Evaluation of Some Traditional Antidiabetic Plants on ...

Medicine Journal 2015; 2(5): 79-87

Published online October 9, 2015 (http://www.openscienceonline.com/journal/med)

Evaluation of Some Traditional Antidiabetic Plants on Testis in the Alloxan-Induced Diabetic Male Albino Rats: Histological, Histochemical, Immunohistochemical and Morphometric Study

Tamer M. M. Abu-Amara1, *

, Abd EL Razek A. Meselhy2, Howada I. Abdel Aziz

3, Amal Said

4

1Histology and Cytology Department, Faculty of Medicine, Al-Azhar University, Cairo, Egypt 2Anatomy and Embryology Department, Faculty of Medicine, Al-Azhar University, Assiut, Egypt 3Histology and Cytology Department, Faculty of Medicine, Suze-Canal University, Ismailia, Egypt 4Zoology Department, Faculty of Science (Girls), Al-Azhar University, Cairo, Egypt

Email address

[email protected] (T. M. M. Abu-Amara)

To cite this article Tamer M. M. Abu-Amara, Abd EL Razek A. Meselhy, Howada I. Abdel Aziz, Amal Said. Evaluation of Some Traditional Antidiabetic

Plants on Testis in the Alloxan-Induced Diabetic Male Albino Rats: Histological, Histochemical, Immunohistochemical and Morphometric

Study. Medicine Journal. Vol. 2, No. 5, 2015, pp. 79-87.

Abstract

Background: Diabetes mellitus is one of the common and widely distributed metabolic diseases all over the world. This disease

is characterized by hyperglycemia that results from defects in insulin secretion, insulin action or both. Previous studies

reported negative complications of diabetes on the male genital system with associated gonadal dysfunction. Different

medicinal plant species were used as a traditional treatment for diabetes mellitus long times ago. Aim of the work: As diabetes

had been reported to affect the male genital system, this work was aimed to investigate the antidiabetic effects of the aqueous

extract of the following herbs: Ambrosia maritima, L. (Damsissa), Chrozophoratinctoria, L. (Sammo), Artemisia annua, L.

(Kaysom) and Artemisia judaica, L. (Sheeh) on the testis’ histology in the alloxan-induced diabetic male albino rats. Material

and Methods: This study was performed on sixty male albino rats with an average 100-110 g body weight. The animals were

divided into six groups (10/cage); Group1 (Control untreated-group), Group 2 (Alloxan-induced diabetic group), Group 3

(diabetic group treated orally with “28.5 mg/kg body wt. twice/day” of the Damsissa extract), Group 4 (diabetic group treated

orally with “28.5 mg/kg body wt. twice/day” of the Sammo extract), Group 5(diabetic group treated orally with “28.5 mg/kg

body wt. twice/day” of the Kaysom extract)and Group 6 (diabetic group treated orally with “28.5 mg/kg body wt. twice/day”

of the Sheeh extract). Results: Considerable improvements in the testicular tissue morphological changes that were observed in

diabetic groups had been detected after treatment with Damsissa and Sammo in comparison to the control group. These

improvements were less obvious after Kaysom and Sheeh treatment. Conclusion: It could be concluded that Damsissa and

Sammo can guard against the negative effects of diabetes on the testis.

Keywords

Diabetes Mellitus, Alloxan, Hyperglycemia, Damsissa, Sammo, Kaysom, Sheeh

1. Introduction

Diabetes mellitus is a serious metabolic disorder with

numerous complications [1]. The increase of the blood

glucose levels leads to many structural and functional

changes in various tissues and organs [2] Experimentally, the

induction of diabetes in male rats had been found to be

associated with altered functions of the reproductive system

[3]. Induction of diabetes in rats was used as an in vivo model

for studying the effects of diabetes on the various organs [4,

5]. Induction of diabetes affects the testicular functions due

to the lack of insulin and subsequently the impairment of

insulin regulatory action on the Leydig and Sertoli cells [6]

This gonadal dysfunction and the subsequent decrease in the

testosterone production lead to insufficiency in

spermatozoids production [7, 8, 9]. Moreover, several

previous studies reported change in the reproductive system

Medicine Journal 2015; 2(5): 79-87 80

structure in diabetic cases [2, 10, 11]. Also, the effect of

diabetes on the changes of the body and testicular weights

have been reported in several studies. For instance, in

diabetic rats, testicular weight was decreased around 20% in

comparison to the healthy rats [12]. A wide variety of

Egyptian folk medicinal plants are used in the treatment of

diabetes [13]. For instance, Damsissa was commonly used in

the treatment of rheumatic pains, asthma, bilharziasis,

diabetes, stomach, and renal troubles [14]. Moreover, several

effects such as anti-inflammatory, anti-oxidative effects, anti-

hyperlipidemia and antihypertensive effects have been

reported for Kaysom treatment [15, 16, 17]. In this study,

was aimed to investigate the antidiabetic effects of the

aqueous extract of the following herbs: Ambrosia maritima,

L. (Damsissa), Chrozophoratinctoria, L. (Sammo), Artemisia

annua, L. (Kaysom) and Artemisia judaica, L. (Sheeh) on the

testis’ histology in the alloxan-induced diabetic male albino

rats.

2. Material and Methods

2.1. Plant Material

The aerial parts of Ambrosia maritima, L. (Damsissa),

Chrozophoratinctoria, L. (Sammo), Artemisia annua, L.

(Kaysom) and Artemisia judaica, L. (Sheeh) were collected

from El-Arbaeen valley, Saint Catherine, Wadi Gebal, South

Sinai, Egypt. The plants were grinded and the aqueous

extracts of them were prepared by boiling 2 g of each of them

with 200ml of tap water for 15 min, left to cool at room

temperature then filtered through filter paper. Later, the

extracts were stored in glass containers in refrigerator. Fresh

extract preparations were done every two days.

2.2. Animals

Sixty male adult albino rats (8-10 weeks/ 100-110 g) were

used in this experiment. The rats were kept under observation

for about 2 weeks before the start of the experiment for

adaptation. Diabetes mellitus was induced in animals by

single dose of alloxan (120 mg/kg B. W. dissolved in saline)

injected intraperitoneally to induce diabetes mellitus in rats18

.

The rats were deprived of food for 16 hours before alloxan

injection. After three days of alloxan injection, the rats were

deprived of food overnight and they were then given glucose

(3g/kg B. W.) by gastric intubation. After 2 hours of oral

glucose administration, blood samples were taken from tail

vein and the fasting blood glucose (FBG) concentration was

determined by means of one touch ultra-glucometer (Johnson

& Johnson Company, USA) and compatible blood glucose

strips. After 2 h of oral glucose administration, the rats’

glucose concentrations “ranging from 180 to 300 mg/dl”

were considered as mild diabetic animals and included in the

experiment.

2.3. Experimental Design

Experimental animals were divided into six groups, ten

each, as follows:

• Group 1 (Control group): Non-diabetic rats.

• Group 2 (Diabetic group): Rats were injected

intraperitoneally with a single dose of alloxan (120

mg/kg dissolved in saline solution).

• Group 3 (Damsissa group): Diabetic rats treated orally

with Ambrosia maritime extract (28.5 mg/kg twice /day)

for 30 days.

• Group 4 (Sammo group): Diabetic rats treated orally

with Chrozophoratinctoria extract (28.5 mg/kg twice

/day) for 30 days.

• Group 5 (Kaysoom group): Diabetic rats treated orally

with Artemisia annua extract (28.5 mg/kg twice /day)

for 30 days.

• Group 6 (Sheeh group): Diabetic rats treated orally with

Artemisia judaica extract (28.5 mg/kg twice /day) for

30 days.

Histological and Histochemical studies: Therats from the

control and treated groups were sacrificed after one month

and small pieces of the testis were taken for the histological

and histochemical studies. The specimens were prepared via

fixation in 10% neutral buffered formalin solution and

Carnoy’s fluid. For histological study, paraffin sections were

stained with Harris’s haematoxylin and eosin (H&E)

19. For

detection of collagen fibers, paraffin sections were stained by

using Mallory’s trichrome stain19

. For histochemical study,

Paraffin sections of 5µm thickness were prepared and stained

with Feulgen stain19

. Later, the stained sections were

examined via light microscope, photographed and all the

detected variations between the three groups on the level of

the microscopic findings had been scientifically discussed.

Immunohistochemical study: Sections of testes were

deparaffinized with xylene, followed by antigen retrieval by

heating in citrate buffer (10 mM, 20 min). This was followed

by endogenous peroxidase blocking in 3% H2O2for 10 min

and incubation with anti-caspase–3 (1:100; Abcam, Ab4051).

After washing the slides with phosphate buffered saline, the

sections were incubated with the related secondary antibodies

at room temperature for 1 h, followed by detection with 3-

amino-9-ethylcarbazole, a chromogen. The slides were

mounted in paramount aqueous mounting medium.

Morphometric analysis: The image analyzer (ImageJ 1.46r)

was used to obtain the following morphometric data:

• The mean diameter of the seminepherous tubules for

the different groups using H&E - stained sections at

400x magnification.

• The mean thickness of the seminepherous tubular

diameters for the different groups using Mallory’s

trichrome-stained sections at 400x magnification.

• The area percentage of the collagen fibers in the

seminepherous tubules for the different groups using

Mallory’s trichrome-stained sections at 400x

magnification.

• The mean apoptotic changes of the seminiferous tubular

germinal cells nuclei for the different groups of the

study using Feulgen-stained sections at 400x

magnification.

• The mean number of the caspase-3+ve expressed cells

81 Tamer M. M. Abu-Amara et al.: Evaluation of Some Traditional Antidiabetic Plants on Testis in the Alloxan-Induced

Diabetic Male Albino Rats: Histological, Histochemical, Immunohistochemical and Morphometric Study

of the seminepherous tubular germinal cells for the

different groups of the study using caspase-3

immnuostained sections at 400x magnification.

Statistical analysis: All statistical analyses were performed

via PAleontological Statistics Version 3.0 (PAST 3.0)

statistical software (Hammer et al., 2001)20

. The obtained

data were expressed as mean ± standard deviation (SD) and

analyzed using analysis of variance (ANOVA)-Bonferroni

with p<0.05 considered statistically significant.

3. Results

3.1. Histological Results

Section of the control adult albino rat testis showed several

seminiferous tubules with interstitial tissue in between

(Fig.1). The seminiferous tubules were lined with

spermatogenic cells in different developmental stages and

mature sperms in the lumen. Seminiferous tubules (S. Ts)

had two cell types: Sertoli cells and spermatogenic cells

(Fig.1). Spermatogenic cells were arranged from the basal to

the adluminal compartments in the following order;

spermatogonia, spermatocytes, spermatids and spermatozoa.

While spermatogonia were basal in position and had small

rounded nuclei, primary spermatocytes were next to it and

had large rounded central nuclei. Next to it, primary

spermatocytes, spermatids are detected (Fig. 1). Spermatids

have smaller nuclei with pale chromatin and an acrosomal

cap. The elongated spermatids were identified via their

elongated deeply stained nuclei. Sertoli cells with ovoid

nuclei were detected in between spermatogonia resting on the

basement membrane (Fig. 1). Within the interstitial

connective tissue (I. T), the blood vessels were surrounded

by polygonal or rounded Leydig cells that had granular

cytoplasm, single or doubled nuclei and appeared singly or in

groups (Fig. 1). The histological investigations of testicular

tissue in the untreated diabetic rats demonstrated irregularity

of the S. Ts shapes with significant decrease in S. Ts

diameters in comparison to the control group (Figs. 2, 6 & T.

1). All the treated groups showed a considerable significant

recovery of the S. Ts diameters in comparison to the diabetic

group (Figs. 2, 6& T. 1). Also, the germinal epithelium

showed obvious disorganization of the germinal epithelium

with abnormal cellular attachment. Moreover, the

spermatogonia cells were the major cell type that was seen

(Figs. 2, 6& T. 1). Also, multinucleated cells with two or

three nucleus were detected in S. Ts (Fig. 2). Moreover, the

interstitial connective tissue had an amorphous material with

marked destruction of the connective tissues with subsequent

widening of the I.T spaces (Figs. 3, 7, 8 & T. 1). All the

treated groups showed a considerable recovery of the I.T to

normal levels with significant increase in the amount of the

collagen fibers in comparison to the diabetic group (Figs. 3, 7,

8 & T. 1).

3.2. Histochemical Results

Fig. 1. A) Light photomicrograph of a section in a rat testicular tissue from the control group. The seminiferous tubules (S.Ts) have ordinary shape, their

epithelium is structurally intact and shows normal association of germ cells. The interstitial connective tissue (I.T) shows normal architecture. H&E (200×).B)

Light photomicrograph of a section in a rat testicular tissue from the control group shows the normal association of the germ cells and normal architecture of

the interstitial tissue. G= Spermatogonium, P=Spermatocytes, D=Spermatid, SP=Sperm, S=Sertoli cells, Ly=Lydig cell, B.V= Blood vessel. H&E (400×).


Feulgen-stained testicular tissue sections of the control

group revealed ordinary S. Ts shapes with intact epithelium

and normal association of the germinal cells (Fig. 4). Within

the untreated diabetic group, marked significant reduction,

apoptotic changes (pyknosis and karyolysis), disorganization

and depletion of the S. Ts germinal epithelium were observed

in comparison to the control group (Figs. 4, 9 & T. 1). In

both Damsissa- and Sammo-treated groups, marked

significant recovery of the germinal epithelium apoptotic

changes were observed in comparison to the diabetic group

(Figs. 4, 9 & T. 1). While in both Kaysom - and Sheeh-

treated groups, mild non-significant recovery of the germinal

epithelium apoptotic changes was observed in comparison to

the diabetic group (Figs. 4, 9 & T. 1).

3.3. Immunohistochemical Results

The immunohistochemical investigations of the testicular

tissue for the control group showed mild expression of

Caspase-3 immunostaining with few caspase-3 +ve cells in

the S. Ts (Figs. 5, 10& T. 1). In diabetic group, a marked

significant increase in the number of caspase-3+ve cells was

observed in the S. Ts in comparison to the control ones (Figs.

5, 10& T. 1). In both Damsissa- and Sammo-treated groups,

mild expression of caspase-3+ve cells were detected with

significant decrease compared with the diabetic group (Figs.

5, 10& T. 1). However, in both Kaysom - and Sheeh-treated

groups, a marked significant increase in the number of

caspase-3+ve cells was observed in the S. Ts in comparison

to the control ones (Figs. 5, 10& T. 1).

Fig. 2. A) Light photomicrograph of a section in a rat testicular tissue from the control group. The seminiferous tubules (S.Ts) have ordinary shape. S.Ts

epithelium is structurally intact and shows normal association of germ cells. B) Light photomicrograph of a section in a rat testicular tissue form untreated

diabetic rat. The S. Ts have irregular shape and the germinal epithelium is disorganized. Depletion of germ cells, pyknotic germ cells (black arrows) and

karyolysis (green arrow) are seen. The giant cell formation with two or three nucleus (red arrow) is seen in the lumen of irregular shaped seminiferous tubule

(ST). C)Light photomicrograph of a section in a rat testicular tissue treated with Damsissa extract. The S.Ts have partial recovery to normal structure. D)

Light photomicrograph of a section in a rat testicular tissue treated with Sammo extract. The S.Ts have partial recovery to normal structure. E) Light

photomicrograph of a section in a rat testicular tissue treated with Kaysom extract. The S.Ts have partial recovery to normal structure. F) Light

photomicrograph of a section in a rat testicular tissue treated with Sheeh extract. The S.Ts have partial recovery to normal structure. H&E (400×).



Fig. 3. A) Light photomicrograph of a section in a rat testicular tissue from the control group shows normal distribution of collagen fibers in the interstitial

tissue (I.T) around the seminiferous tubules. B) Light photomicrograph of a section in a rat testicular tissue form untreated diabetic rat shows marked

reduction of collagen fibers in the I.T around the seminiferous tubules, increase diameter of the interstitial spaces. C) Light photomicrograph of a section in a

rat testicular tissue treated with Damsissa extract shows more or less similar distribution of collagen fibers in the I.T around the seminiferous tubules in

comparison to the control group. D) Light photomicrograph of a section in a rat testicular tissue treated with Sammo extract shows more or less similar

distribution of collagen fibers in the I.T around the seminiferous tubules in comparison to the control group. E) Light photomicrograph of a section in a rat

testicular tissue treated with Kaysom shows mild distribution of collagen fibers in the I.T around the seminiferous tubules in comparison to the control group.

F) Light photomicrograph of a section in a rat testicular tissue treated with Sheeh extract shows more or less similar distribution of collagen fibers in the I.T

around the seminiferous tubules in comparison to the control group. Mallory’s trichrome (200×).

Fig. 4. A) Light photomicrograph of a section in a rat testicular tissue from the control group. The seminiferous tubules (S.Ts) have ordinary shape, their

epithelium is structurally intact and shows normal association of germ cells. B) Light photomicrograph of a section in a rat testicular tissue form untreated

diabetic rat shows marked reduction, pyknosis, disorganization and depletion of the germinal epithelium in the S.Ts. C) Light photomicrograph of a section in

a rat testicular tissue treated with Damsissa extract shows marked recovery of the germinal epithelium to the normal architecture. D) Light photomicrograph

of a section in a rat testicular tissue treated with Sammo extract shows marked recovery of the germinal epithelium to the normal architecture. E) Light

photomicrograph of a section in a rat testicular tissue treated with Kaysom shows mild recovery of the germinal epithelium to the normal architecture. F)

Light photomicrograph of a section in a rat testicular tissue treated with Sheeh extract shows mild recovery of the germinal epithelium to the normal

architecture. Feulgen stain (400×).


Fig. 5. A) Light micrograph of testicular tissue of a rat from the control group. The seminiferous tubules (S.Ts) germinal cells show mild expression of

Caspase-3 immunostaining (black arrows). B) Light micrograph of testicular tissue of a rat from the untreated diabetic rat. The S.Ts germinal cells show

marked expression of Caspase-3 immunostaining (black arrows). C) Light photomicrograph of a section in a rat testicular tissue treated with Damsissa

extract shows mild expression of Caspase-3 immunostaining (black arrows) in the S.Ts germinal cells. D) Light photomicrograph of a section in a rat

testicular tissue treated with Sammo extract shows moderate expression of Caspase-3 immunostaining (black arrows) in the S.Ts germinal cells. E) Light

photomicrograph of a section in a rat testicular tissue treated with Kaysom shows marked expression of Caspase-3 immunostaining (black arrows) in the S.Ts

germinal cells. F) Light photomicrograph of a section in a rat testicular tissue treated with Sheeh extract shows marked expression of Caspase-3

immunostaining (black arrows) in the S.Ts germinal cells.Caspase-3 immunostaining (400×).

Fig. 6. The mean diameters of the Seminepherous Tubules for the different

groups of the study in comparison to the control group.

Fig. 7. The mean thickness of the Seminepherous tubular interstitial

diameters for the different groups of the study in comparison to the control

group.



Fig. 8. The mean area percentage of the collagen fibres of the

Seminepherous tubular interstitial diameters for the different groups of the

study in comparison to the control group.

Fig. 9. The mean apoptotic changes of the Seminepherous Tubular germinal

cells nuclei for the different groups of the study in comparison to the control

group.

Fig. 10. The mean number of the caspase-3+ve cells of the seminepherous tubular germinal cells for the different groups of the study in comparison to the

control group.

Table. 1. Diameters of the seminepherous tubules, thickness of the seminepherous tubular interstitial diameters, percentage of the collagen fibers of the

seminepherous tubular interstitial diameters, apoptotic changes of the seminepherous tubular germinal cells nuclei and number of the caspase-3+ve cells of

the seminepherous tubular germinal cells for the different groups of the study expressed as mean ± SD.

Paramaters Mean diameters

of the

seminepherous

tubules

Mean thickness of

the seminepherous

tubular interstitial

diameters

Mean area percentage of

the collagen fibres of the

seminepherous tubular

interstitial diameters

Mean apoptotic changes

of the seminepherous

tubular germinal cells

nuclei

Mean number of the

caspase-3+ve cells of the

seminepherous tubular

germinal cells Study Groups

Group 1

(Control)

188.11±

13.92

99.89±

7.57

140.03±

23.25

30.073±

1.48

12.00±

1.94

Group 2

(Diabetic)

128.61±

32.17 **a

180.70±

23.50**b

65.97±

16.55**a

14.08±

4.02**a

37.81±

2.78**b

Group 3

(Demcica)

192.78±

10.67**c

90.38±

15.29**d

148.96±

16.79**c

25.75±

6.28**c

15.00±

3.31**d

Group 4

(Samwaa)

179.50±

21.97**c

100.10±

16.51**d

141.48±

20.43**c

22.63±

655.96**c

13.18±

2.92**d

Group5

(Kaysom)

158.13±

19.95**c

69.64±

11.06**d

57.8911±

11.51**a

16.88±

3.48**a

33.90±

2.54**b

Group 6

(Sheeh)

187.43±

20.38**c

84.05±

4.74**d

95.37±

21.31**a

17.55±

2.73**a

33.72±

2.41**b

*Significantly different from the control group (P < 0.05).

**Significantly different from the control group (P< 0.001). aSignificant decrease in the parameters levels of the treated group in comparison to the control group. bSignificant increase in the parameters levels in the treated group in comparison to the control group. cSignificant increase in the parameters levels of the treated group in comparison to the diabetic group. dSignificant decrease in the parameters levels in the treated group in comparison to the diabetic group.


4. Discussion

Diabetes is a common health problem that decrease

sexual functions such as; infertility, adverse effect on

pregnancy outcomes, sexual disinclination, reduction of

clitoral sensitivity and penile erection loss [21, 22]. The

male reproductive dysfunction is a common complication

in the diabetic patients [23, 24]. These dysfunction may be

persistent or temporary according to the degree and the

duration of the disease [23, 24]. Diabetic associated-tissue

injury and its subsequent complications are most probably

induced by free radicals [25] Prior studies on diabetic

subjects showed decreased testosterone levels and

vacuolization in the germinal epithelium (spermatogonia

and spermatocytes) with subsequent decrease in the

testicular weight, sperm number and motility [26, 27].

Other studies pointed to increasing S. Ts thickness and

germ cell depletionin both the diabetic human and rats [28,

29]. Our study results showed similar results as

irregularity of the S.Ts shapes with significant decrease in

S.Ts diameters. Furthermore, similar to several previous

studies results [10, 11, 30], this study’ results showed

degeneration and necrosis of the S. Ts, giant cell

formation and interstitialchanges in the diabetic rats.

These S. Ts changes were significantly improved after

Damsissa- and Sammo-treatment, while no significant

improvement was detected after Kaysom- and Sheeh-

treatment. Moreover, marked significant reduction,

disorganization, apoptotic changes (e.g. pyknosis and

karyolysis) and depletion of the S. Ts germinal epithelium

was detected in our results. Control of apoptosis is

essential factor for the healthy spermatogenesis within the

adult testes [31]. During non-physiological stresses, such

as diabetes, significant induction of apoptotic cell death

may happen [32]. Many herbal extracts or derivatives with

high antioxidant activity are useful for diabetes treatment

and other metabolic syndrome [33]. Thus, antioxidant

therapy is one of the major strategies for diabetes

treatment [33]. Our results showed that Damsissa- and

Sammo have significant protective effects against the

apoptotic changes. This protective effect may be due to

their antioxidant properties that were proved in previous

studies [14, 33]. Abnormal high levels of free radicals and

the simultaneous decline of antioxidant defense

mechanisms may damages the cellular organelles and

enzymes, increases lipid peroxidation and leads to insulin

resistance [34]. Currently, it is proved that, the essential

oils components of Damsissa have antioxidant, anti-

inflammatory and anti-hypoinsulinemic properties [35].

In conclusion, Considerable improvements in the testicular

tissue morphological changes that were observed in diabetic

groups had been detected after Damsissa- and Sammo-

treatment in comparison to the control group. These

improvements were less obvious after Kaysom- and Sheeh-

treatment.

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