Download histomorphometric studies of the aorta, pulmonary trunk and left

1 Department of Anatomy and Cell Biology, Faculty of Basic
Medical Sciences, Obafemi Awolowo University, Ile-Ife, OsunState, Nigeria
2 Department of Physiological Sciences, Faculty of Basic
Medical Sciences, Obafemi Awolowo University, Ile-Ife, OsunState, Nigeria
Original Research Article
HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF
STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS
TREATED WITH MOMORDICA CHARANTIA EXTRACT
O. A. Komolafe1, D. A. Ofusori1, O. S. Adewole1, A. A. Ayoka2, O. A. Ayannuga1
Key words: diabetes, Momordica charantia,
histomorphometry, aorta, pulmonary
trunk, left ventricle
SUMMARY
The present study investigated the effects of
Momordica (M.) charantia on physical changes and on
histomorphometry of the aorta, pulmonary trunk and
left ventricle in streptozotocin-induced diabetic Wistar
rats. Physical changes and body weight were
monitored throughout the experimental period. At the
end of the experiment, stained sections were obtained
for histomorphometry. Results showed a significant
(p<0.05) difference in the mean body weight of the
control and treated groups when compared with
diabetic group. Also, there was a significant increase
(p<0.05) in myocardial thickness and tunics of the
aorta and pulmonary trunk in diabetic animals
compared to other groups and control. In conclusion,
M. charantia exhibit cardiovascular protective
potential possibly via its anti-atherogenic properties.
Corresponding author: O. A. Komolafe, Department of Anatomy and, Cell
Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University,
Ile-Ife, Osun-State, Nigeria
E-mail: [email protected]
Diabetologia Croatica 42-1, 2013
INTRODUCTION
Diabetes mellitus (DM) is a serious metabolic
disorder with micro- and macrovascular complications
that result in a significant morbidity and mortality. The
increasing proportion of the aging population,
consumption of calorie rich diet, obesity and sedentary
lifestyle have led to a tremendous increase in the
number of diabetics worldwide (1). Diabetes mellitus
is one of the most important world health problems,
especially in developing countries where the
prevalence and incidence rates are highest. Diabetic
patients are particularly prone to cardiovascular
diseases including hypertension, atherosclerosis,
diabetic cardiomyopathy, congestive heart failure and
cardiac autonomic neuropathy (2). Coronary
atherosclerosis and cardiomyopathy occur as a result
of the metabolic abnormalities associated with
diabetes (3).
Momordica (M.) charantia (Linn Family:
Cucurbaceae) is one of the popular herbs that grows in
different regions of Nigeria. It is commonly called
bitter melon, bittergourd, balsam pear. Bittergourd is
known in some tribes of Nigeria as Ejirin wewe
(Yoruba) Okban, Ndeme (Igbo) and Garafun (Hausa).
It is a slender, climbing annual vine with long-stalked
leaves and yellow, solitary male and female flowers
23
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
borne in the leaf axils. The fruit looks like a warty
gourd, usually oblong and resembling a small
cucumber. The young fruit is emerald green, turning to
orange-yellow when ripe. At maturity, the fruit splits
into three irregular valves that curl backwards and
release numerous reddish-brown or white seeds
encased in scarlet arils. The Latin name Momordica
means “to bite,” referring to the jagged edges of the
leaves, which appear as if they have been bitten. All
parts of the plant, including the fruit, taste very bitter
(4).
Various parts of M. charantia such as the seed, fruit
and even the whole plant have been reported to have
beneficial effects in the prevention and treatment of
many diseases in folkloric medicine, especially in the
treatment of DM in individuals with non-insulin
dependent diabetes (5,6). It has hypoglycemic
properties as it significantly suppressed the rise in
blood glucose concentrations in albino rats (5,7). The
first clinical study into the influence of the fresh juice
of bittergourd on the management of DM was
performed by Akhtar in 1981. These findings
suggested the intervention would effectively treat all
symptoms of diabetes including polyuria, polydipsia,
and polyphagia. Sarkar et al. (1996) and Miura et al.
(2001) indicated that the fresh bittergourd juice caused
a significant reduction in plasma glucose
concentration and an improvement in the response to
oral glucose load. Bitter melon contains an array of
biologically active plant chemicals including
triterpenes, proteins and steroids. In addition, a protein
found in bitter melon, momordin, has clinically
demonstrated anticancerous activity against Hodgkin’s
lymphoma in animals. Other proteins in the plant,
alpha- and beta-momorcharin and cucurbitacin B,
have been tested for possible anticancerous effects
(11). In some studies, at least three different groups of
constituents found in all parts of bitter melon have
clinically demonstrated hypoglycemic (blood sugar
lowering) properties or other actions of potential
benefit against DM (12). These chemicals that lower
blood sugar include a mixture of steroidal saponins
known as charantins, insulin-like peptides, and
alkaloids. The hypoglycemic effect is more
pronounced in the fruit of bitter melon where these
24
chemicals are found in greater abundance. The present
study investigated the effects of M. charantia on
physical changes observed in rat models of diabetes
and on the histomorphometry of the aorta, pulmonary
trunk and left ventricle of the heart in streptozotocin
(STZ)-induced diabetic Wistar rats and compared the
effects with those of glimepiride, an oral blood
glucose-lowering drug of the sulfonylurea class (13).
MATERIALS AND METHODS
Animal care
Forty healthy adult Wistar rats of both sexes, mean
weight 134.4 g, were used for the experiment. The rats
were bred in the animal holding of College of Health
Sciences, Obafemi Awolowo University, Ile-Ife. They
were maintained on standard rat pellet (Capsfeed,
Ibadan, Nigeria) and water was provided ad libitum.
The animals were randomly assigned into five groups
A, B, C, D and E of eight rats each.
- group A, control (normal rats)
- group B, experimentally induced diabetic rats
administered 10% Tween 80
- group C, experimentally induced diabetic rats
treated with methanolic extracts of Momordica
charantia dissolved in10% Tween 80 for two
weeks (withdrawal group)
- group D, experimentally induced diabetic rats
treated with methanolic extracts of Momordica
charantia dissolved in 10% Tween 80 for four
weeks
- group E, diabetic rats treated with a standard
diabetic drug (2 mg/kg of glimepiride) dissolved
in 10% Tween 80 for four weeks
Physical changes were observed throughout the
experimental period and body weight was also
monitored. The animals received humane treatment as
outlined in the Care and Management of Laboratory
Animals published by the National Institute of Health
(NIH, 1985).
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
Plant material
Administration of extract and anti-diabetic drug
Mature leaves of M. charantia (Cucurbitaceae) were
collected during the raining season from suburban
villages of Ile-Ife metropolis in Osun State of Nigeria.
The leaves were taken to the Herbarium at the
Department of Botany, Obafemi Awolowo University,
Ile-Ife to confirm identification and a voucher
specimen (No. UHI 16510) was placed in the
Herbarium.
Methanolic extracts of the leaves of M. charantia
(100 mg/kg) were dissolved in 10% Tween 80 and
administered daily (orally) by gastric intubation to the
rats in groups C and D for 2 and 4 weeks, respectively.
The standard antidiabetic drug (glimepiride 2 mg/kg)
was administered to group E rats for four weeks (15),
while those in group B were left untreated.
Preparation of methanolic extract of Momordica
charantia
Leaves of M. charantia were air dried and powdered
in a warring blender. Then, 765 g of the powdered
leaves were extracted in 1950 mL of absolute
methanol for 72 hours with intermittent shaking and
filtered. The filtrate was concentrated in vacuo at 35
ºC using a vacuum rotary evaporator (Büchi
Rotavapor R110, Switzerland). The extract was
partitioned between water and dichloromethane, the
dichloromethane fraction (5.94%) was oven-dried at
37 ºC and stored until it was ready to be used. The
aqueous portion obtained was very little. Aliquot
portions of the extract were weighed and dissolved in
10% Tween 80 for use on each day of the experiment.
Induction of diabetes
Diabetes mellitus was experimentally induced in
groups B, C, D and E by a single intraperitoneal
injection of 65 mg/kg body weight of streptozotocin
(Tocris Bioscience, UK) dissolved in 0.1M sodium
citrate buffer (pH 6.3). Diabetes was confirmed in
animals 48 hours after induction by determining
fasting blood glucose level using a digital glucometer
(Accu-chek® Advantage, Roche Diagnostic,
Germany) consisting of a digital meter and test strips
using blood samples obtained from the tail vein of the
rats. Group A animals were given equal volume of
citrate buffer used on dissolving STZ intraperitoneally.
Diabetologia Croatica 42-1, 2013
Sacrifice of the animals
At the end of the experimental period, all animals
were physically observed and anesthetized by
chloroform inhalation. A midline incision was
performed at the thoracic region. The organs were
dissected out, weighed and fixed in 10% formol saline
processed histologically for routine hematoxylin and
eosin staining.
Histomorphometry
All histomorphometric studies were carried out on an
Olympus research microscope (Leica Galen III,
Germany) with a linear scale-ocular micrometer
inserted into the eyepiece. The ocular micrometer was
calibrated with 1-mm stage micrometer (Graticles
Tonbridge, Kent, England). Histologically stained
sections were used for morphometric analysis of the
aorta, pulmonary trunk and left ventricle of the control
and diabetic groups to estimate the thickness of each
of the tunica intima, media, adventitia and
myocardium.
RESULTS
Physical observation
The control group appeared presumably healthy as
evident in their physical agility, which was
characterized by healthy fur and pinkish eyeball
without any evidence of alopecia. The feeding and
fluid intakes were also appreciably normal. In the
diabetic group, there was alopecia on the head, neck
and some other parts of the body. Other observable
physical changes included polyuria, which was
25
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
characterized by wetness of the ventral and lateral
body surfaces of the animals. The animals were less
active as compared with the controls, which suggested
a sign of ill health. Animals that were treated with M.
charantia therapy and glimepiride, i.e. group D and E,
respectively, presented a milder physical abnormality
as compared with the diabetic group. Withdrawal of
the extract in group C did not cause much change in
the physical appearance when compared with the
group that completed the extract treatment for four
weeks (Fig. 1).
GROUP D
Figure 1. One animal from each group (groups A, B, C,
D and E) is shown.
GROUP E
Changes in body weight
GROUP A
GROUP B
GROUP C
26
Prior to the commencement of the research work
(day 0), body weight of all animals in both control and
experimental groups showed no significant difference
(p>0.05). By the end of day 7, after DM induction,
body weight increased significantly in animals from
groups A, D and E, and decreased significantly in
animals from groups B and C. By day 14 of the
experiment, body weight of the rats in all groups
appeared to be constant. By day 21 of the experiment,
there was a significant (p<0.05) increase in body
weight of the animals. By day 28 of the experiment
(commencement of extract administration), there was
a significant increase in the mean body weight of the
animals in groups A, D and E compared to animals in
groups B and C, which showed a decrease in the mean
body weight. By the end of day 35 of the experiment,
the animals in groups A, C and D significantly differed
in the mean body weight when compared with the
mean body weight of group B rats. The animals in
group E showed a relatively constant body weight, as
shown in Table 1. By day 42 of the experiment, there
was a significant increase in body weight of the rats in
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
groups A, C, D and E, while body weight of the rats in
group B reduced significantly. By day 49 of the
experiment, the animals in groups A, D and E
maintained a nonsignificant difference in the mean
body weight, while the mean body weight in group B
and C rats decreased significantly by 1.97% and
2.54%, respectively. At the end of the experimental
period (day 56), there was a significant (p<0.05)
decrease in the mean body weight of diabetic group
(121.25±6.11)
as
compared
with
control
(172.50±7.79). Withdrawal of the extract from group
C rats showed a nonsignificant difference (p>0.05) in
the mean body weight when compared with diabetic
group. The rats in groups D and E presented a
significant increase (p< 0.05) in the mean body weight
when compared with the rats in groups A, B and C
(Table 1).
Relative organ weight
There was a significant (p<0.05) increase in the
relative heart weight of diabetic group (0.44±0.10)
when compared with control group (0.31±0.08) and a
nonsignificant (p>0.05) decrease in the relative
pancreas weight of diabetic group (0.29±0.08) as
compared with control group (0.34±0.09). However,
there was a significant (p<0.05) decrease in the
relative weights of the heart and pancreas in the
animals that received M. charantia extract therapy for
four weeks (group D) when compared with diabetic
group (group B). The relative heart weight in group C
(withdrawal group) increased significantly (p<0.05),
while the relative weight of the pancreas decreased
nonsignificantly (p>0.05) when compared with group
E. The rats in group E treated with antidiabetic drug
(glimepiride) showed a significant decrease in the
relative weights of the heart and pancreas when
compared with diabetic rats (Table 2).
Table 1. The effect of Momordica charantia on body weight (g)
Group A
Group B
Group C
Group D
Group E
0
Day
110.13 ± 2.90a
148.00 ± 16.78b
155.00 ± 7.79b
130.63 ± 4.67ab
128.25 ± 4.06ab
7
126.88 ± 4.52ab
128.13 ± 12.50ab
105.00 ± 5.77a
137.86 ± 9.00b
139.00 ± 9.54b
126.88 ±
4.53ab
128.75 ±
12.24ab
105.00 ±
5.77a
137.86 ±
9.00b
139.00 ± 9.54b
145.00 ±
4.33a
135.63 ±
6.91a
135.00 ±
7.91a
146.67 ±
7.60a
170.00 ± 10.21b
149.38 ±
5.30a
132.50 ±
7.50a
128.00 ±
9.17a
180.00 ±
5.48b
176.67 ± 3.33b
35
158.13 ±
6.47b
127.50 ±
7.20a
130.00 ±
5.77a
183.33 ±
6.28c
176.67 ± 1.67bc
42
162.50 ± 7.13b
126.88 ± 6.68a
131.67 ± 7.27a
188.33 ± 6.91c
181.67 ± 1.67bc
49
166.25 ± 7.30b
124.38 ± 6.58a
128.33 ± 6.01a
191.67 ± 6.41c
185.00 ± 2.89bc
56
7.79b
6.11a
5.00a
3.27c
190.00 ± 5.00bc
14
21
28
172.50 ±
121.25 ±
130.00 ±
204.17 ±
Values are given as mean ± SEM for body weight on days coded as 0, 7, 14, 21, 28, 35, 42, 49 and 56 in each group; a, b, c, d, ab, bc, cd, abc within column signify that the means
with different letters differ significantly at p<0.05, while the means with the same letters do not differ significantly at p<0.05 (one-way ANOVA with Duncan multiple range test).
Table 2. The effect of Momordica charantia on the
relative organ weight (g) in streptozotocin-induced
diabetic rats
RWH
0.08ab
RWP
0.34 ± 0.09b
Group A
0.31 ±
Group B
0.44 ± 0.10b
Group C
0.35 ±
0.13b
Group D
0.21 ± 0.08ab
0.16 ± 0.06abc
Group E
0.12 ± 0.08ab
0.10 ± 0.06a
0.29 ± 0.08b
0.10 ±
0.07ab
Values are given as mean ± SEM for relative weight of heart and relative weight of
pancreas coded RWH and RWP, respectively, in each group; a, b, ab, abc within
column signify that the means with different letters differ significantly at p<0.05, while
the means with the same letters do not differ significantly at p<0.05 (one-way ANOVA
with Duncan multiple range test).
Diabetologia Croatica 42-1, 2013
Morphometry
Aorta
Tunica intima of the diabetic group increased
significantly (p<0.05) when compared with groups A
(control), C (withdrawal group), D (treated with M.
charantia for four weeks) and E (treated with
glimepiride for four weeks). However, tunica media of
groups B, C, D and E showed a significant decrease
(p<0.05) when compared with control group (group
27
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
Table 3. The effect of Momordica charantia on the
tunics of the aorta
Group
Tunica intima Tunica media
(µm)
(µm)
Tunica
adventitia
(µm)
Table 4. The effect of Momordica charantia on the
tunics of pulmonary trunk
Group
Tunica intima Tunica media
(µm)
(µm)
Tunica
adventitia
(µm)
Group A
0.25±0.00a
2.25±0.23a
2.88±0.07a
Group B
0.56±0.06b
4.38±0.41b
6.00±0.57b
Group C
0.25±0.00a
1.94±0.33a
3.88±0.66a
1.75±0.14a
Group D
0.31±0.06a
2.13±0.22a
3.13±0.16a
1.56±0.06a
Group E
0.25±0.00a
1.94±0.06a
3.69±0.11a
Group A
0.25±0.00a
2.38±0.22b
3.63±0.16b
Group B
0.53±0.11b
1.13±0.24a
3.81±0.15b
Group C
0.25±0.00a
1.63±0.13a
1.88±0.07a
Group D
0.25±0.00a
1.31±0.60a
Group E
0.25±0.00a
1.50±0.10a
Values are given as mean ± SEM for each morphometric parameter, i.e. tunica intima,
media and adventitia in each group; a, b, within column signify that the means with
different letters differ significantly at p<0.05, while the means with the same letters do
not differ significantly at p<0.05 (one-way ANOVA with Duncan multiple range test)
A). Tunica adventitia in the diabetic group showed a
nonsignificant decrease (p>0.05) when compared with
control group. There was a significant reduction in
groups C, D and E as compared with diabetic and
control group (Table 3).
Values are given as mean ± SEM for each parameter: tunica intima, media and
adventitia in each group; a, b within column signify that the means with different letters
differ significantly at p<0.05, while the means with the same letters do not differ
significantly at p<0.05 (one-way ANOVA with Duncan multiple range test).
Table 5. The effect of Momordica charantia on the
thickness of the myocardium in the left ventricle
Group
Myocardium (µm)
Group A
176.25±12.14a
Group B
368.75±23.11c
Group C
221.87±10.67b
Group D
220.62±3.87b
Pulmonary trunk
Group E
230.00±6.77b
The three tunics (intima, media and adventitia) of the
diabetic group increased significantly (p<0.05) when
compared with groups A (control), C (withdrawal
group), D (treated with M. charantia for four weeks)
and E (treated with glimepiride for four weeks).
However, the three tunics of groups A, C, D and E
showed a nonsignificant difference (p>0.05) when
compared (Table 4).
Values are given as mean ± SEM for myocardium in each group; a, b, c, within column
signify that the means with different letters differ significantly at p<0.05, while the
means with the same letters do not differ significantly at p<0.05 (one-way ANOVA with
Duncan multiple range test) (n=10).
Myocardium
The thickness of the myocardium in diabetic group
increased significantly (p<0.05) when compared with
groups A (control), C (withdrawal group), D (treated
with M. charantia for four weeks) and E (treated with
glimepiride for four weeks). However, the thickness of
the myocardium in groups C, D and E showed a
nonsignificant difference (p>0.05) when compared
(Table 5).
DISCUSSION
Diabetes has been reported to be associated with
profound alterations in glucose transport function and
microanatomy of visceral organs, leading to an
28
increased risk of coronary heart disease (16-19). The
reduced polyuria and alopecia noticed in the M.
charantia treated group in this investigation buttressed
the nontoxic and promising effects of M. charantia,
which is in correlation with other investigators (8,20).
As the extract treatment commenced, M. charantia
was observed to have significantly increased the mean
body weight of animals in group D as compared with
animals in groups B and C, which showed a decrease
in their mean body weight. This observation supports
previous studies such as that carried out by Shetty et
al. (21) who report a weight gain in diabetic rats
following treatment with M. charantia. Garau et al.
(22) also report an improved weight after diabetic rats
were treated with M. charantia. Other investigations
on the benefit of M. charantia on body weight are
documented (23,24).
The relative weight of the heart in group D (diabetic
rats treated with M. charantia for four weeks) and
group E (diabetic rats treated with glimepiride for four
weeks) was observed not to differ significantly as
O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
compared with the control, whereas the relative heart
weight increased significantly (p<0.05) in diabetic
group (group B). The reason for this may be due to an
increase in cholesterol deposit, as diabetes is known to
be associated with hyperlipidemia (25). Following the
withdrawal of the extract treatment in group C
animals, the relative heart weight increased by
66.67%. This may be due to systemic depletion of M.
charantia, thus creating an avenue for cholesterol
deposit. The relative weight of the pancreas, which
decreased significantly in diabetic group, may be due
to a reduction in β cell mass by STZ induction.
Selective destruction of beta cells can be obtained by
injecting STZ or alloxan (26). Streptozotocin is a
DNA-alkylating agent and alloxan is a generator of
oxygen free radicals, both causing extensive DNA
damage (26,27). Their selectivity is thought to be due
to better uptake by beta cells. Bouwens and Rooman
(26) state that intravenous administration of 100
mg/kg STZ on the day of birth in rats reduces total
beta-cell mass by ∼90% in 48 h. Twenty days later,
<40% of the normal beta-cell mass is restored (26,28).
These animals can maintain normoglycemia up to a
certain body weight, but at the age of ∼6 weeks they
become glucose intolerant (26).
Morphometric findings in this study showed that the
three tunics of the aorta and pulmonary trunk were
significantly thicker in diabetic rats when compared
with control group. The thickness of the tunica intima
may be due to lipid build up within these vessels to
form atherosclerotic plaques, which may prevent the
free flow of blood. This observation is consistent with
previous studies (29-31). These effects were abrogated
with the administration of M. charantia extract and
glimepiride for four weeks. The reason for the
significant increase of tunica media and adventitia in
diabetic group is yet to be understood. Further
investigation is, however, ongoing to ascertain the
cause. The thickness of the myocardium in diabetic rat
may be due to blood viscosity associated with
hyperglycemia, thus leading to left ventricular
hypertrophy. This finding corroborates a previous
study (32).
This suggests that M. charantia exhibits a
cardiovascular protective potential, possibly via its
antiatherogenic properties.
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O. A. Komolafe, D. A. Ofusori, O. S. Adewole, A. A. Ayoka, O. A. Ayannuga / HISTOMORPHOMETRIC STUDIES OF THE AORTA,
PULMONARY TRUNK AND LEFT VENTRICLE OF STREPTOZOTOCIN-INDUCED DIABETIC WISTAR RATS TREATED WITH
MOMORDICA CHARANTIA EXTRACT
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