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in atherosclerosis, thrombosis or injury to the great vessels
in the extremities, severe crushing injury and surgery [1-3] . It
can lead to limb edema, skeletal muscle dysfunction, and
necrosis, and may further result in the dysfunction and
structural damage to other organs such as the heart, lungs,
brain, small intestines, and so on [4,5] . Therefore, protection
against ischemia-reperfusion injury has become an important
focus of research in clinical work.
In recent years, many narcotics have been used to reduce
the damage caused by ischemia-reperfusion [6-8] . The
new anti-cholinergic drug penehyclidine hydrochloride
(PHC), reported to have protective effects against organ
injury [9-11] , was developed by the Institute of Pharmacology
and Toxicology in China (Academy of Military Medical Sciences)
to minimise side effects harmful to the cardiovascular
system [12,13] . To date, no reports on the protective effects
of PHC against LIR in the small intestines have been published.
In this study, the serum and small intestinal tissue
diamine oxidase (DAO), plasma endotoxin (reflecting the
barrier function of the small intestinal mucosa), superoxide
dismutase (SOD), and myeloperoxidase (MPO) activity of
the small intestinal tissue, as well as serum tumor necrosis
factor-α (TNF-α) and interleukin (IL)-10 levels (reflecting
damage to the small intestine) were examined in rats.
MATERIALS AND METHODS
Experimental animals
One hundred and eight healthy 6-mo-old male Wistar
rats weighing 220-250 g were provided by the Medical
Experimental Animal Center of the Gansu College
of Traditional Chinese Medicine, China. The rats were
randomly divided into 3 groups: the sham-operation
group (group S), limb ischemia-reperfusion group (group
LIR), and penehyclidine hydrochloride post-conditioning
group (group PHC). Each group was divided into subgroups
(n = 6 in each group) according to ischemicreperfusion
time, i.e. immediately (T1), 1 h (T2), 3 h (T3),
6 h (T4), 12 h (T5), and 24 h (T6).
Animal model
The LIR model was established as follows: the rats were
fasted 12 h preoperatively with unlimited drinking water;
and exposed to 2% isoflurane until the loss of righting
reflex, and fixed onto a sit-board on the operating table.
The posterior limbs of the rats were ligated with elastic
rubber bands above the greater trochanter to completely
block the blood flow. Group S was anesthetized, but did
not undergo ligation. Group LIR was ligated until complete
ischemia of the lower limbs, and released after 3 h
to restore blood flow to the posterior limbs; reperfusion
was conducted for 1, 3, 6, 12, and 24 h. In group PHC, the
rubber band was released after 3 h of ischemia, and then
0.15 mg/kg PHC was injected into the tail veins, followed
by reperfusion for 1, 3, 6, 12 and 24 h. After the reperfusion
at pre-set time points (T1-T6), the rats were sacrificed
under deep isoflurane anaesthesia. All experiments were
conducted according to the protocols approved by the
Lanzhou University Animal Care and Use Committee.
WJG|www.wjgnet.com
Zhang Y et al . Effects of penehyclidine hydrochloride in ischemia-reperfusion
About 5 mL of blood was drawn from the inferior vena
cava of the rats. The blood was centrifuged at 3000 r/min
for 15 min to separate the serum, which was stored at
-20℃ for further target detection. The small intestines
of the rats were quickly removed up to 5 cm from the
ileocecal valve and washed three times with normal saline.
Then, 0.5 g of the small intestines were ground into tissue
homogenate in a glass homogeniser; after centrifugation
at 3500 r/min for 20 min, the resultant supernatant was
diluted into a 10% solution with normal saline and stored
at -20℃ for further target detection. The remaining small
intestines were fixed in 10% formalin, embedded in paraffin,
stained with hematoxylin and eosin, sectioned, and observed
for pathological changes under optical microscope.
Evaluation of changes in the barrier function of the small
intestinal mucosa
The rat small intestinal tissue and serum DAO were detected
using a spectrophotometer with an automatic biochemical
analyser (OLYMPUS-AU5400; kit provided by
the Nanjing Jiancheng Bioengineering Institute, China).
Plasma endotoxin was measured using a quantitative
chromogenic substrate assay (Xiamen TAL Experimental
Plant Co., Ltd.).
Evaluation of damage mechanism in the small intestine
SOD and MPO activities in the rat small intestinal tissue
were measured by colorimetry (Nanjing Jiancheng Bioengineering
Institute). Serum TNF-α and IL-10 were measured
by enzyme-linked immunosorbent assay (Wuhan Boster
Biological Technology, Ltd., China).
Statistical analysis
All data were reported as mean ± SD. Statistical significance
was determined by one-way analysis of variance using
SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL,
USA).
RESULTS
Rat small intestinal pathological changes
The small intestinal microstructure of the group S rats
were normal: the villi were lined with normal epithelial cells,
the interstitium was congestion-free, and gland morphology
was normal. The small intestinal tissue of the group
LIR rats had obvious pathological changes: the villi were
malpositioned, atrophied, and shorter and thicker; loose interstitial
edema with lymphocytic infiltration was observed,
and lymph node follicles and submucosal lymphatic vessels
were filled with lymphocytes. The small intestinal microstructure
of the group PHC had certain pathologic changes
that were less pronounced than in the group LIR.
Changes in the barrier function of the small intestinal
mucosa
DAO is an enzyme synthesised primarily in gastrointestinal
mucosal cells. The intestinal tissue and serum levels of
DAO have been used as an indicator of the integrity and
functional mass of the intestinal mucosa [14,15] . When intes-
255 January 14, 2011|Volume 17|Issue 2|