|
Department
of Gastroenterology, Ruijin Hospital, Shanghai Second Medical
University, Shanghai 200025, China
Dr. Yao Zong Yuan, graduated from Shanghai Second Medical University
as Ph.D in 1991, Professor of gastroenterology, major in pancreatic
disease and gastrointestinal motility, having 30 papers published.
Presented at the meeting of 11th Asian Pacific
Congress of Gastroe
nterology, 8th Asian Pacific Congress of Digestive
Endoscopy, Hong Kong, 10-14
March, 2000
Correspondence to: Dr. Yao Zong Yuan,
Department of Gastroenterology, Ruijin Hospital, Shanghai Second
Medical University, Shanghai 200025, China
Telephone:
0086-21-64370045
Ext.665242,
Fax. 0086-21-64333548
Email. yyz28@hotmail.com
Received:
2000-06-06
Accepted: 2000-06-29
Subject
headings: pancreatitis; lung/injuries;
apoptosis; protein
P53; immunohistochemistry; platelet activating factor
Yuan YZ, Gong ZH, Lou KX, Tu SP, Zhai ZK, Xu JY. Involvement of
apoptosis of alveolar epithelial cells in acute pancreatitis-
associated lung injury. World J
Gastroentero, 2000;6(6):920-924
INTRODUCTION
Acute pancreatitis in a common and potentially fatal disease
which is associated
with considerable morbidity and a mortality rate of approximately
30%; its path
ogenesis remains unknown[1].
The cause of death is often due to multiple organ failure which
frequently complicates severe acute pancreatitis. In particular,
acute lung injury often occurs at an early stage of severe acute
pancreatitis and develops into adult respiratory distress syndrome (ARDS)[2,3].
Numerous
studies have been done to clarify the mechanisms of acute lung
injury in AHNP. Activated leukocytes and inflammatory mediators,
such as interleukin
１and
tumor necrosis factor α-
have been suggested to play a predominant role in the development of
acute lung injury in AHNP[4-6].
Further investigation have found that pretreatment of mice with
antineutrophile serum results in reduction of severity of
pancreatitis and complete prevention
of lung injury[7].
More recently, Hofbauer et al
[8-10]postula
ted that the pancreatitis-associated
lung injury (PALI) might be dependent on the continued generation
and action of platelet-activating
factor (PAF). Yamaguchi e
t al[11]have
suggested that the PAF antagonist TCV-309
effectively prevented cytokine-induced
neutrophil chemoattractant expression by bronchoalve
olar macrophages and subsequent PALI. Furthermore, attenuation of
pancreatitis and prevention of PALI could be achieved with
recombinant PAF acetylhydrolase. P-selectin
is a key determinant of leukocyte recruitment, which is upregulated
in the pulmonary endothelium during acute pancreatitis[12].
However, detailed mechanisms of acute lung injury in AHNP has not
been elucidated.
In the current study, sodium taurocholate-induced
pancreatitis rat models was used to investigate the apoptosis of
alveolar epithelial cells and expression of apoptosis-regulated
gene in PALI, and the relationship between TGFβ-1
and apoptosis.
MATERIAL AND METHODS
Material and chemicals
Sodium taurocholate was obtained
from Sigma Chemical Co.(USA). In situ cell death detection
kit, POD was purchased from Borehinger Mahheim Co. (Germany),
protinase K was from Merch Co.(USA), the purified goat anti-bax
polyclonal antibody and mouse anti-p53
monoclonal antibody were from Santa Cruz Biotechnology Inc.(USA),
the goat anti-TGFβ-1
polyclonal antibody was from Promega Co.(USA), and immunochemical SP
kit was from Biotech Co.(USA). Other materials and chemicals were
obtained from commercial sources.
Animals
Male Wistar rats, weighing
200g-250g, were used. Animals were bred and housed in standard cages
in a climate-controlled
room with an ambient temperature of 22℃±2℃
and a 12-hour
light/dark cycle. They were fed standard laboratory chow, given
water at libitum, and fasted overnight before each
experiment.
Models for pancreatitis
Male Wistar rats were anesthetized
with 2.5%
pentobarbital (0.1mL/100g
body weight intraperitoneally). A midline laparotomy was performed,
followed
by the ligation of the bile-pancreatic
ducts close to the liver and duodenum. Then pancreatic duct was
retrogradely injected 5% sodium taurocholate (0.1mL/100g
body weight) for 1 minute, and stagnant for 4 minutes. For control,
sham operation or retrograde infusion of normal saline into the
pancreatic duct was performed as described above, 2, 5 and 14 hours
after sodium taurocholate infusion, laparotomy was performed again
and blood samples were collected aseptically from the abdominal
aorta. The pancreas and right lung were rapidly removed and fixed in
10% neutral phosphate buffered formalin for histological study.
Pancreatitis was confirmed by measuring amylase levels before and
after the experiment and by histological examination.
Serum amylase activity
Serum amylase activity was
measured by a chromogenic method with the phadeba amylase test.
Detection of apoptosis
Morphological examination Paraffin-embedded
pancreas and lung samples were sectioned (4μm),
stained with hematoxylin/ eosin (H &
E), and examined by an experienced
morphologist who was unaware of the sample identity[13,14].
TUNEL on light microscope TdT-mediated
dUTP nick-end
labeling (TUNEL) was performed according to the method of Gavrieli et
al[15,16
]with
some modification. Briefly, the sections were deparaffinized by
heatin
g for 20 minutes at 60℃,
rehydrated in descending concentrations of ethanol (100%, 95%, 90%,
80%) and then immersed in double.distilled
water (DDW). After rehydration, the sections were incubated with
20mg/L proteinase K for 15 minutes at room temperature. The slides
were washed in DDW for 2 minutes 4 times and covered with 0.3%
hydrogen peroxidase in methanol for 30 minutes at room temperature
to inactivate endogenous peroxid
ase. The slides were rinsed with phosphate-buffered
saline (PBS, pH 7.4)
5 minutes three times and incubated the slides to 50μL
TUNEL reaction mixture for 60 minutes at 37℃.
After washing in PBS, the slides
were incubated with 50μL
Converter-POD
for 30 minutes at 37℃.
The reaction products were visualized by immersion in diaminobenzide
solution. For positive controls, TUNEL was performed after
deoxyribonuclease treatment. For negative controls, TUNEL was
performed with labeling solution instead of TUNEL reaction mixture.
Observations and photographs were made using an Olympus microscope.
TUNEL positive nuclei were counted in fields (×40)
magnification chosen at random, and the number of labeled nuclei per
total nuclei in those fields was expressed as the apoptotic index
(AI).
Immunohistochemical staining Immunohis-tochemical
staining was performed to explore the expression of apoptosis-regulated
gene bax and p53, as well as TGFβ-1.
Consecutive 4μm
paraffin-embedded
tissue sections were subjected to immunostaining with streptavidin
peroxidase technique. The sections were deparaffinized, rehydrated
in descending concentrations of ethanol and then digested by
incubation with 0.1%
typsin for 20 minutes at 37℃.
Tissue sections were submerged for 15 minutes in 0.1M
PBS containing 0.1%
Triton X-100.
Endogenous peroxidase activ
ity was blocked by incubating the slides with 0.3%
(vol/vol) H2O2 in methanol, followed by
washing in PBS. The sections were incubated for 30 minutes at room
temperature with normal goat serum before overnight incubation at 4℃
with either goat anti-bax
polyclonal antibody diluted 1∶50
in PBS, mouse anti-p53
monoclonal antibody diluted 1∶50
in PBS or goat anti-TGFβ-1
polyclonal antibody diluted 1∶100
in PBS. After washing in PBS, slides were treated with biotinylated
link immnoglobulins biotinylated
goat anti-mouse
IgG antibody was used for bax and p53 staining. The slides were then
incubated for 30 minutes with streptavidin-biotin-peroxidase
complex and were visualized by a 10 minutes application of
diaminobenzidine substrate. The sections were counterstained with
hematoxylin to identify nuclei and observed under a light
microscope. It was judged that positive staining nuclei were brown,
and immunolocalized nuclei. The positive rate of bax and p53 were
counted in fields (40×)
magnification chosen at random, and the number of positive staining
nuclei per total nuclei in those fields was expressed as the
positive rate.
Statistical analysis
Results were expressed as the mean
and standard error of the mean (SEM). The significance of changes
was evaluated by using Student＇s
t
test when the data consisted of only two
groups or by analysis of variance (ANOVA) when comparing three or
more groups. A P value ＜0.05
indicated a significant difference.
RESULTS
Acute hemorrhagic necrotizing
pancreatitis
AHNP was manifested by a rise in
serum amylase activity and morphological evidence. In all animals a
marked elevation of serum amylase levels was observed
at 2, 5, and 14 hours after sodium taurocholate infusion. The
morphological changes were observed after sodium taurocholate
infusion and included obvious acinar cell necrosis, fat necrosis,
extensive neutrophils infiltration, microthrombosis of pancreatic
vessels and massive intralobular hemorrhage. In addition, 1 rat
(25%) died before the end of the experiment (14 hours after sodium
taurocholate infusion), whereas other animals survived the entire
observation period.
Histological findings
The morphological changes observed
in lung included pronounced interstitial edema associated with
massive neutrophils infiltration, alveolar wall thickening
and foci of vascular thrombosis in large vessels. Simultaneously,
increase of pulmonary microvascular permeability was found.
Evidence of apoptosis
Morphological examination In the H
&
E staining, alveolar epithelial cells
presented pyknotic nuclei, cell shrinkage, condensatio
n of chromatin and formation of apoptotic bodies, etc. which were
the typical morphological features of apoptosis.
Figure
1(PDF) AI of alveolar
epithelial cells in sodium taurocholate-induced
pancreatitis-associated
lung injury. AHNP was induced by
retrograde injections into the pancreatic ducts of 5% sodium
taurocholate (0.1mL/100g
body wt) in rats. Results shown are the MEAN±SEM
for four or more animals in each group. Asterisks indicates bP＜0.01
when the AI of alveolar epithelial cells at 5 hours after induction
of AHNP compared with 2 and 14 hours after induction of AHNP.
Figure 2(PDF)
The positive rate of bax and p53 protein in alveolar epithelial
cells after sodium taurocholate-induced
pancreatitis-
associated lung injury. AHNP was induced
by retrograde injections into the pancreatic ducts of 5% sodium
taurocholate (0.1mL/100g
body wt) in rats. Results shown were the MEAN±SEM
for four or more animals in each group. Asterisks indicates aP＜0.05
when the positive rate of bax protein in
alveolar epithelial cells at 5 hours after induction of AHNP
compared with 2 and 14 hours and the positive rate of p53 protein in
alveolar epithelial cells at 2 hours after induction of AHNP
compared with 5 and 14 hours.
Figure 3(PDF)
The positive rate of TGFβ-1
in alveolar epithelial cells after sodium taurocholate-induced
pancreatitis-associated
lung injury. AHNP was induced by retrograde injections into the
pancreatic ducts of 5% sodium taurocholate(0.1mL/100g
body wt) in rats. Results shown were the MEAN±SEM
for four or more animals in each group. There
was no significant difference among the positive rate of TGFβ-1
at 2, 5 and 14 hours after induction of AHNP.
TUNEL staining As
shown in Figure 1, TUNEL selectively labeled
alveolar epithelial cells nuclei. The apoptotic index of alveolar
epithelial cells at 2, 5, and 14 hours after induction of AHNP were
0.33%±0.7%,
0.89%±0.06%
and 0.54%±0.08%,
respectively. Moreover, the apoptotic index of alveolar epithelial
cells at 5 hours after induction of AHNP was significantly higher
than those of 2 and 14 hours(P＜0.01).
Immunohistochemical detection of bax and p53 protein
As shown in Figure 2, the positive rate of bax protein in
alveolar epithelial cells at 2, 5 and 14 hours after induction of
AHNP were 0.38%±0.11%,
0.67
%±0.13%
and 0.39%±0.03%,
respectively. Similar to AI of alveolar epithelial cells, the
positive rate of bax protein in alveolar epithelial cells at 5 hours
after induction of AHNP was similarly markedly higher than those of
2 and 14 hours (P＜0.05).
In this study, the positive rate of p53 protein in alveolar
epithelial cells at 2, 5, and 14 hours after induction of AHNP were
0.72%±0.29%,
0.28%±0.08%
and 0.32%±0.04%,
respectively.
The positive rate of p53 protein in alveolar epithelial cells
significantly decreased at 5 and 14 hours after induction of AHNP
compared with that of 2 hours (P＜0.05).
Immunohistochemical detection of TGFβ-1
Immun-ohistochemical
staining of TGFβ-1
was scarcely detected in normal lung tissues. As shown in Figure 3,
although the positive rate of TGF β-1
in alveolar epithelial cells at 2, 5, and 14 hours after induction
of AHNP were 0.35%±0.14%,
0.39%±0.15%
and 0.48%±0.12%,
respectively, no significance difference was observed among them.
DISCUSSION
Acute lung injury and adult respiratory distress syndrome (ARDS)
are frequently
observed during the course of severe acute pancreatitis. The
pathogenesis of PALI remained unclear. Bhatia et al[
17]reported
that neurogenic factors, such as substance P, increased release of
proinflammatory midiators from the pancreas leading to increase lung
injury by acting via NK1R on acinar cells. Recent studies[18]have
suggested that intercellular adhesion molecule 1 (ICAM-1)
expression on pulmonary microva
scular endot helial cells during pancreatitis contributed to the
evolution of PALI by promoting neutrophil sequestration within the
lung. Moreover, treatment with monoclonal antibodies against ICAM-1
signifi
cantly reduced the lung injury in severe acute pancreatitis[19].Very
recently, Tsukahara et al[20]noted
that alveolar macrophages w
ere activated by PLA2, mainly by sPLA2 type Ⅱ,
and produced a large amount
of NO that contributed to lung injury in acute pancreatitis. Further
studies had suggested that the lung injury was reduced by the
administration of the PLA2 inhibitor. Recent studies
showed that the absence of T and B lymphocyt
es could prevent severe pulmonary injury resulting from acute
pancreatitis, this indicated that systemic lymphocyte activation
modulated the systemic respo
nse, in particular, pulmonary injury caused by acute pancreatitis[21].
Apoptotic cell death of renal tubules and hepatocytes have been
reported to be involved in the mechanisms of renal failure and liver
failure during severe acute pancreatitis[22,23].
Although numerous trials had been done to clarify the mechanisms of
pancreatitis-associated
lung injury, there was no literature referring to involvement of
apoptosis in pancreatitis-associated
lung injury.
Apoptosis
was initially confirmed as a specific form of cell death that served
to eliminate excessive or unwanted cell types during embryogenesis
and normal tissue growth[24],
but had been recently clarified to be induced in cellular injury
with inflammatory disease[25].
It was generally believed that apoptosis was a genetically regulated
form of cell death, and pyknotic nuclei and formation of apoptotic
bodies were the typical features of apoptosis[26,27].
In the current work, we selected two apoptosis-regulated
gene bax and p53, which were confirmed to be involved in the control
of apoptosis.Proapoptotic gene bax, one of the members of apoptosis-regulated
gene bcl-2
family, was a dominant inhibitor of bcl-2
and promoted cell apoptosis[28-30].
Tumor supressor gene p53 was a special related gene with activation
of cell-cycle
arrest and apoptosis[31,32].
A very important role of normal or wild type p53 as a “guardian
of the genome”
was capable of inducing apoptosis upon DNA damage. Recent studies
suggested that activation of NF-kappa
B activity augmented p53-induced
apoptosis[32].
On the contrary, mutant p53 failed to initiate apoptosis process, so
mutant p53 was thought to have effect on apoptosis inhibition. More
recently, Buckley et al[33]
demonstrated that apoptosis via induction of p53, p21, and bax
proteins was seen in alveolar epithelial cells cultured from
hyperoxic rats, and apoptosis might be a protective michanism that
limits lung injury[34].
TGFβ-
family in mammalian cells was comprised of at least three homologous
polypeptides that could regulate a variety of cellular functions,
including proliferation and differentiation processes[35,36].
Recently, some investigators reported that TGFβ-1
could induce apoptosis in normal and malignant cells[37,38].
It was demonstrated that the induction of apoptosis by TGF-β-1
was due to the regeneration of reactive oxygen species in the cells,
the involvement of caspase family proteases[39-41],
induction of p53 and bax[37],
or expression of TIEG (TGFβ-
inducible early gene)[42-44]
. In this study, we sought to
explore the potential relationship between TGFβ-1
and apoptosis of alveolar epithelial cells in pancreatitis-associated
lung injury.
We found that the apoptotic index of
alveolar epithelial cells at 5 hours after induction of AHNP was
significantly higher than those of 2 and 14 hours (P＜0.01),
and the positive rate of bax protein in alveolar epithelial cells at
5 hours was markedly higher than those of 2 and 14 hours after
induction of AHNP (P＜0.05).
Interestingly, the positive
rate of bax protein in alveolar epithelial cells was paralleled to
the AI of alveolar epithelial cells in pancreatitis-associated
lung injury. The positive rate of p53 protein in alveolar epithelial
cells dramaticaly decreased at 5 and 14 hours after induction of
AHNP compared with 2 hours(P＜0.05).
Similarly, the positive rate of p53 protein in alveolar epithelial
cells was intended to parallel to the AI of alveolar epithelial
cells in PALI. Immunostaining of TGFβ-1
was detected
at 2, 5 and 14 hours after induction of AHNP, no significant
difference was observed among them. Our data demonstrated that
apoptosis of alveolar epithelial cells might be mediated by
apoptosis-regulated
gene bax and p53, but TGFβ-1
was not implicated in apoptosis of alveolar epithelial cells.
In summary, apoptosis of alveolar epithelial cells might be
involved in PALI, and the mechanisms of apoptosis probably
correlates with the expression of
apoptosis-regulated
gene bax and p53 but is not related with the expression
of TGFβ-1.
Recent studies[45]have
suggested that mild pancreatitis was found to be associated with
extensive apoptotic acinar cell death while severe pancreatitis was
noted to involve extensive acinar cell necrosis but very little
acinar cell apoptosis. More recently, some investigators[46-48]have
found that induction of apoptosis in pancreatic acinar cells
attenuates the severity of experimental acute pancreatitis.
Furthermore, Kaiser et al[45]have
demonstrated that inhibition of apoptosis by administration of
cyclohexamide could enhance the severity of pancreatitis. These data
have led to the concept that pharmacological induction of apoptosis
in pancreatic acinar cell injury to reduce inflammatory reaction
provides a new therapeutic strategy for the treatment of acute
pancreatitis. Very recently, we found that the mechanisms
of SSa in treating acute pancreatitis might be induction of
apoptosis in pancreatic acinar cell injury to reduce inflammatory
response[49].
Future studies for clarifying the initiators, regulators,
genetic control and signal transductal pathway of apoptosis of
alveolar epithelial cells, would be
able to reveal the pathogenesis of PALI, and further elucidate the
role of apoptosis of alveolar epithelial cells in PALI.
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