstrated significant improvement in
the above parameters at 24 h after trauma-hemorrhage. In
contrast to DPN, treatment of rats with ER-␣ agonist PPT did
not attenuate the above parameters of lung injury after traumahemorrhage.
DPN acts as an agonist on both ER subtypes but has 70-fold
higher relative binding affinity and 170-fold higher relative
estrogenic potency in transcription assays with ER-␤ than with
DISCUSSION
Previous studies have shown that lung injury is significantly
increased in male animals after trauma-hemorrhage (2). In
contrast, female rats in the proestrus state, a state in which
plasma levels of estradiol were found to be the highest, showed
attenuation of lung injury after trauma-hemorrhage (2). However, ovariectomized females displayed depression in lung
function after trauma-hemorrhage, similar to those observed in
males (2). Thus it appears that female sex steroids have
protective effects on attenuation of lung injury after traumahemorrhage.
AJP-Lung Cell Mol Physiol • VOL
Fig. 6. Lactate dehydrogenase (LDH) activity in the BALF from sham operation with vehicle (sham), trauma-hemorrhage with vehicle (T-H⫹Veh),
trauma-hemorrhage with propyl pyrazole triol (T-H⫹PPT), trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and trauma-hemorrhage with
17␤-estradiol (T-H⫹E2). Data are shown as means ⫾ SE of 6 rats in each
group. *P ⬍ 0.05 compared with sham, T-H⫹DPN, and T-H⫹E2; #P ⬎ 0.05
compared with T-H⫹Veh.
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Fig. 5. Total protein content in the BALF from sham operation with vehicle
(sham), trauma-hemorrhage with vehicle (T-H⫹Veh), trauma-hemorrhage
with propyl pyrazole triol (T-H⫹PPT), trauma-hemorrhage with diarylpropiolnitrile (T-H⫹DPN), and trauma-hemorrhage with 17␤-estradiol (T-H⫹E2).
Data are shown as means ⫾ SE of 6 rats in each group. *P ⬍ 0.05 compared
with sham, T-H⫹DPN, and T-H⫹E2; #P ⬎ 0.05 compared with T-H⫹Veh.
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ESTROGEN RECEPTOR AND LUNG INJURY AFTER TRAUMA-HEMORRHAGE
AJP-Lung Cell Mol Physiol • VOL
recent study has shown that administration of PPT or DPN
alone in sham groups did not produce any deleterious or
salutary effects (37). Because PPT or DPN administration by
themselves did not influence organ function in sham animals,
administration of PPT or DPN alone was therefore not carried
out in this study.
In conclusion, our results suggest that similar to E2, DPN,
which is an ER-␤ agonist, also provides protection in the lung
after trauma-hemorrhage. Furthermore, we found that downregulation of iNOS likely plays a significant role in the DPNmediated attenuation of lung injury after trauma-hemorrhage.
Additional studies using ER-␤ knockout mice under these
conditions will provide further support to this notion.
GRANTS
This study was supported by National Institute of General Medical Sciences
Grant R37-GM-39519.
REFERENCES
1. Ameredes BT, Zamora R, Gibson KF, Billiar TR, Dixon-McCarthy B,
Watkins S, and Calhoun WJ. Increased nitric oxide production by
airway cells of sensitized and challenged IL-10 knockout mice. J Leukoc
Biol 70: 730 –736, 2001.
2. Ananthakrishnan P, Cohen DB, Xu DZ, Lu Q, Feketeova E, and
Deitch EA. Sex hormones modulate distant organ injury in both a
trauma/hemorrhagic shock model and a burn model. Surgery 137: 56 – 65,
2005.
3. Baker AE, Brautigam VM, and Watters JJ. Estrogen modulates microglial inflammatory mediator production via interactions with estrogen
receptor ␤. Endocrinology 145: 5021–5032, 2004.
4. Baue AE, Durham R, and Faist E. Systemic inflammatory response
syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF): are we winning the battle? Shock 10: 79 – 89,
1998.
5. Chaudry IH, Samy TSA, Schwacha MG, Wang P, Rue LW III, and
Bland KI. Endocrine targets in experimental shock. J Trauma 54: S118 –
S125, 2003.
6. Cuzzocrea S, Mazzon E, Sautebin L, Serraino I, Dugo L, Calabro G,
Caputi AP, and Maggi A. The protective role of endogenous estrogens in
carrageenan-induced lung injury in the rat. Mol Med 7: 478 – 487, 2001.
7. Cuzzozrea S, Santagati S, Sautebin L, Mazzon E, Calabro G, Serraino
I, Caputi AP, and Maggi A. 17␤-Estradiol anti-inflammatory activity in
carrageenan-induced pleurisy. Endocrinology 141: 1455–1463, 2000.
8. Deitch EA. Multiple organ failure: pathophysiology and potential future
therapy. Ann Surg 216: 117–134, 1992.
9. Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking J,
Nathan CF, Peitzman AB, Billiar TR, and Tweardy DJ. Essential role
of induced nitric oxide in the initiation of the inflammatory response after
hemorrhagic shock. J Exp Med 187: 917–928, 1998.
10. Hierholzer C, Menezes JM, Ungeheuer A, Billiar TR, Tweardy DJ,
and Harbrecht BG. A nitric oxide scavenger protects against pulmonary
inflammation following hemorrhagic shock. Shock 17: 98 –103, 2002.
11. Homo-Delarche F, Fitzpatrick F, Christeff N, Nunez EA, Bach JF,
and Dardenne M. Sex steroids, glucocorticoids, stress and autoimmunity.
J Steroid Biochem Mol Biol 40: 619 – 637, 1991.
12. Jarrar D, Kuebler JF, Rue LW III, Matalon S, Wang P, Bland KI, and
Chaudry IH. Alveolar macrophage activation after trauma-hemorrhage
and sepsis is dependent on NF-␬B and MAPK/ERK mechanisms. Am J
Physiol Lung Cell Mol Physiol 283: L799 –L805, 2002.
13. Kiang JG, Lu X, Tabaku LS, Bentley TB, Atkins JL, and Tsokos GC.
Resuscitation with lactated Ringer solution limits the expression of molecular events associated with lung injury after hemorrhage. J Appl Physiol
98: 550 –556, 2005.
14. Kimura Y, Hirooka Y, Sagara Y, Ito K, Kishi T, Shimokawa H,
Takeshita A, and Sunagawa K. Overexpression of inducible nitric oxide
synthase in rostral ventrolateral medulla causes hypertension and sympathoexcitation via an increase in oxidative stress. Circ Res 96: 252–260,
2005.
15. Kuiper GGJM, Carlsson B, Grandien J, Enmark E, Haggblad J,
Nilsson S, and Gustafsson JA. Comparison of the ligand binding spec290 • MAY 2006 •
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ER-␣ (19). PPT, on the other hand, is a selective agonist for the
ER-␣ subtype and binds to ER-␣ with high affinity, displaying
410-fold binding selectivity over ER-␤ (29). Previous studies
have shown that there are differences in the tissue distribution
of the ER subtypes (15). Consistent with these findings, we
also found differences in ER-␣ and -␤ distribution in various
organs. Our findings suggest that ER-␤ mRNA expression is
higher than ER-␣ mRNA expression in the lung (data not
shown). Therefore, the described differences between the ER
subtypes in tissue distribution might contribute to the selective
action of ER agonists in different tissues (15). Thus our results
provide evidence that after trauma-hemorrhage, E2-induced
attenuation of lung injury is mediated via ER-␤ activation.
iNOS has been previously shown to be overexpressed in
rodent lung after hemorrhagic shock (13, 23). Hierholzer and
colleagues (9) have also reported that iNOS inhibition resulted
in a marked reduction of lung injury produced by hemorrhagic
shock. These findings support the view that an enhanced
formation of NO from iNOS plays an important role in lung
injury after hemorrhagic shock (9). In agreement with those
studies, our present study also shows that there is a positive
correlation between lung injury and iNOS expression. In addition, under stressful conditions, iNOS can directly produce
superoxide radicals (14). It is therefore possible that iNOS in
the lung produces both NO and superoxide concurrently.
It is known that iNOS can be transcriptionally regulated in
rodent cells (7). The complexity of iNOS promoter suggests
that a substantial number of factors may participate in its
regulation (16, 22). By interacting with AP-1 or NF-␬B components, estrogen could directly influence iNOS mRNA synthesis (17). Furthermore, a few studies have examined the
effects of E2 and gender on iNOS expression in the lung (6, 7).
Ovariectomy increased the level of iNOS in female lung, and
this could be attenuated by E2 replacement therapy (6). Additional studies have shown that E2 treatment reduced the level
of iNOS in male rats (21). Consistent with these findings, we
observed downregulation of lung iNOS after trauma-hemorrhage with DPN or E2 administration. These findings corroborate previous studies by Baker and colleagues (3), which
showed that ER-␤ knockout mice display increased iNOS
expression after endotoxemia compared with wild-type mice.
Together, these findings suggest that DPN or E2 might attenuate lung injury after trauma-hemorrhage through downregulation of iNOS.
It could be argued that the present study utilized measurement at a single time point, i.e., at 24 h after treatment, and thus
it remains unclear whether the salutary effects of E2 or DPN
are sustained for periods of time longer than 24 h after
treatment. In this regard, our previous studies have shown that
if the improvement in organ functions by any pharmacological
agent is evident at 2, 5, or 24 h after treatment, those salutary
effects are sustained for prolonged intervals and also improve
the survival of animals (5). Thus although a time point other
than 24 h was not examined in this study, based on our
previous studies, it would appear that the salutary effects of E2
or DPN on the measured parameters in the lung would be
evident even if one measured those effects at another time
point after trauma-hemorrhage.
It can also be argued that we should have administered PPT
or DPN alone in sham groups in these studies to determine
whether that per se has any adverse effects. In this regard, our
ESTROGEN RECEPTOR AND LUNG INJURY AFTER TRAUMA-HEMORRHAGE
16.
17.
18.
19.
21.
22.
23.
24.
25.
26.
27.
AJP-Lung Cell Mol Physiol • VOL
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
inflammatory protein-2 expression in sepsis. J Immunol 169: 2093–2101,
2002.
Song Y, Ao L, Raebum CD, Calkins CM, Abraham E, Harken AH,
and Meng X. A low level of TNF-␣ mediates hemorrhage-induced acute
lung injury via p55 TNF receptor. Am J Physiol Lung Cell Mol Physiol
281: L677–L684, 2001.
Stauffer SR, Coletta CJ, Tedesco R, Nishiguchi G, Carlson K, Sun J,
Katzenellenbogen BS, and Katzenellenbogen JA. Pyrazole ligands:
structure-affinity/activity relationships and estrogen receptor-␣ selective
agonists. J Med Chem 43: 4934 – 4947, 2000.
Szabo C and Billiar TR. Novel roles of nitric oxide in hemorrhagic
shock. Shock 12: 1–9, 1999.
Tsai BM, Wang M, Clauss M, Sun P, and Meldrum DR. Endothelial
monocyte-activating polypeptide II causes NOS-dependent pulmonary
artery vasodilation: a novel effect for a proinflammatory cytokine. Am J
Physiol Regul Integr Comp Physiol 287: R767–R771, 2004.
Wang P, Hauptman JG, and Chaudry IH. Hepatocellular dysfunction
occurs early after hemorrhage and persists despite fluid resuscitation.
J Surg Res 48: 464 – 470, 1990.
Wang W, Wang P, and Chaudry IH. Intestinal alkaline phosphatase:
role in the depressed gut lipid transport after trauma-hemorrhagic shock.
Shock 8: 40 – 44, 1997.
Wichmann MW, Zellweger R, DeMaso CM, Ayala A, and Chaudry
IH. Mechanism of immunosuppression in males following trauma-hemorrhage: critical role of testosterone. Arch Surg 131: 1186 –1191, 1996.
Wichmann MW, Zellweger R, DeMaso CM, Ayala A, and Chaudry
IH. Enhanced immune responses in females, as opposed to decreased
responses in males following haemorrhagic shock and resuscitation. Cytokine 8: 853– 863, 1996.
Wu LL, Tang C, and Liu MS. Altered phosphorylation and calcium
sensitivity of cardiac myofibrillar proteins during sepsis. Am J Physiol
Regul Integr Comp Physiol 281: R408 –R416, 2001.
Yu HP, Shimizu T, Choudhry MA, Hsieh YC, Suzuki T, Bland KI,
and Chaudry IH. Mechanism of cardioprotection following traumahemorrhagic shock by a selective estrogen receptor-␤ agonist: upregulation of cardiac heat shock factor-1 and heat shock proteins. J Mol Cell
Cardiol 40: 185–194, 2006.
Yu HP, Yang S, Choudhry MA, Hsieh YC, Bland KI, and Chaudry
IH. Mechanism responsible for the salutary effects of flutamide on cardiac
performance following trauma-hemorrhagic shock: upregulation of cardiomyocyte estrogen receptors. Surgery 138: 85–92, 2005.
290 • MAY 2006 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.246 on June 18, 2017
20.
ificity and transcript tissue distribution of estrogen receptor ␣ and ␤.
Endocrinology 138: 863– 870, 1997.
Kunz D, Mühl H, Walker G, and Pfeilschifter J. Two distinct signaling
pathways trigger the expression of inducible nitric oxide synthase in rat
renal mesangial cells. Proc Natl Acad Sci USA 91: 5387–5391, 1994.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, and Evans
RM. The nuclear receptor superfamily: the second decade. Cell 83:
835– 839, 1995.
Meldrum DR, McIntyre RC, Sheridan BC, Cleveland JC Jr, Fullerton
DA, and Harken AH. L-arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric
oxide synthase-dependent mechanism. J Trauma 43: 888 – 893, 1997.
Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS,
and Katzenellenbogen JA. Estrogen receptor-␤ potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and
their acetylene and polar analogues. J Med Chem 44: 4230 – 4251, 2001.
Moore FA, Sauaia A, and Moore EE. Post injury multiple organ failure:
a bimodal phenomenon. J Trauma 2: 501–513, 1996.
Noris M, Todeschini M, Zappella S, Bonazzola S, Zoja C, Corna D,
Gaspari F, Marchetti G, Aiello S, and Remuzzi G. 17␤-Estradiol
corrects hemostasis in uremic rats by limiting vascular expression of nitric
oxide synthases. Am J Physiol Renal Physiol 279: F626 –F635, 2000.
Perrella MA, Yoshizumi M, Fen Z, Tsai JC, Hsieh CM, Kourembanas
S, and Lee ME. Transforming growth factor-␤1, but not dexamethasone,
down-regulates nitric-oxide synthase mRNA after its induction by interleukin-1␤ in rat smooth muscle cells. J Biol Chem 269: 14595–14600,
1994.
Pittet JF, Lu LN, Geiser T, Lee H, Matthay MA, and Welch WJ. Stress
preconditioning attenuates oxidative injury to the alveolar epithelium of
the lung following haemorrhage in rats. J Physiol 538: 583–597, 2001.
Remmer DE, Wang P, Cioffi WG, Bland KI, and Chaudry IH.
Testosterone receptor blockade after trauma-hemorrhage improves cardiac
and hepatic functions in males. Am J Physiol Heart Circ Physiol 273:
H2919 –H2925, 1997.
Samy TSA, Schwacha MG, Cioffi WG, Bland KI, and Chaudry IH.
Androgen and estrogen receptors in splenic T lymphocytes: effects of
flutamide and trauma-hemorrhage. Shock 14: 465– 470, 2000.
Sir O, Fazal N, Choudhry MA, Goris RJ, Gamelli RL, and Sayeed
MM. Role of neutrophils in burn-induced microvascular injury in the
intestine. Shock 14: 113–117, 2000.
Skidgel RA, Gao XP, Brovkovych V, Rahman A, Jho D, Predescu S,
Standiford TJ, and Malik AB. Nitric oxide stimulates macrophage
L1009