Effect
of Tangweian Jianji on the Biomechanical and Morphometric Remodeling of
Colon and Rectum in STZ Induced Diabetic Rats
Hong Sha, Xiao-Lin
Tong, Gui-Fang Liu, Zhong Zhen, Peng-Min Chen, Hans Gregersen, JingBo Zhao
Hong Sha, Peng-Min Chen, Institute of Clinical
Medicine, China-Japan Friendship Hospital, Beijing 100029, China
Xiao-Lin Tong, Gui-Fang
Liu, Zhong Zhen, Guang’anmen
Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
JingBo Zhao, Mech-Sense,
Aalborg Hospital, DK 9000 Aalborg, and Institute of Clinical Medicine, Aarhus
University, DK8200 Aarhus N, Denmark
Hans Gregersen, Sino-Danish
Centre for Education and Research, Aarhus, Denmark and Beijing, China
Correspondence to: Jingbo
Zhao, Mech-Sense, Aalborg Hospital, DK 9000 Aalborg, and Institute of Clinical
Medicine, Aarhus University, DK8200 Aarhus N, Denmark. jz@rn.dk
Telephone: +
45-99326907
Fax:
+45-99326801
Received: March 3, 2012
Revised: March 29, 2012
Accepted: April 5, 2012
Published
online:
July 21, 2012
ABSTRACT
AIM: The aim of the study was to investigate the effect of TWAJJ on the
biomechanical and morphometrical remodeling of colon and rectum in
streptozotocin (STZ) induced diabetic rats.
METHODS: The colonic and rectal segments obtained from diabetic
(DM), TWAJJ treated diabetic (TH, high dosage: 10 g/kg; TL, low dosage: 5 g/kg)
and normal (CON) rats were used for the study. Blood glucose and serum insulin
levels were measured. At the end of experiment, the circumferential length, the
wall thickness and the opening angle were measured from the digitized images of
those segments and residual strain was computed. Circumferential and
longitudinal stresses and strains were computed from the length, diameter and
pressure data and from the zero-stress state geometry for the colonic
segment.
RESULTS: The glucose and insulin levels did not differ among DM,
TH and TL groups. Wet weight, wall thickness, cross-sectional wall area,
opening angle, and absolute values of residual strain of colonic and rectal
segments in DM group were significantly higher than those in CON group (P<0.05
and P<0.01), whereas those parameters in TH group but not in TL group
were significantly lower than those in DM group (P<0.05, P<0.01).
Furthermore, the circumferential and longitudinal stiffness of the colonic wall
increased in DM group compared those with CON group. TH but not TL treatment
could significantly decrease the colonic wall stiffness in both directions (P<
0.01).
CONCLUSION: Although TWAJJ (high dose) treatment could not
significantly correct the changes of blood glucose and serum insulin levels in
the diabetic rats; it could partly restore the morphometric and biomechanical
remodeling of lower GI tract caused by diabetes.
© 2012 Thomson research. All rights reserved.
Key words: Diabetes rats;
Lower gastrointestinal tract; Tangweian Jianji; Biomechanics; Morphometry;
Remodeling
Sha H, Tong XL, Liu GF, Zhen Z, Chen PM, Gregersen H,
Zhao J, Effect of Tangweian Jianji on the Biomechanical and Morphometric
Remodeling of Colon and Rectum in STZ Induced Diabetic Rats. Journal of Gastroenterology and
Hepatology Research 2012;
1(6): 86-91 Available from: URL: http://www.ghrnet.org/index./joghr/
INTRODUCTION
Diabetic gastrointestinal disorder (DGID) is a common
complication of diabetes. Up to 76% of diabetic patients showed significant
gastrointestinal (GI) symptoms including dysphagia, early satiety, reflux,
constipation, abdominal pain, nausea vomiting and diarrhea[1,2]. The
motility disturbances of colon and rectum were seen in human diabetes[3-4].
Delayed transit of colon was reported in IDDM patients and the symptoms related
closely to the region impaired[5]. The mean total colonic transit
time of the diabetic patients was significantly longer than that in healthy
subjects. Furthermore, the diabetic patients with constipation showed longer
total, left and recto-sigmoid colonic transit times than those without
constipation[6].
The GI tract is functionally subjected to dimensional changes. Hence,
biomechanical properties such as the stress-strain relationships are of
particularly importance[7]. The biomechanical properties are crucial
for GI motor function because peristaltic motion that propels the food through
the GI tract is a result of interaction of the passive and active tissue forces
and the hydrodynamic forces in the food bolus. The remodeling of the mechanical
properties reflects the changes in the tissue structure that determine a
specific motor dysfunction. Previous study demonstrated that experimental
diabetes induces colon morphological and biomechanical remodeling[8].
Following the development of diabetes, the colonic wall becomes thicker and the
stiffness of the wall increases in a time-dependent manner. Therefore, the
diabetic GI morphological and biomechanical remodeling plays an important role
in DGID and has become the new perspective of diabetic GI pathogenesis[9].
The western medicine treatment of DGID mainly focused on symptomatic
control with improvement of gastric motility, promoting agents and supportive
measures based on blood glucose control[2]. However, as its
pathogenesis is unclear, the basic paresis factors still exist. Although these
therapy methods can partially improve the clinical symptoms, it can not
fundamentally reverse the diabetes-induced changes with very high relapse rate.
Clinical practice showed that TWAJJ could improve the DGID significantly with
low relapse rate, but the mechanism is not fully understood. Therefore, the aim
of the present study was to investigate if TWAJJ treatment can improve the
morphometric and biomechanical remodeling of colon and rectum in STZ-induced
diabetic rats.
MATERIALS AND METHODS
Animal model and groups
Forty male Sprague Dawley (SD) rats weighing 220-250g
were included in this study. Thirty rats were made diabetic by a single tail
vein injection of 40 mg/kg streptozotocin (STZ, Sigma-Aldrich, China).This dose
of STZ resulted in a random blood glucose level ≥16.7 mmoL/L in 90% of rats after 7 d of injection. The remaining 10% of
rats were excluded from this study. Twenty-seven STZ induced diabetic rats were
subdivided into three groups (n=9 in each group), i.e., diabetic control
group (DM); high dose of TWAJJ group (TH) and low dose of TWAJJ group (TL).
Another 10 rats of similar age and body weight from the same vendor were used
as non-diabetic control group (CON). The rats in DM, TH, TL and CON groups were
fed with the same isocaloric diet.
Drugs and administration methods
TWAJJ is composed of Atractylodes macrocephala,
Citrus aurantium, Wine rhubarb, Pinellia and Ginseng provided by Guang’anmen
Hospital, China Academy of Chinese Sciences. The medicine was directly injected
into stomach lumen by gastric lavage once daily from the beginning of
experiment. The dosage was 10 g/kg for TH and 5 g/kg for TL respectively. The
rats of DM and CON groups were only poured the physiological saline.
Experimental procedures
Body weight and blood glucose levels were measured at
2-weeks intervals after initiating the experiment. The insulin level was
measured at the end of the experiment.
The experimental period was 60 d. At the ending of the experiment, the
rats were fasting overnight and anesthetized with 4% Chloral hydrate (10 mL/kg,
ip). Following laparatomy, the middle part of colon and whole rectum were
harvested. After gently cleaning the lumen of the segments with saline, the
length and the wet weight were measured. The colonic segment were divided into
two parts, the proximal 1 cm long segment were used for zero-stress state
experiment. The remaining part was used for distension test. The rectal segment
was only used for the zero-stress state experiment.
Zero-stress state experiment
For obtaining data on the zero-stress state, three
1-2 mm wide colonic and rectal rings were cut and placed in the Krebs solution
with EGTA (100 mg/L) at room temperature. The composition of Krebs solution
(mmol/L) is: NaCl, 118; KCl, 4.7; NaHCO3, 25; NaH2PO4,
1.0; MgCl, 1.2; and ascorbic acid, 0.11. A photograph was taken of the
cross-section of the rings by camera (Canon, Japan) and it is presented as the
no-load state. Then each ring-shaped segment was cut radially from the opposite
mesentery site and the photographs were taken about 60 min after the radial
cutting to allow viscoelastic creep to take place. This is presented as the
zero-stress state.
Distension test
The distal end of the remaining colonic segment was
tied with a suture and the proximal end was cannulated with a tube for the
distension experiment. After preconditioning of the segment two times from 0 to
20 cmH2O, it was inflated with Krebs solution using a
step-wise distension protocol up to 20 cmH2O (0, 1, 2, 3, 5,
10, 15 and 20 cm H2O). The segment conformed to a cylindrical
geometry during the distensions. Each pressure lasted for 2 m and then the
outer diameter and length of the segment was photographed by the camera (Canon,
Japan).
Mechanical data analysis
The morphometric data were obtained from digitized
images of the segments in the zero-stress, no-load and pressurized states.
Measurements were undertaken using image analysis software (Sigmascan ver. 4.0,
Sigma Corp.,
The measured data was used for computation of
biomechanical parameters defined as:
Residual Green's strain at the mucosal surface: [1]
Residual Green's strain at the serosal
surface: [2]
The stress and strain of the colonic segment in the
pressurized state were determined under assumptions that the wall was
homogenous and the organ shape was cylindrical. Calculation was done from
knowing the no-load state dimensions, the outer diameters and lengths of the
specimen at varying pressures, and assuming incompressibility of the wall. The
longitudinal stretch ratio, ; the luminal radius, ; the wall thickness, ; the mucosal
circumferential length, ; the serosal
circumferential length, ; the mid-wall
circumferential length, ; the circumferential
stretch ratio, (where the middle-wall circumferential
length at zero-stress state, ) were computed. Then the
Kirchhoff's stress and Green's strain in a wall at a given pressure were
computed according to the following equations:
Circumferential Kirchhoff's stress: [3]
Longitudinal Kirchhoff's stress:
[4]
Circumferential mid-wall Green's strain: [5]
Longitudinal Green's strain:
[6]
DP is the transmural pressure
difference. The
longitudinal mid-wall stretch ratio was referenced to the no-load state because
tissue strips could not be cut for obtaining the zero-stress state in
longitudinal direction. However, the longitudinal mid-wall length in rat
intestine does not differ between the no-load and zero-stress states [10].
Statistical analysis
The data were representative of a normal distribution
and accordingly the results were expressed as means±SEM. The stress-strain
curve for each direction was fitted using the exponential function
equation
.
S* and E* are the stress and strain at
a physiological reference level[7]. The
constants a and b from the above exponential function were used for the
statistical evaluation of the stress-strain data. Analysis of variance was used
to detect the differences of different parameters in different groups
(Sigmastat 2.0TM). The results were regarded as significant when p<0.05.
RESULTS
The blood glucose, serum insulin levels and body weight
The blood glucose, serum insulin levels and body
weight at the ending of experiment were shown in Figure 1. The blood glucose
level was about 4-fold higher in DM group compared with CON group (Figure 1A, p<0.01).
Whereas the serum insulin level was significantly lower in DM group compared
with CON group (Figure 1B, p<0.01). The body weight in DM group
decreased nearly 50% compared with that in CON Group (Figure 1C). Compared with
DM group, the blood glucose level did not significantly decrease (Figure 1A, p>0.05)
and serum insulin level did not significantly increase (Figure 1B, p>0.05)
in TH and TL groups. It indicated that both the low and high dosage of TWAJJ
did not obviously affect the blood glucose and serum insulin level. However,
after treatment with high dosage of TWAJJ could partially increased the body weight
compared with that of DM group (Figure 1C, p<0.05)
The weight/cm to body weight ratio, wall thickness and wall area
The wet weight per unit length to body weight ratio
(Figure 2A), no-load wall thickness (Figure 2B) and cross-section wall area
(Figure 2C) of colonic and rectal segments were significantly increased in DM
group compared with CON group (p<0.01). After treatment with TH,
those parameters significantly decreased in both of two segments (Figure 2, p<0.05
and p<0.01); however those parameters did not significantly change in
TL group (p>0.05) with exception of wall thickness of colon (Figure
2B, p<0.05).
Opening angle and residual strain
At the end of the experiment, the opening angle of
both colonic and rectal segments was significantly increased in DM group
compared with CON group (Figure 3A, p<0.05). Treatment with high
dosage of TWAJJ significantly decreased the opening angle (Figure 3A, p<0.05);
whereas the opening angle did not change in TL group (p>0.05).
The similar pattern as the opening angle was found for the inner
residual strain in both segments and outer residual strain for rectal segment
(Figure 3B), i.e., the absolute value of residual strain of both colonic and
rectal segments was significantly increased in DM group compared with CON group
(Figure 3B and 3C, p<0.05). Treatment with high dosage of TWAJJ (TH
group) could partially recover the changes of residual strain (Figure 3B and
3C, p<0.05), but not in TL group.
Stress-strain distribution
At the end of the experiment, the stress-strain
analysis showed that both the circumferential and longitudinal stress-strain
curves of colonic segment (Figure 4A and 4B) in DM group shifted to the left
compared with those in CON group. It indicated that the colonic wall became
stiffer due to diabetes. Computation of constant a showed the significant
difference between DM group and CON group (Figure 5A and 5B, p<0.05).
High dosage of TWAJJ (TH) treatment significantly decreases the stiffness of
colonic wall in both circumferential (Figure 4A, 5A, p<0.05) and
longitudinal (Figure 4B, 5B, p<0.05) direction. Low dosage of TWAJJ
treatment (TL) did not show the improvement of the stiffening of colonic wall
caused by diabetes (Figure 4 and 5, p>0.05).
DISCUSSION
According
to WHO report; it is expected to reach 370 million DM patients by 2030. The
serious impact caused by diabetes and its complications are not only on the
patients themselves but also the heavy financial burden to the state and
families[11]. DGID is a common complication of diabetes[2].
The ano-rectal symptoms in the diabetic patients correlated better with the
manometric abnormalities[4]. The manometric abnormities may relate
to the changes of biomechanical properties in the diabetes[9].
Previous study showed that prominent proliferation and biomechanical remodeling
of colonic wall was occurred in experimental diabetes[8]. The main
findings were found at the present study that the morphometric and
biomechanical remodeling occurred in STZ-induced diabetic rats. This confirmed
the findings of previous study[8]. Furthermore, it was demonstrated
that TWAJJ (high dose) treatment could partly restore the morphometric and
biomechanical remodeling of colonic and rectal segments caused by diabetes.
In the view of
traditional Chinese medicine, DGID is in the category of fullness, epigastric
pain, vomiting, diarrhea, and constipation concurrent with diabetes. The major
syndrome of DGID is spleen deficiency and qi stagnation. The principle
treatment is to strengthen spleen and move stagnation. TWAJJ is a clinic
practical Chinese medicine compound on the diabetic gastrointestinal
dysfunction[12-14] composed of several herbs, such as Atractylodes
macrocephala, Cictrus aurantium, Pinellia downbear and Ginseng. Each of these
herbs has some specific effects[15-20]. However, the TWAJJ seems
mainly to effect on up-GI tract, such as gastroparesis. We also in another
study (un-published data) demonstrated that the treatment with high dosage of
TWAJJ could partially improve the morphomeric and biomechanical remodeling of
Up-GI tract. This indicated the mechanism of TWAJJ effect on DGID is at least
partially through this pathway. Therefore, it is also interesting to
investigate if TWAJJ can through the same pathway to affect the lower GI tract
in the diabetes.
The colon and rectum are tubular organs. One
important function is transportation of the content by the wall movement;
therefore the biomechanical properties of the wall are important
for the function[21]. The
biomechanical properties of the colon and rectum depend on its structure and
can be evaluated by opening angle, residual stress and strain, and
stress-strain relationship[8]. The previous studies demonstrated the
colonic wall remodeled during the development of the diabetes. The present
study confirmed the previous findings that the morphometric and biomechanical
remodeling of colonic wall occurred in STZ-induced diabetic rats. Furthermore,
it was also showed the remodeling was occurred in the rectal segment. Although
the treatment with TWAJJ did not significantly changes the blood glucose and
serum insulin levels, high dosage of TWAJJ does partially improve the
morphometric and biomechanical remodeling caused by diabetes. Improvement of
the morphometric remodeling expresses as reducing the wall thickness and area.
Improvement of biomechanical remodeling expresses as reducing the opening
angle, the absolute value of residual strain and decreasing the wall stiffness.
The present study clearly indicated that the high dosage of TWAJJ could partially
improve the biomechanical remodeling of lower GI wall in the STZ-induced
diabetic rats.
The morphological changes, such as increasing wall thickness caused by
diabetes will change the relative location of mechanosensitive afferents in the
GI tract[22-23]. The alterations of residual stress and wall
stiffness in the diabetes will change the tension and stress distribution in
the location of mechanosensitive afferents. Therefore the morphometric and
biomechanical remodeling of GI wall in the diabetes in this way will affect the
GI sensory and motor function. High dosage of TWAJJ could partially correct the
disorders of morphometric and biomechanical remodeling caused by diabetes;
therefore can improve the motor and sensory dysfunction of diabetic GI tract
through this mechanism.
In this study, we adopted STZ-induced diabetic rat model. In order to
get high glucose level and obvious remodeling of GI tract, we did not treat
with insulin for the diabetic rats. However, it is well known that in the
nowadays very common settings where patients are insulin- treated for diabetes
type 1, or non-insulin dependent diabetes type 2. Therefore, we will consider
including the group of type-1 diabetic rats treated with insulin and adapt the
type-2 diabetic animal model in the future studies. Furthermore, in order to
overcome the possible variations among the different groups and to investigate
the effect of TWAJJ on different time point during the development of diabetes,
the data in different study groups of rats at baseline and at certain time
intervals after initiating the experiment should be taken into account in the
future studies.
In conclusion, High dosage of TWAJJ treatment could partly improve the
biomechanical and morphometric remodeling of colonic and rectal wall in the
diabetes. This indicated that the TWAJJ not only improve the up-GI disorders in
the diabetes, but also may improve the low GI disorders in the diabetes.
Therefore, it is possible to explore the new application of TWAJJ to the diabetic
patients in the clinics. Furthermore, it is important to develop some Chinese
herbs, such as TWAJJ, to improve the morphometric and biomechanical remodeling,
and then further improves GI dysfunction caused by diabetes.
ACKNOWLEDGEMENTS
The study was financially supported by National Key
Basic Research Program of China (973),No.2010CB530600. Mingyi Yuan and
Jia-Cheng Zhang are thanked for help to do animal experiments.
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Peer reviewers: I. Michael Leitman, MD, Chief Of
General Surgery, Albert Einstein College Of Medicine-Beth Israel Medical
Center, 10 Union Square East, 2M, New York, USA; Claudio Chiesa, MD, Institute
of Translational Pharmacology, National Research CouncilVia del Fosso del
Cavaliere,100, 00133-Rome, Italy.
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