|
Xin
Wang, Zong-You Zhang, Mei Lan, Ji-Yan Miao, Xue-Gang Guo, Yong-Quan
Shi, Yan-Qiu Zhao, Jie Ding, Kai-Cun Wu, Dai-Ming Fan, Institute of
Digestive disease, Xijing Hospital, Fourth Military Medical
University, Xi'an 710032, Shaanxi Province, China
Yue-Xia Zhong, Emergency Department, Tangdu Hospital, Fourth Military
Medical University, Xi'an 710038, Shaanxi Province, China
Ju Lu, Class EE 87, Department of Electronic Engineering, Tsinghua
University, Beijing 100084, China
Bo-Rong Pan,Oncology Center, Xijing Hospital, Fourth Military
Medical University, Xi'an 710032, Shaanxi Province, China
Supported by National Natural Science Foundation of China,
No.39970901
Correspondence to: Prof. Dai-Ming Fan, Institute of Digestive
Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an
710033, Shaanxi Province, China. daimfan@pub.xaonline.com
Telephone: +86-29-3375221 Fax: +86-29-2539041
Received 2001-08-23 Accepted 2001-09-05
Abstract
AIM:
To
investigate the effect of L-NAME on nitric oxide and
gastrointestinal motility alterations in cirrhotic rats.
METHODS:
Rats
with cirrhosis induced by carbon tetrachloride were randomly divided
into two groups, one(n=13)receiving
0.5mg·kg-1 per day of N G-nitro-L-arginine methyl ester
(L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas
the other group (n=13) and control (n=10) rats were
administrated the same volume of 9g·L-1 saline. Half
gastric emptying time and 2h residual rate were measured by SPECT,
using 99mTc-DTPA-labeled barium sulfate as test meal.
Gastrointestinal transition time was recorded simultaneously. Serum
concentration of nitric oxide (NO) was determined by the kinetic
cadmium reduction and colorimetric methods. Immunohistochemical SABC
method was used to observe the expression and distribution of three
types of nitric oxide synthase (NOS) isoforms in the rat
gastrointestinal tract. Western blot was used to detect expression
of gastrointestinal NOS isoforms.
RESULTS:
Half
gastric emptying time and trans-gastrointestinal time were
significantly prolonged(124.0±26.4min; 33.7±8.9min;72.1±15.3min; P<0.01),
(12.4±0.5h; 9.5±0.3h; 8.2±0.8h; P<0.01), 2h residual
rate was raised in cirrhotic rats than in controls and cirrhotic
rats treated with L-NAME(54.9±7.6%,13.7±3.2%, 34.9±10.3%, P<0.01).
Serum concentration of NO was significantly increased in cirrhotic
rats than in the other groups (8.20±2.48)μmol·L-1
, (5.94±1.07)μmol·L-1 ,and control (5.66±1.60)μmol·L-1
, P<0.01. NOS staining intensities which were mainly
located in the gastrointestinal tissues were markedly lower in
cirrhotic rats than in the controls and cirrhotic rats after treated
with L-NAME.
CONCLUSION:
Gastrointestinal
motility was remarkably inhibited in cirrhotic rats, which could be
alleviated by L-NAME.Nitric oxide may play an important role in the
inhibition of gastrointestinal motility in cirrhotic rats.
Wang
X, Zhong YX, Zhang ZY, Lu J, Lan M, Miao JY, Guo XG, Shi YQ, Zhao YQ,
Ding J, Wu KC, Pan BR, Fan DM. Effect of L-NAME on nitric oxide and
gastrointestinal motility alterations in cirrhotic rats. World J
Gastroenterol 2002;8(2):328-332
INTRODUCTION
Investigation
in gastrointestinal motility has promoted the interest of
researchers in the functional changes in gastrointestinal motility
of cirrhotic patients[1-8]. It is validated that nitric
oxide (NO) plays a pivotal role in neural transduction and
gastrointestinal motility regulation as a neurotransmitter and
messenger molecule with various physiological functions[9-19].
However, the relationship between NO and functional changes in
cirrhotic gastrointestinal motility is not yet clear[20-27].
We used nitric oxide synthase (NOS) -specific inhibitor to treat
cirrhosis model rats and observe changes in their gastrointestinal
motility so as to disclose the role of NO in such changes and to
provide experimental basis for diagnosis of cirrhotic
gastrointestinal motility abnormalities.
MATERIALS
AND METHODS
Preparation
of animal model
Male SD rats were provided by the Experimental Animal Center, the
Fourth Military Medical University, weighting (250±50)g, fed with
standard granule food, and randomly divided into model group(26
rats) and normal control group (10 rats). CCl4 toxic
cirrhosis model preparation: rats in the model group received
subcutaneous injection of CCl4 (3mL·kg-1)
twice a week for 12 weeks; and rats in the control group received
olive oil (3mL·kg-1) twice a week for 12 weeks. At the
end of 12 weeks, cirrhosis model group was randomly subdivided into
treated group and untreated group. Normal SD rats were served as
controls, each group having 10 rats. Rats in the treated group were
given 0.5mg·kg-1 d-1 NG-nitro-L-arginine
methyl ester (L-NAME, Sigma product, USA), a NOS inhibitor, through
intragastric administration for 10 days, and the untreated group and
control group received gastric delivery of 9g·L-1 NaCl
once a day for 10 days. Hepatic tissue samples were normally fixed
with 40g·L-1 polyformaldehyde, prepared into paraffin
wrapped sections, stained with HE and observed under light
microscope.
Analysis
of gstrointestinal motility
Examination of gastric emptying Experimental animals were fast for
over 12h without receiving drugs or food that might influence
gastric motility during the previous 2 weeks.!Each rat received 2mL
BaSO4 mixed with 3.7GBq·L-1 99mTc-DTPA
through the esophagus into the stomach in 2min, and was placed lying
on the back. SPECT (Type CT, Sophycamera Co, France) detector was
focused on the abdomen of the animals with the whole stomach as the
Region of Interest (ROI); radioactivity was recorded and images were
displayed for 60s as the total radioactivity. Gastric images were
then taken at 15, 30, 45, 60, 90 and 120min, delineating the ROI;
the curve of time-half gastric emptying was drawn and gastric
residual radioactivity rates at different time points were
calculated by computer.
Total
gastrointestinal transition time (TGIT) After
isotopic scanning, the time of BaSO 4 excretion through anus was
recorded.
Alterations
of NO and NOS
Assay of serum NO2ˉ/NO3ˉcontent
Rats
were decapitated and the blood was collected and quietly placed for
60min. After 4000r·min-1 centrifugation for 15min, the
supernatant was used for NO2ˉ/NO3ˉ
measurement. Operation was performed following the instructions of
the reagent kit (purchased from Institute of Nuclear Medicine,
General Hospital of Chinese PLA).
Expression
of NOS in gastrointestinal tract Immunohistochemical
method was used with immunohistochemical reagents from Wuhan Boshide
Biotech Co Ltd. Five rats were randomly selected from each of the
three groups. Each rat was anesthetized with 10g·L-1
sodium pentabarbitol (50mL·kg-1 ) through abdominal
cavity injection and perfused with 40g·L-1
polyformaldehyde for 1.5h. Gastric, small intestinal and colonic
tissues were immediately taken out and immersed in 200g·L-1
sucrose solution at 4℃for
24h until the tissues sank to the bottom. Then the tissues were
cryotomized at -20℃
into slices 14-16μm thick. The tissues were rinsed three times
with 0.01mmol·L-1 PBS for 5min; treated with peroxide
and methanol for 15min; vibrated and washed with PBS for 5min three
times; and blocked with normal bovine serum for 30min. Rabbit
anti-NOS1 (1:100 rabbit polyclonal antibody), anti-NOS2
(1:50 rabbit polyclonal antibody), anti-NOS3 (100 rabbit
polyclonal antibody) were added and the solution was incubated at 4℃
overnight. Then it was vibrated and washed with PBS three times for
5min. Biotinized sheep-anti-rabbit lgG was added and the solution
was let react at 37℃
for 30min. The system was washed again with 0.01mmol·L-1
PBS for 5min×3; SABC was added and let react at 37℃
for 30min; and washed with 0.01mmol·L-1 PBS for 5min×4.
The stain was developed with DAB and stained with lignin. The sample
was normally dehydratized till transparent and sealed with neutral
resin. Microscopic observation was done and photo taken under
microscope.
Determination
of NOS in gastrointestinal tract with Western blot Animals fasted
12h before experiment without water deprivation. Three rats each
from treated group, untreated group and control group were randomly
chosen, decapitated, and eviscerated immediately to obtain the
stomach, small intestine and colon, which were rinsed in ice-water
containing 0.1mmol·L-1PMSF, frozen in liquid nitrogen
and moved into -70℃
refrigerator for preservation. The whole process should be finished
within 5min. Tissue lysis liquid was prepared with ion-free water
containing 0.1mmol·L-1 PMSF. The gastrointestinal
tissues were weighed and homogenized (3500r·min-1, 5s×5)
in ice-bathing, the mass/volume ratio of tissue to tissue lysis
liquid being 1:5. Centrifugation was then performed at 12500r·min-1
for 10min; the supernatant was separated and preserved at -70℃;
the protein concentration in the extract was measured by the
Bradford method.
Eighty
g·L-1 PAGE gel was prepared and 2×SDS loading buffer
was added to the protein samples, followed by heating at 100℃
for 3min. Centrifugation was performed again and protein samples of
the same amount were added. Electrophoresis was done with 20mA
current; and the gel was stained with Coomassie brilliant blue.
Separation of protein extracts of the stomach, small intestines and
colons of the rats was accomplished with 80g·L-1 SDS-PAGE.
After electrophoresis, electric transfer of the proteins onto NC
membrane was done with a constant current of 0.8mA·cm-2
for 1h using semi-dry electric transfer device (Beijing 61 Factory).
Transfer buffer ingredients (25mmol·L-1 Tris-Hcl,
192mmol·L-1 glycine, 10g·L-1 SDS, 200g·L-1
methanol, pH8.3. TBS pH7.5+50g·L-1 non-fat milk+0.5g·L-1NP-40)
were used for blocking the sample for 2h at RT. Primary antibody NOS1,
NOS2 and NOS 3 (1:100 rabbit multiclonal antibody) was
diluted with TBS buffer containing 1g·L-1 BSA and added.
The sample was incubated at 4℃
for 16-18h and rinsed in TBS three times for 10min. HRP linked
sheep-anti-rabbit secondary antibody (1:400, Boshide Co.) was
diluted with TBS, added to the sample and let react for 2h at RT.
The sample was then rinsed in TBS+1 g·L-1 NP-40 10min×5
and developed with DAB.
Statistical
analysis
Analysis of variance was conducted using NOSA statistics program
(Fourth Military Medical University), and the results were presented
in form of mean±SD.
RESULTS
Establishment
of rat model with cirrohosis
Model rats had hepatomegaly and splenomegaly, the liver became hard,
the edge turned blunt, and the surface was not smooth, with nodules
of varied sizes. Light microscopy showed hepatocyte regeneration,
fat degeneration, proliferation of collagen fibers, and pseudo-lobulation.
Gastrointestinal
motility
Gastrointestinal motility for barium Gastric semi-solid emptying:
after intragastric administration of BaSO4 containing 99mTc-DTPA,
abdominal radioactivity images were taken in 8 rats in normal
group(Figure 1A), untreated group (Figure 1B) and treated group
(Figure 1C) each at 5, 30, 90 and 120min. The images showed that
gastric emptying of cirrhotic rats was slowed down, and after
treatment it was accelerated. The residual rate of gastric
semi-solid substance of rats was observed dynamically.
Gastric
emptying of cirrhotic rats was slowed down, the 2h residual rate
being increased significantly. After L-NAME treatment, the gastric
emptying was accelerated, the 2h residual rate decreased
significantly (Figure 2 and Table 1).
Figure
2(PDF)Residual rate curves of gastric semi-solid substance of rats
Table
1 Half-emptying
time and 2h residual rate (n=8)
|
Groups
|
GET1/2(min)
|
RR2h(%)
|
|
Control
|
33.7
|
13.7
|
|
Untreated
|
124.0
|
54.9b
|
|
Treated
|
72.1
|
34.9
|
bP<0.01,
vs control and treated. GET1/2: half-emptying
time; RR2h: 2h residual rate
Gastrointestinal
transition time TGIT of rats in untreated group (12.4±0.5)h was
significantly longer (P<0.01) than that in the control
group (9.5±0.3)h, whereas TGIT of rats in treated group (8.2±0.8)h
was significantly shorter (P<0.01). Figure 3 shows that
gastrointestinal transition of barium sulfate apparently slowed down
in cirrhotic rats, and was significantly accelerated after L-NAME
treatment; and barium sulfate was excreted in 6h after delivery.
Figure
1
Gastric emptying for barium in rats. A: Normal; B: Cirrhotic; C:
Cirrhotic treated with L-NAME
Figure
3
X-ray analysis of rat gastrointestinal motility. A, B: Control rats
at 5min and 6h; C, D: Untreated rats at 5min and 6h; E, F: Treated
rats 5min and 6h
Figure
4
Expression and distribution of NOS in rat gastrointestinal tract
with cirrhosis.
Alterations of NO and NOS
Serum concentration of NO The serum NO2ˉ/NO3ˉ
concentrations were (8.20±2.48)μmol·L-1 , (5.94±1.07)μmol·L-1,
and (5.66±1.60)μmol·L-1 in the rats of untreated
group, normal control group, and treated group, respectively. It was
apparent that NO concentration in the untreated group was
significantly higher than in other groups (P<0.01).
Expression
of NOS in rat gastrointestinal tract NOS immunohistochemical
staining showed that NOS1, NOS2 and NOS 3 had
similar distribution in gastrointestinal mucosal lamina propria
layer, principally in neutrophiles, monocytes, macrophages and some
lymphocytes of gastrointestinal mucosal lamina propria layer
interstitial. NOS1 existed mainly in intermuscular nerve
bundles in the gastrointestinal wall, endocrine cells in the mucosal
layer, macrophages in gastrointestinal mucosal lamina propria layer
interstitial, and some lymphocytes. In normal rats, NOS positive
cells were mainly located in the lower third part of the gastric
mucosal layer, in the intermuscular nerve bundles and villus
interstitial of small intestine, and pervasively in colonic mucosal
villus interstitial (Figure 4). In cirrhotic rats, NOS positive
cells decreased significantly in the whole gastrointestinal tract
and intermuscular nerve bundle.These two indexes in cirrhotic rats
treated with L-NAME were significantly higher than those in
untreated rats.
Western
blot analysis Protein electrophoresis showed that the sampling
amounts of gastric, small intestinal and colonic proteins of rats in
the three groups were the same, and that the protein composition in
the small intestine was quite different from that in the stomach and
the colon. Western blot showed that NOS expression decreased
significantly in the gastric and colonic tissues of cirrhotic rats,
and it returned to normal after treated with L-NAME. NOS was not
detected in the small intestine in either groups.
Figure
5
Expression of NOS1 in stomach(1,2,3), intestine(4,5,6)
and colon(7,8,9) of rats 1,4,7: Control; 2,5,8: Cirrhotic; 3,7,9:
Treated
DISCUSSION
NO
plays an important role in gastrointestinal physiological activities
as well as in the pathogenesis and progress of many severe diseases[27-31].
It is involved in the regulation of gastrointestinal smooth muscle
contraction and secretion of water and salt of intestinal epithelial
cells[32,33]. It mediates endotoxin-induced inhibition of
gastric acid secretion, protects gastrointestinal mucosa, sustains
mucosal blood flow, inhibits neutrophile adhesion to vascular
endothelium and blocks platelet adhesion; prevents macrophage
activation. NOS is the rate-limiting enzyme of NO synthesis, which
exists pervasively in gastrointestinal tissues, including epithelia,
fibroblasts, macrophages, inherent and infiltrating lymphocytes,
neutrophiles, monocytes, smooth muscle cells, endocrine cells, and
intramuscular ganglia. The kinds and densities of NOS positive cells
diverse at different regions[34-40]. NOS can be
classified into 3 types according to biological characters and
encoding genes: neuronal type nNOS (NOS1), endothelial
type eNOS (NOS3) and induced type iNOS (NOS2).
There are 50% homology between them. NOS1 primarily
exists in neural and epithelial cells. NOS NOS2 was
firstly separated from macrophages and later discovered to exist in
other kinds of cells such as vascular smooth muscle cells. NOS3
mainly exists in vascular endothelial cells. According to the
activity dependence on Ca2+/CaM, NOS has two subtypes:
constructive NOS (cNOS), including NOS1 and NOS3
whose activity is regulated by Ca2+/CaM, and induced NOS,
including NOS2 whose enzymatic activity is not dependent
on Ca2+/CaM but needs inducing factors. The cNOS
primarily exists in normal vascular endothelial cells, and is also
found in adrenal gland cells, platelets, fibroblasts, PMNs, brain
cells and certain non-cholinergenic, non-adrenalergenic synapses[41,42].
NOS
expression in gastrointestinal tissues differs in certain
pathological situations. In abdominal inflammation, positive cells
on the small intestinal wall mainly exist in the mucosal lamina
propria layer, over 80% of the positive cells are CD45 positive
inflammatory cells, about 15% are CD3 positive T lymphocytes, and
epithelial cells are all negative. In ulcerative colitis,
iNOS positive cells are mainly intesitinal epithelial cells, while
mucosal inherent cells are all negative. The status in cirrhosis is
not known yet[43-45]. NO is the major inhibitory
neurotransmitter released by non-adrenaline, non-cholergenic
neurons, which is closely related to gastrointestinal motility and
pathology. Gastric physiological expansion and intestinal
peristalsis are regulated by NO, which can directly inhibit
gastrointestinal smooth muscle contraction and retard
gastrointestinal motility. NOS inhibitor can promote ascites
re-absorption and urinary sodium excretion of cirrhotic rats, and
the rats' colonic motility recovery after abdominal operations. We
prepared a toxic cirrhotic rat model, used radioactive isotopic
method to determine gastric emptying functions of the rats, and
recorded the total gastrointestinal transition time (TGIT). The
results showed that TGIT of the rats in untreated group was
significantly longer than that of the rats in the control group and
the treated group, while gastric emptying was significantly slower
in the former. It suggested the dysfunction of gastrointestinal
motility in cirrhosis.
Cirrhotic
patients are prone to develop endotoxemia due to floratranslocation,
enhanced absorption of endotoxins and reduced hepatic
detoxification. Endotoxins stimulate vascular endothelial cells,
activate NOS, and consequently increasing NO synthesis. The serum NO
concentration in cirrhotic patients rose significantly[46-48],
and the same findings were observed in cirrhotic rat's model in our
experiment. However, immunohistochemical staining revealed the
different distribution of NOS1, NOS2 and NOS3
as described above. The quantity of NOS positive cells in cirrhotic
rat gastrointestinal tissues was significantly lower than that in
rats treated with L-NAME and normal control, so was NOS staining
intensity in nerve bundles. Western blot was used to examine the
expression of the three types of NOS in gastric, small intestinal
and colonic tissues of rats and the same results were obtained as we
did through NOS immunohistochemistry. These results indicate that
local synthesis of NO is regulated by many factors in cirrhotic rat
gastrointestinal tissues[49].
NOS-specific
competitive inhibitor L-NAME was used to treat cirrhotic rats and
the results were as follows: TGIT of untreated cirrhotic rats was
significantly longer than that of normal or treated cirrhotic rats,
and gastric emptying in the former group was significantly slower.
L-NAME treatment significantly accelerated gastric emptying and
reduced TGIT. The sreum NO concentration in cirrhotic rats was
elevated, and L-NAME treatment reduced the serum NO concentration
and gastrointestinal NO synthesis as well. These results indicate
that NO contributes greatly to cirrhotic gastrointestinal motility
dysfunction. NOS inhibitor L-NMMA can reduce the duration of small
intestinal digestive interval MMC I phase as well as the total
duration of MMC, whereas the occurrence frequency of MMC was raised
and small intestinal motility was enhanced. This might be one of the
mechanisms of L-NAME enhancing cirrhotic rat gastrointestinal
motility[50].
Disorder
of cirrhotic gastrointestinal motility is a multi-factor disease.
Our research showed that the gastrointestinal motility of cirrhotic
rats was significantly inhibited, which was demonstrated by slowed
gastric emptying and prolonged gastrointestinal transition time. As
NO activity in the serum and tissues of cirrhotic rats was
comparatively high, we used NOS-specific inhibitor to treat the rats
and removed such inhibition, and found that NO played an important
role in cirrhotic gastrointestinal motility dysfunction. Thus, we
conclude that drugs inhibiting NO synthesis would be clinically
conducive to alleviate the gastrointestinal motility dysfunction of
cirrhotic patients and could consequently reduce the occurrence of
cirrhosis-related complications.
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