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Introduction
Almost 40 years have passed since the clinical
symptoms of acute lung injury (ALI) and its more severe form,
acute respiratory distress syndrome (ARDS), were first
described by Ashbaugh et al in
1967[1]. Despite the strategies for treating ALI/ARDS being extensively investigated, the
overall mortality of ALI/ARDS still remains high at
approximately 30%_70%[2]. Treatment options are limited and most
of them are supportive; the only intervention that can
reduce the mortality rate is the use of low tidal volume
mechanical ventilation[3]. While drug therapy for ARDS/ALI is
disappointing, to date, no pharmacological intervention has
been proven to be effective to the survival rate of
ARDS/ALI patients clinically[4]. Extensive neutrophil influx into the
lungs, the production of pro-inflammatory mediators, and
damage to lung epithelial and endothelial surfaces are
the main characteristics of
ALI[5,6].
Clinically, the development of ALI/ARDS is complex and
it is rare for only 1 single instigating factor to cause
ALI/ARDS[7,8]. Many researchers suggest that 2-hit models are
more appropriate to reflect the real situation present in ALI
patients[9_12]. Our previous results have shown that rats
with lipopolysaccharide (LPS) intraperitoneal (ip) injection
and HCl aspiration had significant lung injury when
compared to the saline solution control group (only LPS
injection group, or only HCl instillation group).
Raloxifene, a selective estrogen receptor modulator
(SERM), has now been widely used in the prevention and
treatment of osteoporosis in postmenopausal
women[13]. Recently, raloxifene has been reported to inhibit
interleukin-6 (IL)-6 production as well as having an anti-inflammatory
role in a murine model induced by
LPS[14,15]. It was also found to have protective effects on the development of
carrageenan-induced edema and
pleurisy[16]. Although early
studies have shown that estrogens have some salutary
effects on ALI, and these effects may be mediated via
ER-β[17,18], we did not come across raloxifene usage or experimental
studies on ALI/ARDS. In the present study, we used the 2-hit
model to investigate whether raloxifene had some protective
effects on ALI.
Materials and methods
Animals Male Sprague-Dawley rats (Grade II), each
weighing between 180 and 210 g, were purchased from the
Animal Center of Zhejiang University school of Medicine
(Hangzhou, China). All the animals were housed in air-filtered,
temperature-controlled units with access to food and water
ad libitum. All experimental protocols were approved by the
animal care committee and all experiments were done in
conformity with the Guiding Principles for Research Involving
Animals of Zhejiang University School of Medicine.
Experimental protocol The rats were divided into 3
groups: the raloxifene-LPS-HCl group (n=10),
LPS-raloxifene-HCl group (n=10), and the placebo group
(n=10). All the rats were challenged with ip administration of LPS
(Escherichia coli, serotype 0111, B4, Sigma, St Louis, MO, USA) at the
dose of 5 mg/kg. Raloxifene (Evista, Lilly, SA, Spain) was
solubilized in normal saline solution and was orally
administered 1 h before and 14 h after the LPS ip injection into the
raloxifene_LPS_HCl and LPS_raloxifene_HCl groups at the
dose of 30 mg/kg, respectively. Administration was done by
gentle gavage with a ball-tipped (18 gauge) needle, while the
placebo group received nothing. Sixteen hours after the LPS
ip injection, all the animals were anesthetized with an ip
injection of sodium pentobarbital (40 mg/kg)
and placed in a 60° inclined position. The femoral artery was cannulated
and connected to a pressure transducer to record the mean
arterial pressure (MAP) on a polygraph recorder (Mindary
Company, Shenzhen, China). The trachea was surgically
exposed and all the animals received a direct intratracheal
(IT) injection of HCl (pH 1.2; 0.5 mL/kg). Blood gas samples
(0.3 mL each) were obtained before HCl instillation and at 30,
90, 240 min after the instillation and replaced by the same
volume of saline solution. The samples were analyzed using
a blood gas analyzer (OMNI C, Roche, Roswell, GA, USA).
MicroPET examination Two hundred and ten minutes
after HCl IT, 15 rats (5 in each group, respectively) were
randomly selected and underwent microPET examination.
PET was performed using a microPET R4 rodent model
scanner (CTI Concorde Microsystems, Knoxville, TN, USA),
which was equipped with a microPET manager for data
acquisition in the list mode and Acquisition Sinogram and
Image Processing (ASIPro) for preparing sinograms and image
reconstruction. The scanner had a computer-controlled bed,
a 10.8 cm transaxial, and a 7.8 cm axial field of view (FOV)
with image resolution at <1.8 mm. Fluorodeoxyglucose (FDG)
was prepared with a specific activity of 500 Ci/mmol at the
Department of Nuclear Medicine, Zhejiang University School
of Medicine. Before the examination, rats were
re-anesthetized and injected with 10.56 MBq (0.3 mCi) FDG through the
femoral artery cannulation, then 30 min later, the rats were
placed at the center of the FOV of the microPET R4 scanner
and underwent a 10 min static examination. The images were
reconstructed by a maximum-a-posteriori probability (MAPP)
algorithm. The ratio of the regions of interest (ROI) in the
right lung to the muscle was calculated for each scan using
ASIPro. These ROI were drawn and arranged by one of the
authors who had extensive experience in manual ROI
definition and was blinded to the results. The corrections for dead
time, random scattering, and attenuation were performed for
all the scans.
Wet/dry weight ratio At the end of the experiment, all the
animals were killed by an injection of sodium pentobarbital.
The right lung was excised and weighed, placed in an oven
at 50 oC for 24 h, and then reweighed to determine the
wet/dry (W/D) weight ratio. The left lung was used for the
histopathological examination.
Histology The left lung was placed in 4% formalin,
embedded in paraffin, and stained with hematoxylin-eosin (HE).
According to an arbitrary 4-grade
scale[19], all the sections were examined and graded by a pathologist who knew
nothing about the experimental conditions of each individual
animal. Briefly, the sections were assessed by the airway
epithelial necrosis, intra-alveolar edema, hyaline membranes,
hemorrhage, and the recruitment of inflammatory cells to the
air space. Each characteristic was scored 0 to 3 (0=absent;
1=mild; 2=moderate; and 3=prominent).
Statistical analysis Data were expressed as mean±SD
and analyzed by SPSS 13.0 statistical software (SPSS,
Chicago, IL, USA). ANOVA with repeated measurements
analysis was used to compare samples obtained at several
time-points from the same animals. Independent samples
t-test was used to determine which group was significantly
different. The correlation between 2 variables was measured
by Bivariate test. A P-value less than 0.05 was considered
statistically significant.
Results
Blood gas analysis of
pO2 The
pO2 remained normal and there were no differences between the 3 groups immediately
prior to tracheal acid instillation. As expected, the
pO2 showed an initial decline in all animals 30 min after acid instillation,
but the pO2 in the placebo group decreased more markedly
and there were significant differences compared with that in
the LPS-raloxifene-HCl group (P<0.01). As time passed, the
pO2 in the LPS-raloxifene-HCl group slowly returned to
normal ranges[20]
(pO2: 80_120 mmHg;
pCO2: 35_45 mmHg; pH:
7.40_7.50; and MAP: 70_110 mmHg), while that of the placebo
group at the end of the experiment was 58.81±10.27 mmHg
(Figure 1).
pH The pH values differed between the
LPS-raloxifene-HCl group and the placebo group
(P<0.05). In the placebo group, the pH value remained below the normal range,
although it reached 7.273±0.054 at 90 min after instillation,
while that of the LPS-raloxifene-HCl group gradually returned
to the normal range after acid instillation and finally
reached 7.380±0.064 (7.243±0.108 in the placebo group). However,
there were no significant differences between the
raloxifene-LPS-HCl group and the placebo group (Figure 2).
pCO2 In the LPS-raloxifene-HCl group, the average value
of the pCO2 was 40.07±7.79 mmHg, while in the placebo group
that value was 50.75±13.03 mmHg, much higher than that in
LPS-raloxifene-HCl group (P<0.05; Figure 3). There were
also no significant differences between the
raloxifene-LPS-HCl group and the placebo group.
MAP There were no differences between the three
groups in initial measurements of MAP. However, after
HCl instillation, MAP in the LPS-raloxifene-HCl group remained stable, while MAP in placebo-treated group and
raloxifene-LPS-HCl group showed a marked decrease. There
were significant differences between the
LPS-raloxi-fene-HCl group and the placebo group
(P<0.01; Figure 4).
MicroPET The ratio of ROI in the right lung to the muscle
was 9.01±1.58 in the placebo group, significantly higher than
that (4.67±1.33) of the LPS-raloxifene-HCl group
(P<0.01; Figure 5). There were no significant differences between the
raloxifene-LPS-HCl group and the placebo group.
W/D weight ratio The W/D weight ratio was 5.886±0.257 in the placebo group, 5.335±0.198 in the
LPS-raloxifene-HCl group, and 5.766±0.203 in the raloxifene-LPS-HCl group.
There were significant differences between the placebo group
and the LPS-raloxifene-HCl group (P<0.01).
Histology An histological examination revealed lung
injury of varying degrees in all animals in the 3 groups, but
were more prominent in the placebo group. Briefly, the mean
lung injury score in the placebo group was 12.6±0.97, much
higher than that (8.20±1.23) of the LPS-raloxifene-HCl group
(P<0.01). That score in the raloxifene-LPS-HCl group was
11.4±1.65, no significant difference when compared with the
placebo group (Figure 6).
Correlation The ratio of the ROI between the right lung
and the muscle correlated significantly with the histological
lung injury score (r=0.824, P<0.001; Figure 7).
Discussion
In acute lung injury, the predominant inflammatory cells
are neutrophils[21], which play an important role in the
development of most cases of ALI[22] . When acute lung injury
occurs, neutrophils adhere to the injured capillary
endothe-lium, marginate into the air spaces, and are stimulated by
cytokines, such as TNF-α. The activated neutrophils in
return begin to release leukotrienes, oxidants, proteases, and
other inflammatory mediators[21,23]. In short, it is the
adhesion and activation of neutrophils that produce lung
injury[24,25]. The activated neutrophils also take up
[18F]FDG at an accelerated rate after
[18F]FDG injection, thus generating a PET
imaging signal[26]. As can be seen in the present study, an
increasing number of reports are appearing in the
field[27_30]. [18F]FDG PET/CT has become a useful tool for evaluating
pulmonary lesions. Furthermore, the pulmonary
transcapil-lary escape rate for 68 Ga-transferrin obtained by PET has
been shown to be the only method which correlates with
lung damage histopathologically[31,32], although PET is
expensive and not readily available at all times or at all
institu-tions.
One of the most commonly used procedures to quantify
neutrophils during ALI is bronchoalveolar lavage (BAL), but
BAL can only detect neutrophils which penetrate into the air
spaces. Other activated neutrophils may be
neglected, furthermore, part of the neutrophils in BAL might not be
activated and are not involved in the inflammatory process.
So when the number of neutrophils in BAL is used as the
indicator of activated neutrophils, it is inaccurate and may
underestimate the influx of neutrophils in the lungs, while
[18F]FDG PET imaging is more accurate when at least using
increased glucose uptake as the criterion for neutrophil
activation[26]. The expression of CD11b, which is a marker of
neutrophil activation in vivo during
ALI[33], by flow cytometry examination again is less accurate compared with
[18F]FDG PET because it may also neglect the activated neutrophils
which do not penetrate into the air spaces. Furthermore,
BAL is invasive, while [18F]FDG PET is non-invasive and
can be easily accepted clinically.
Raloxifene, a benzothiophene derivative which has
anti-estrogenic properties in breast tissues, a neutral effect on
the endometrium, and potentially beneficial estrogen-like
effects in non-reproductive tissues, such as
bone[34], was also shown to have an anti-inflammatory role in acute
inflammation. Esposito et
al[16] demonstrated that raloxifene
plays a protective role in the development of
carrageenan-induced edema and pleurisy. They speculated that this
effect may be caused by reducing inducible
pro-inflammatory enzymes (COX-2 and iNOS) and by decreasing the
infiltration of neutrophils into the inflamed paw and pleural cavity.
Suuronen et al[15] showed that SERM, raloxifene, and
tamoxifen, but not 17β-estradiol, suppressed the secretion
of IL-6 and nitric oxide and induced a significant,
concentration-dependent anti-inflammatory response both in rat
primary microglial cells and in mouse N9 microglial cells which
were induced by LPS.
In the present study, we found that raloxifene also had
an anti-inflammatory role during ALI. The results of the
[18F]FDG microPET showed that the rats in the placebo group
had significant influx of activated neutrophils in the lungs,
while the rats receiving raloxifene treatment 2 h before the
instillation of HCl exhibited diminished influx. It is well known
that leukocyte activation at the site of inflammation is
fundamental in the inflammatory process and raloxifene exerted a
marked inhibition on leukocyte infiltration and activation in
this 2-hit acute lung injury model. Oral raloxifene can also
maintain MAP stability and improve pulmonary gas exchange.
The W/D weight ratio, indicating pulmonary edema, was
much lower in the LPS-raloxifene-HCl group. These data
indicate that the oral administration of raloxifene offers a
statistically significant protective effect on ALI.
Findings in the histological examination also showed that
raloxifene had significant anti-inflammatory effects in the
2-hit model when administered 2 h before HCl IT. Neutrophil
infiltration, hemorrhage, hyaline membranes, alveolar edema,
and airway epithelial necrosis were common and prominent
in the placebo group, but were rare in the LPS-raloxifene-HCl
group. Furthermore, our findings also showed that there
was a significant correlation between
[18F]FDG uptake and lung damage histopathologically. Therefore, we concluded
that the degree of histological lung injury can be roughly
assessed with [18F]FDG PET.
The half-decay period of raloxifene in human is 27.7 h,
but it is much shorter in rats. Therefore, there were no best
blood drug level in the rats of the raloxifene-LPS-HCl group
and raloxifene showed no protective effect on ALI at 17 h
after administration. The pharmacology of raloxifene is very
complex. It has recently been reported that raloxifene could
block NF-κB biological activity through the modulation of
the ER-α association with p65 in multiple
myeloma[35], and because ER-α are also highly expressed in lung
tissues[36], we hypothesized that raloxifene can also suppress
NF-κB activity during ALI through the modulation of
ER-α and thus exert a protective effect on ALI. This hypothesis needs to
be further studied and verified.
In conclusion, oral raloxifene 2 h before HCl instillation
can decrease neutrophil influx, suppress neutrophil
activa-tion, improve pulmonary gas exchange, and maintain MAP
stability in the 2-hit ALI model, which may therefore have an
important therapeutic role on ALI patients clinically.
Furthermore, the oral administration was of interest since
oral use by humans is desirable compared to other methods
of drug administration.
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