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Original Paper
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
Received: June 8, 2017
Accepted after revision: July 31, 2017
Published online: September 20, 2017
Pharmacotherapy with Thymoquinone Improved
Pancreatic β-Cell Integrity and Functional Activity,
Enhanced Islets Revascularization, and Alleviated
Metabolic and Hepato-Renal Disturbances in
Streptozotocin-Induced Diabetes in Rats
Adel Galal El-Shemi a, d Osama Adnan Kensara b Aiman Alsaegh a
Mohammed Hasan Mukhtar c
Keywords
Diabetes · Thymoquinone · Survivin · Caspase-3 · Vascular
endothelial growth factor · Cluster of differentiation 31 ·
Interleukin-1beta · Interleukin-10 · Rats
Abstract
Aims: This study is aimed at evaluating the antidiabetic effects of thymoquinone (TQ) on streptozotocin (STZ)-induced
diabetes in rats, and exploring the possible underlying
mechanisms. Methods: Diabetes was induced in adult male
Wistar rats by intraperitoneal injection of freshly prepared
STZ (65 mg/kg). After disease induction, 42 rats were equally
assigned to: controls, STZ-diabetic group, and STZ-diabetic
group treated with oral TQ (35 mg/kg/day) for 5 weeks. Fasting blood glucose levels were determined weekly, and the
animals were euthanized at day 38 post-STZ injection. Blood
samples were assessed for glucose-insulin homeostasis parameters (plasma glucose, glycated hemoglobin, serum insulin, homeostatic model assessment of insulin resistance,
and insulin sensitivity index) and lipid profile. Resected pancreases were subjected to histological examination and im-
© 2017 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/pha
munohistochemical or enzyme-linked immunosorbent assay assessment to determine the pancreatic expression of
insulin sensitizing β-cells, anti-apoptotic protein “survivin,”
apoptosis-inducer “caspase-3,” prototypic angiogenic factors (vascular endothelial growth factor [VEGF] and endothelial cluster of differentiation 31 [CD31]), pro- and anti-inflammatory cytokines (interleukin-1beta [IL-1β] and interleukin-10 [IL-10], respectively), thiobarbituric acid reactive
substances (TBARS), total glutathione (GSH), and superoxide
dismutase (SOD). The hepato-renal statuses were assessed
biochemically and histologically. Results: Therapy with TQ
markedly improved the integrity of pancreatic islets, glucose-insulin homeostasis-related parameters, lipid profile
parameters, and hepato-renal functional and histomorphological statuses that collectively were severely deteriorated
in untreated diabetic group. Mechanistically, TQ therapy efficiently increased insulin producing β-cells, upregulated
survivin, VEGF, CD31, IL-10, GSH and SOD, and downregulated caspase-3, IL-1β, and TBARSs in the pancreatic tissues
of STZ-diabetic rats. Conclusions: These findings prove the
anti-diabetic potential of TQ and its efficacy in regenerating
pancreatic β-cells and ameliorating pancreatic inflammation
Dr. Adel Galal El-Shemi
Associate Professor of Pharmacology Department of Laboratory Medicine
Faculty of Applied Medical Sciences, Umm Al-Qura University
PO Box 7607, Holy Makkah (Saudi Arabia)
E-Mail dr_adel_elshemy2006 @ yahoo.com and agshemi @ uqu.edu.sa
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a
Department of Laboratory Medicine, and b Department of Clinical Nutrition, Faculty of Applied Medical Sciences,
and c Department of Medical Biochemistry, Faculty of Medicine, Umm Al-Qura University, Mecca, Saudi Arabia;
d
Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt
© 2017 S. Karger AG, Basel
Introduction
Diabetes mellitus (DM) is a multifactorial metabolicendocrine disorder with steadily increasing global incidence, socioeconomic burden, and serious comorbidities; expecting to affect 592 million people worldwide by
the year 2035 [1]. Progressive failure of pancreatic β-cell
to produce insulin on demand and development of peripheral insulin resistance, associated with persistent hyperglycemia, disturbance of fat and protein metabolism
and other biochemical and cellular deteriorations, are the
ultimate hallmarks of all subcategories of DM [2, 3]. Furthermore, intense apoptotic death of β-cells, impaired islet vascularity, and islet inflammation (insulitis) are the
major contributors for β-cell dysfunction, depletion and
mass reduction in both types I and II DM [4–6]. Hyperglycemia-induced imbalance between pro-apoptotic and
anti-apoptotic proteins with subsequent β-cell apoptosis
has been strongly suggested [4, 5]. Similarly, generation
of inflammation, lipid peroxidation, and oxidative stress,
largely secondary to uncontrolled hyperglycemia, have
also been found to play a crucial role in induction and
exacerbation of β-cell failure and apoptosis, and in the
development and exacerbation of organ injury in diabetic patients [7, 8].
From therapeutic standpoint, the current treatment
options of DM, which are insulin-based therapy and noninsulin oral anti-hyperglycemic drugs, are unfortunately
unable to concurrently restore pancreatic β-cell mass and
functional activity, improve peripheral insulin activity,
and recover diabetic complications in an efficient manner
[9–11]. In addition, these medications have several proven dose-dependent serious side effects, such as hypoglycemia, edematous reactions, pancreatitis, fractures, tremors, lactic acidosis, increased risk of hepatitis and cancer
etc., that further hinder their anti-diabetic effectiveness
[9–11]. Therefore, there is an essential medical demand
to modify and improve the current anti-diabetes treatment strategies. For instance, add-on therapy with natural agents with well-proven anti-diabetic and tissue protective properties have been strongly suggested [12]. To
this end, thymoquinone (TQ); the main active compound
of Nigella sativa with proved pharmacological and bio10
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
logical benefits on various disease modalities, seemed be
the most promising remedy in this setting [13–16].
Several preclinical studies and some clinical reports
have demonstrated the favorable euglycemic control, antioxidant, anti-inflammatory, immunomodulatory, antidyslipidemic, anti-atherogenic, and broad tissue protective effects of TQ, which collectively appear to be of value
in improving DM therapy and alleviating its serious
multi-organ complications [15–21]. In addition, a large
body of reports has evidenced that TQ is a highly safe
natural agent with no serious toxicity [13, 14]. Therefore,
the present study was designed to examine and highlight
the anti-diabetic activity of TQ in rats with diabetes induced by streptozotocin (STZ), and more specifically, to
get a better understanding of the underlying anti-diabetogenic mechanisms that could be mediated by TQ. In
this regard, it is well established that STZ is a highly diabetogenic toxin that preferentially passes into- and rapidly destructs pancreatic β-cells. In addition, STZ has
been reported to have broad spectrum diabetogenic roles,
promote the development of insulin resistance, and act as
a donor of free radicals and pro-inflammatory mediators,
which in turn further damage the pancreatic β-cells and
different body organs [22–24].
Materials and Methods
Chemicals, Reagents, and Antibodies
Both TQ and STZ were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Primary antibodies against rat insulin, cluster
of differentiation 31 (CD31), and survivin were purchased from
Santa-Cruz Biotechnology Inc. (Burlingame, CA, USA). Enzymelinked immunosorbent assay (ELISA) kits of rat insulin, glycosylated hemoglobin (HbA1c), and caspase-3 were purchased from
Cusabio Biotech (Hubei, China), while ELISA kits of rat interleukin-1beta (IL-1β), interleukin-10 (IL-10), and vascular endothelial
growth factor (VEGF) were purchased from R&D systems (Minneapolis, MN, USA). The assay kits of thiobarbituric acid reactive
substances (TBARS), superoxide dismutase (SOD), and glutathione (GSH) were purchased from Cayman Chemical CO. (Ann Arbor, MI, USA). Other reagents and chemicals, unless otherwise
stated, were obtained from Sigma-Aldrich Co. (St. Louis, MO,
USA).
Animals, Induction of Diabetes, and Treatment Approach
All animal experiments and procedures were approved by the
local Ethics Committee of Faculty of Applied Medical Sciences,
Umm Al-Qura University, KSA, and were carried out in accordance with the National Institutes of Health guide for the care and
use of laboratory animals (NIH Publications No. 8023, revised
1978). Adult male Wistar rats of body weight (b.w.) 200–220 g
were housed in well-ventilated polyvinyl metabolic cages under
controlled conditions of temperature (23 ± 2 ° C) and 12 h lightdark cycle. The animals were allowed free access to tap water and
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and oxidative stress, and highlight its novelty in repressing
apoptosis of β-cells and enhancing islet revascularization in
STZ-diabetic rats. Further studies are required to support
these findings and realize their possible clinical significance.
Blood Sampling, Estimation of Blood Glucose-Insulin
Homeostasis-Related Parameters, and Biochemical Analysis of
Lipid Profile and Hepato-Renal Function
At the end of the experimental period (i.e., day 38 post-STZ
injection), the rats of different groups fasted for 16 h and then
euthanized under anesthesia with i.p. injection of a combination
of ketamine (60 mg/kg b.w.) and xylazine (10 mg/kg b.w.). Two
blood samples were withdrawn from the vena cava of each rat.
One sample was collected into a tube containing EDTA-anticoagulant and used for assessment of fasting plasma glucose (FPG)
and HbA1c levels, and concentrations of lipid profile-related parameters (total cholesterol, low-density lipoprotein, triglycerides, very low-density lipoprotein and high-density lipoprotein).
The second blood sample was collected into a plain tube without
anticoagulant, centrifuged at 4,000 rpm for 10 min at 4 ° C, and
its clear serum was harvested and stored at –20 ° C until used to
measure the levels of serum insulin and biomarkers of the liver
and kidney functions (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, blood urea nitrogen, and urea). Concentrations of FPG, lipid profile, and hepato-renal function biomarkers were measured using the standard
biochemical laboratory method (Cobas e411 system; Roche Diagnostics International Ltd., Switzerland), while HbA1c and serum insulin were measured using their commercial ELISA kits
(Cusabio®, China) and a fully automated ELISA system (DYNEX,
Technologies MRX II, VA, USA), following the manufacturers’
instructions. To assess the functional status of insulin, both homeostatic model assessment of insulin resistance (HOMA-IR)
and insulin sensitivity index (ISI) were calculated using the following 2 formulae: HOMA-IR = (FPG × Fasting serum insulin)/22.5 and ISI = 1/(FPG × Fasting serum insulin), respectively
[28].
Antidiabetic Role of TQ in STZ-Diabetic
Rats, and Its Underlying Mechanisms
Histopathology of Pancreas, Liver and Kidney, and
Immunostaining of Pancreatic Tissues for Insulin, Survivin,
and CD31 Positive Cells
Immediately after withdrawing blood samples, the pancreas,
liver, and kidneys of each rat were excised, washed in sterile-cold
PBS to remove blood clots, and then, a small portion of each organ
fixed in 10% neutral formalin solution and embedded in paraffin.
Five micrometer tissue sectioning slices were stained with hematoxylin and eosin (H&E) and examined under light microscopy by
a pathologist in a blinded fashion. Pancreatic tissue sections of all
animal groups were also subjected to immunohistochemical (IHC)
analysis to stain the pancreatic cells expressing insulin, endothelial marker CD31, or anti-apoptotic protein “survivin.” After deparaffinization, rehydration, antigen retrieval, and endogenous
peroxidase blocking steps [27], the tissue sections were blocked for
30 min with normal blocking serum and then incubated overnight
at 4 ° C with 1:100 dilution of primary antibodies (Santa-Cruz Biotechnology Inc., Burlingame, CA, USA) against rat insulin (Catalog No. SC-7839), CD31 (Catalog No. SC-1506), and survivin
(Catalog No. SC-17779). After washing, the slides were incubated
with appropriate combinations of secondary antibodies, followed
by Avidin/Biotin complex reagent and 3,3′-diaminobenzidine
chromogen substrate (Vector Laboratories, Burlingame, CA,
USA). The sections were counterstained with Meyer’s hematoxylin, dehydrated using ethanol and xylene. After immunostaining,
microscopic examination and digital imaging of the sections were
done by 2 observers who were blind to the animal group of the sections.
ELISA Assessment of Pancreatic Tissue Homogenates
A specimen of each harvested pancreas was homogenized (1:6
w:v) in RIPA lysis buffer containing proteinase inhibitor cocktail
(Santa-Cruz Biotechnology Inc., Burlingame, CA, USA). The homogenates were centrifuged at 10,000 rpm for 10 min at 4 ° C, and
their clear supernatants were used to measure the concentrations
of IL-1β, IL-10, VEGF, and caspase-3, as well as the levels of GSH,
SOD, and TBARSs, by using commercial ELISA kits and following
the protocols and guidelines of their manufactures. A fully automated ELISA system (DYNEX, Technologies MRX II, VA, USA)
was used, and all samples and standards were assayed in duplicate.
Statistical Analysis
Data are expressed as mean ± SEM, and statistical analyses were
performed using SPSS software version 17.0 (SPSS Inc., Chicago,
IL, USA). Differences among the groups were analyzed using oneway analysis of variance followed by Mann-Whitney U test to compare between groups. Differences were considered significant at
p < 0.05 and highly significant at p < 0.01.
Results
Biochemical, Histological, and IHC Evidence of the
Antidiabetic Activity of TQ Therapy on STZ-Diabetic
Rats
This study showed that FBG is markedly increased in
STZ-diabetic rats throughout the experimental period
compared to normal control non-diabetic rats (Fig. 1a).
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
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a commercial standard lab rodent pelleted diet, and were kept for
1 week for acclimatization prior to the experiment. For induction
of diabetes, the rats were fastened overnight and then intraperitoneally (i.p.) injected with a single dose of STZ (65 mg/kg b.w.),
freshly dissolved in 0.1 mol/L fresh cold citrate buffer of pH 4.5
[25]. During the first 24 h post-STZ-injection, the rats were provided with 5% glucose in their drinking water to avoid possible
hypoglycemic death that could occur as a result of rapid destruction of islets β-cells with excess release of pancreatic insulin stores.
Next, 3 days post-STZ injection, the induced diabetes was confirmed via measuring fasting blood glucose (FBG) level of a tail
vein blood sample using ACCU-CHEK Digital Glucometer
(Roche Diagnostics GmbH, Mannheim, Germany), and rats with
FBG threshold of ≥200 mg/dL were considered diabetic and enrolled in the study [26]. After disease induction, a total of 42 rats
were randomly and equally arranged into three groups (n = 14 per
group) as follows: group 1 – normal control rats (NC group) that
received the same volume of citrate buffer alone; group 2 – STZdiabetic untreated rats (STZ group); and group 3 – STZ-diabetic
rats treated with TQ (STZ + TQ group), in which TQ (35 mg/kg
b.w. per day) was freshly prepared in 0.1% dimethyl sulfoxide and
diluted in normal saline daily. The dosage schedule of TQ was selected based on our previous pilot study and our recently published work [27]. TQ was orally administered by gastric gavage for
5 consecutive weeks. Throughout the experimental period, FBG
levels of the tail vein blood samples of all animal groups were
weekly measured.
500
)%*PJG/
400
350
300
##
##
450
#
**
##
**
**
#
**
**
**
ns
250
*
200
*
150
*
*
*
100
50
a
0
Day 3
Week 1
Week 2
Week 3
Week 4
Week 5
#
**
b
8
*
6
STZ + TQ
2
0
NC
STZ
4
NC
STZ
STZ + TQ
Fig. 1. Effect of thymoquinone (TQ) therapy on fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in streptozotocin (STZ)-diabetic rats. Induced diabetes was confirmed by
measuring FBG level of a tail vein blood sample at day 3 post-STZ
injection. Rats with FBG threshold of 200 mg/dL were considered
diabetic. A total of 42 rats were enrolled and randomly and equally arranged into normal control (NC) group, STZ-diabetic un-
treated (STZ) group, and STZ-diabetic treated with TQ (STZ +
TQ) group, in which oral TQ (35 mg/kg) was daily administered
for 5 consecutive weeks. a Values of FBG of tail vein blood samples
throughout the experimental period. b Values of HbA1c at day 38
post-STZ injection. * p < 0.05 and ** p < 0.01 vs. NC group; # p <
0.05 and ## p < 0.01 vs. STZ + TQ group.
Similarly, HbA1c, a pivotal biomarker for persistent hyperglycemia and progression of DM, was significantly elevated in the STZ group when matched with its value in
the normal animals (Fig. 1b). Besides, fasting serum insulin level was significantly decreased in the STZ group
compared to that in the normal control group (Fig. 2a),
reflecting the incapability or incompetence of pancreatic
β-cells of STZ-diabetic rats to produce insulin. On the
contrary, daily treatment of these STZ-diabetic rats with
35 mg/kg TQ over 5 consecutive weeks exhibited remarkable normalization of their hyperglycemia and elevated
HbA1c (Fig. 1a, b, respectively) and significantly increased their lowered serum insulin levels (Fig. 2a). Theoretically, the observed effects of TQ therapy on the altered blood glucose and insulin levels could be achieved
by either direct protective/regenerative effect on β-cells
integrity and functional activity to produce insulin and/
or subduing peripheral insulin resistance, this in turn enhanced the glucose uptake by different body cells. In ad-
dition to its ameliorating effect on STZ-induced insulinopenia (Fig. 2a), TQ increased the peripheral insulin sensitivity. This was evidenced by the significantly increased
value of ISI (Fig. 2b) with concomitant reduction of
HOMA-IR (Fig. 2c) in the STZ/TQ group when matched
with STZ group.
The histopathological observations were also consistent with the biochemical findings and further supported
the potent protective/regenerative property of TQ on the
destructive effects mediated by STZ on pancreatic islets.
As demonstrated in Figure 3a, H&E staining of the pancreatic tissue sections of non-diabetic normal control rats
showed the normality of their pancreatic islets in size and
cellular components (white arrow), and these islets are
surrounded by a fine capsule and embedded within the
deeply stained pancreatic acinar cells (Fig. 3a). On the
contrary, the pancreatic islets of STZ group were remarkably reduced in their size and showed marked demarcation, disrupted cellular components, and extensive ne-
12
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+E$FnjJP/
10
##
10
**
5
0
*
NC
STZ
STZ + TQ
b
0.5
0.4
0.3
0.2
0.1
0
#
#
*
NC
Fig. 2. Effect of thymoquinone (TQ) therapy on fasting serum in-
sulin level, insulin sensitivity index (ISI) and homeostatic model
assessment of insulin resistance (HOMA-IR) in streptozotocin
(STZ)-diabetic rats. At day 38 post-STZ injection, rats in normal
control (NC), diabetic untreated (STZ), and diabetic treated with
TQ (STZ + TQ) group were fasted, euthanized under anesthesia,
Pancreas (H&E)
NC
HOMA-IR
15
ISI
,QVXOLQnj8P/
a
20
STZ
STZ
STZ + TQ
c
6
5
4
3
2
1
0
**
*
NC
STZ
STZ + TQ
and then their blood samples were used to measure the concentration of their serum insulin (a) as well as fasting plasma glucose
(FPG) levels. Additionally, to assess insulin sensitivity and resistance, ISI (b) and HOMA-IR (c) were calculated based on the values of their serum insulin and FPG. * p < 0.05 and ** p < 0.01 vs.
NC group; # p < 0.05 and ## p < 0.01 vs. STZ + TQ group.
STZ + TQ
BV
b
c
NC
STZ
STZ + TQ
d
e
f
Insulin-positive cells
a
ogy and regeneration of insulin-producing β-cells in streptozotocin (STZ)-diabetic rats. At the end of the experimental period, the
pancreatic tissues of normal rats (NC), diabetic untreated rats
(STZ), and diabetic rats treated with TQ (STZ + TQ) were subjected to histopathological (H&E; a–c) examination and immunohistochemical staining (IHC) of insulin-positive β-cells (d–f).
a A representative H&E staining of the pancreatic tissues of NC
group showed the normality of their islets (white arrow) in size and
cellular components, and these pancreatic islets were markedly reduced in size, demarcated and showed disrupted cellular components and extensive necrotic changes in diabetic untreated (STZ
Antidiabetic Role of TQ in STZ-Diabetic
Rats, and Its Underlying Mechanisms
group; b). By contrast, treatment of diabetic rats with TQ (STZ +
TQ group; c) efficiently restored the size and shape of their pancreatic islets and ameliorated most of the histomorphological deteriorations that were induced by STZ. d A representative IHC for
insulin-positive β-cells (brown color) that occupy most of the islet
mass of NC group (yellow arrow). However, these insulin-positive
cells were intensely reduced in pancreatic islets of diabetic untreated (STZ) group (e) as a result of their destruction by STZ. On the
contrary, therapy with TQ had successfully endeavored to replenish and restore the mass and functional activity of these insulin
producing β-cells (f). Original magnification: ×200.
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
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Fig. 3. Effect of thymoquinone (TQ) therapy on pancreatic histol-
STZ
STZ + TQ
a
b
c
CD31-positive cells
NC
d
*
300
200
#
**
100
0
NC
STZ
STZ + TQ
Fig. 4. Angiogenic and revascularization effect of thymoquinone
(TQ) therapy on pancreatic islets in streptozotocin (STZ)-diabetic rats. The expression pattern of two important angiogenic and
vascularizing factors, endothelial cell CD31 (a–c) and vascular endothelial growth factor (VEGF; d) were measured by immunohistochemical staining and enzyme-linked immunosorbent assay, respectively, in the harvested pancreatic tissues of normal rats (NC),
diabetic untreated rats (STZ), and diabetic rats treated with TQ
(STZ + TQ). Compared to NC group (a, d), the pancreatic islets
of STZ group (yellow arrow; b) showed profound diminishing in
cells positivity stained with CD31 (red-brown colored) and also a
significant decrease in their pancreatic concentration of VEGF
(d). However, therapy with TQ had successfully replenished the
intra-pancreatic expression pattern of both CD31 (c) and VEGF
(d) in these diabetic rats. a–c Original magnification: ×200. d * p <
0.05 and ** p < 0.01 vs. NC group; # p < 0.05 vs. STZ + TQ group.
crotic changes (Fig. 3b), and treatment of these diabetic
rats with TQ efficiently succeeded to ameliorate most of
the histomorphological deteriorations of STZ on pancreatic islets (Fig. 2c).
In coherency, the results of IHC staining of insulin
positive cells (Fig. 2d–f) also proved a clear evidence of
the regenerative/restoring effect of TQ on the mass of
functionally active insulin synthetizing β-cells. As demonstrated in Figure 2d, insulin positive cells (brown color) occupy most of the pancreatic islets (yellow arrow)
of normal control animals, and these cells are intensely
lowered in the pancreatic islets of the STZ group
(Fig. 2e); thus confirming the destructive and depletion
effect of injected STZ on pancreatic β-cells. Interestingly, therapy with TQ had endeavored to replenish
and restore the mass and functional activity of
insulin-producing β-cells in these STZ-diabetic rats
(Fig. 2f).
TQ Therapy-Mediated Angiogenic and Anti-Apoptotic
Effects on Pancreases of STZ-Diabetic Rats
There is a compelling body of evidence that impaired
islets vascularization and progressive β-cell apoptosis are
the pivotal contributors for the progressive β-cell failure
and mass depletion in the different subcategories of DM.
Hence, to explore this, the expression patterns of two important angiogenic and vascularizing factors, represented
by endothelial cell CD31 and VEGF, as well as expression
levels of the anti-apoptotic/cell surviving protein, survivin, and its antagonism “caspase-3” that acts as executioner, inducer and driver of cellular apoptosis, were measured in the pancreatic tissues of different animal groups,
using IHC for CD31 and survivin, and ELISA or VEGF
and caspase-3. As demonstrated in Figure 4, islets cells
(yellow arrow) stained with CD31 (red-brown colored;
Fig. 4b), and also the concentration of VEGF (Fig. 4d),
were diminished in the pancreatic tissues of STZ diabetic
14
Pharmacology 2018;101:9–21
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VEGF, pg/mL
400
Caspase-3, ng/mL
STZ + TQ
b
c
BV
a
d
STZ
16
14
12
10
8
6
4
2
0
##
**
*
NC
STZ
STZ + TQ
Fig. 5. Anti-apoptotic effect of thymoquinone (TQ) therapy on islet
cells in streptozotocin (STZ)-diabetic rats. Intra-pancreatic expression pattern of a cell surviving/anti-apoptotic protein “survivin” in
adjunction with its antagonism apoptosis-inducer “caspase-3” were
measured by immunohistochemical staining and enzyme-linked
immunosorbent assay, respectively. Compared to NC group (a, d),
cells positivity stained with survivin (red-brown colored) were
weekly expressed in pancreatic islets (yellow arrows) of STZ group,
and on the contrary, there was an overexpression of capsase-3 in
their pancreatic tissues (d). Noticeably, treatment of these diabetic
rats with TQ (STZ + TQ group) was associated with increased intraislet survivin (c), but downregulated the pancreatic caspase-3 in
these diabetic rats. a–c Original magnification: ×200. d * p < 0.05
and ** p < 0.01 vs. NC group; ## p < 0.01 vs. STZ + TQ group.
rats compared with those of STZ + TQ group (Fig. 4c, d,
respectively). Likewise, the pancreatic islets of STZ group
(Fig. 5b; yellow arrows) had a weak staining towards survivin (red-brown colored cells) and, on the contrary, they
had an overexpression of capsase-3 (Fig. 5d) compared to
the levels of STZ + TQ group (Fig. 5c, d, respectively).
Based on these findings, it can be suggested that, via upregulation of survivin, VGEF, and CD31 and downregulation of capsase-3, therapy with TQ successfully exhibited
concordance intra-islet angiogenic, proliferative and antiapoptotic effects to promote the regeneration and restoration of the functional activity of their β-cells that were remarkably deteriorated in this STZ diabetes model.
TQ Therapy-Mediated Anti-Inflammatory
and Antioxidant Effects and Suppressed Lipid
Peroxidation in Pancreases of STZ-Diabetic Rats
To identify further mechanistic insights behind the
antidiabetic effects of TQ, the levels of IL-1β, a key in-
flammatory cytokine which directly triggers β-cell dysfunction and apoptotic death; IL-10, an anti-inflammatory cytokine that prevents insulitis and reduces severity of diabetes; GSH and SOD, predominant antioxidant
defense elements; and TBARS, a strong index of lipid
peroxidation and oxidative stress, were measured in the
pancreatic tissues of the different animal groups using
ELISA assays. As shown in Figure 6, there were significant elevations in the levels of IL-1β (Fig. 6a) and TBARS
(Fig. 6e), and significant reductions in the levels of IL-10
(Fig. 6b), GSH (Fig. 6c), and SOD (Fig. 6d) in the pancreatic tissues of STZ group compared with normal control group. Nevertheless, all these changes were significantly reversed with TQ therapy, whereby the pancreatic tissues of STZ + TQ group demonstrated
upregulation of IL-10 (Fig. 6b), GSH (Fig. 6c) and SOD
(Fig. 6d), and downregulation of IL-1β (Fig. 6a) and
TBARS (Fig. 6e) compared with STZ-diabetic untreated
group.
Antidiabetic Role of TQ in STZ-Diabetic
Rats, and Its Underlying Mechanisms
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
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Survivin-positive cells
NC
##
**
,/SJP/
NC
STZ
STZ + TQ
b
TBARS,
njPROPJSURWHLQ
c
e
80
SOD,
8PJSURWHLQ
GSH,
njPROPJSURWHLQ
100
60
40
##
20
**
0
NC
STZ
STZ + TQ
d
400
350
300
250
200
150
100
50
0
350
300
250
200
150
100
50
0
#
**
NC
STZ
STZ + TQ
*
##
**
NC
STZ
STZ + TQ
##
200
**
150
100
*
50
0
NC
STZ
STZ + TQ
Fig. 6. Thymoquinone (TQ) therapy-suppressed pancreatic lipid
peroxidation and mediated antioxidant and anti-inflammatory effects on streptozotocin (STZ)-diabetic rats. At day 38 post-STZ
injection, the levels of interleukin (IL)-1β (a), the key driver inflammatory cytokine, IL-10 (b); a well-known anti-inflammatory
cytokine, reduced glutathione (GSH; c) and superoxide dismutase
(SOD; d); as indices of antioxidant status and thiobarbituric acid
reactive substances (TBARS; e); as an index of lipid peroxidation/
oxidative stress were measured in the resected pancreases of normal rats (NC), diabetic untreated rats (STZ), and diabetic rats
treated with TQ (STZ + TQ). * p < 0.05 and ** p < 0.01 vs. NC
group; # p < 0.05 and ## p < 0.01 vs. STZ + TQ group.
Anti-Dyslipidemic and Hepato-Renal Protective
Effects of TQ Therapy on STZ-Diabetic Rats
As shown in Table 1, the STZ-diabetic rats that were
left without treatment showed significant elevations in
their plasma levels of total cholesterol, low-density lipoprotein, triglycerides, and very low-density lipoprotein;
however, a decrease was observed in high-density lipoprotein, compared to the normal control group; and
these disturbed lipid profile parameters were significantly improved when the diabetic rats were treated with TQ.
In a similar line, the biochemical and histological examinations (Table 1; Fig. 7) reflected that diabetic untreated
rats developed clear injury and impairment in their livers
and kidneys, evidenced by significant increases in their
hepato-renal sero-biomarkers (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and
ALB as biomarkers of liver function; and creatinine,
blood urea nitrogen, and urea as biomarkers of renal
function), and such hepato-renal changes and impairments were forcefully attenuated by TQ therapy (Table 1; Fig. 7). As demonstrated in Figure 7a, the histological examination of the livers of normal control group
showed the normal architecture and histomorphology of
their hepatic lobules (L) with hepatocytes normally arranged from the portal vein (V). On the contrary, livers
of diabetic untreated rats (Fig. 7b) had inflammatory/necrotic hepatic lobules (circular dotted lobule) with congested portal vein (red arrow) and inflammatory cellular
infiltration (black arrows). Similarly, histological examination of kidney tissue sections of STZ-diabetic rats
(Fig. 7e) revealed the presence of a significant degree of
necrotic and vacuolization of renal tubular epithelial
cells and tubular atrophy (black arrows) with thickening
of glomerular basement membrane and condensation of
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,/DŽSJP/
a
1,200
1,000
800
600
400
200
0
Table 1. Biochemical findings of plasma lipid profile and serum biomarkers of hepato-renal function at day 38,
post-STZ injection
Parameter
NC group
Plasma levels of lipid profile parameters, mg/dL
TC
66.3±8.2
LDL
19.8±3.2
TG
43.6±8.4
VLDL
8.72±1.7
HDL
37.8±3.3
Serum levels of liver function biomarkers
AST, IU/L
93.6±5.1
ALT, IU/L
41.2±3.7
ALP, IU/L
104.6±12.9
ALB, g/dL
4.6±0.4
Serum levels of kidney function biomarkers, mg/dL
CRE
0.29±0.02
BUN
21.3±4.1
Urea
42.8±3.2
STZ group
113.5±11.2**, #
39.4±5.8**, #
231.0±16.5**, ##
46.2±7.3**, ##
26.9±4.1*, #
141.6±7.3*, #
76.7±5.2*, #
189.3±45.4*, #
3.1±0.2*, #
0.65±0.04**, #
40.7±6.6*, #
81.4±9.5**, #
STZ + TQ group
73.9±9.8
23.4±4.1
88.2±9.5§
17.6±1.9§
33.9±3.8
111.5±5.6
53.5±4.0
136.8±9.5§
4.2±0.2
0.34±0.02
27.5±5.3
50.4±7.2
glomerular matrix (yellow arrows), when compared with
renal tubules (T) and glomeruli (G) of normal control
group (Fig. 7d). Evidently, most of these hepato-renal
histopathological changes were obviously hidden when
these diabetic rats were treated with TQ (Fig. 7c and f,
respectively).
The current study investigated the antidiabetic role
mediated by TQ therapy on STZ-model of experimental
diabetes in rats. Data revealed that daily therapy of STZdiabetic rats with oral TQ (35 mg/kg) for 5 consecutive
weeks markedly protected their pancreatic islets and significantly improved their altered blood glucose, HbA1c,
and insulin levels. In addition, TQ therapy significantly
improved peripheral insulin activity, as evidenced by the
normalization of ISI and HOMA-IR values [28], lipid
profile parameters, and hepato-renal function and histomorphology in STZ-diabetic rats. Most importantly, TQ
therapy exhibited potent regenerative effects on the integrity and mass of insulin-producing β-cells as well as in
revascularization of impaired islets vasculature in these
STZ-diabetic rats.
Mechanistically, the reported antidiabetic activity of
TQ therapy in this diabetic model could be achieved by
more than one mechanism. In this regard, it is well
known that gradual β-cell depletion and mass reduction,
due to imbalance between β-cell death and renewal, is a
hallmark of type I and type II DM, and apoptosis of pancreatic β-cells is the main contributor for β-cell depletion and mass reduction in diabetic patients [4, 5, 29].
Multiple studies have investigated whether β-cell apoptosis in DM is due to the abnormal overexpression of
apoptosis inducers with downregulation of anti-apoptotic proteins [4, 5, 29], and highlighted the crucial roles
mediated by a multifunctional anti-apoptotic/cell surviving protein, known as survivin, in preventing β-cell
apoptosis, maintaining their survival, and enhancing
their renewal and mass expansion [29–32]. Chronic hyperglycemia and glucotoxic stress of DM lead to predominant reduction in survivin expression in pancreatic
islets [30–32]. By contrast, aberrant overexpression of
caspase-3, the well-known key driver of apoptosis-inducing death in mammalian cells, has been significantly
Antidiabetic Role of TQ in STZ-Diabetic
Rats, and Its Underlying Mechanisms
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
Discussion
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STZ, streptozotocin; NC, normal control rats; STZ, diabetic untreated rats; STZ + TQ, diabetic rats treated
with oral thymoquinone (35 mg/kg/day) for 5 consecutive weeks; TC, total cholesterol; LDL, low-density lipoprotein; TG, triglycerides; VLDL, very low-density lipoprotein; HDL-C, high-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; ALB, albumin; CRE, creatinine;
BUN, blood urea nitrogen.
* p < 0.05, § p < 0.05, and ** p < 0.01 vs. NC group; # p < 0.05 and ## p < 0.01 vs. STZ + TQ group.
NC
STZ
STZ + TQ
a
b
c
NC
STZ
STZ + TQ
e
f
Liver (H&E)
V
Kidney (H&E)
L
G
d
kidneys. At day 38 post streptozotocin (STZ)-injection, the resected
livers and kidneys of rats in normal control (NC), diabetic untreated
streptozotocin (STZ), and diabetic treated with thymoquinone (TQ)
(STZ + TQ) group were examined histologically by H&E staining.
a Photomicrographs of livers of NC group showed the normal architecture and histomorphology of their hepatic lobules (L) with hepatocytes normally arranged from the portal vein (V). b Photomicrographs of livers of STZ group showed inflammatory/necrotic hepatic lobules (circular dotted lobule) with congested portal vein (red
arrow) and inflammatory cellular infiltration (black arrows). d Photomicrographs of kidney tissues of NC group with normal histomorphology pattern of their renal tubules (T) and glomeruli (G). e Photomicrographs of kidney tissues of STZ group revealed the presence
of necrotic and vacuolization of renal tubular epithelial cells and tubular atrophy (black arrows) with thickening of glomerular basement membrane and condensation of glomerular matrix (yellow arrows). Evidently, most of these hepato-renal histopathological
changes were obviously hidden when these diabetic rats were treated
with TQ (c, f, respectively). Original magnification: ×200.
implicated in β-cell apoptosis [33], and the ability of survivin to directly inhibit caspase-3 has been previously
evidenced [34]. Interestingly, data of the present study
are in harmony whereas therapy with TQ exhibited profound restoration of survivin expression with concomitant downregulation of caspase-3 levels in the pancreatic tissues of STZ-diabetic rats; proposing the antiapoptotic role mediated by TQ against the peculiar β-cell
cytotoxic-, apoptotic-, and destructive effects induced by
STZ in this model [22–24]. Although it has been shown
[27, 35] that TQ is a potent antineoplastic agent that induces apoptosis in cancer cells, its distinguished antiapoptotic properties, via decreasing apoptotic drivers
and increasing anti-apoptotic proteins, have also been
observed in several non-cancerous modalities, suggesting its paradoxical anti-apoptotic and apoptotic dual
roles in non-cancerous and cancerous cell injuries, respectively [19, 36–39].
Second, in the current study we have found that therapy with TQ upregulated the pancreatic expression of two
important angiogenic factors, CD31 and VEGF that were
markedly repressed in STZ-diabetic untreated rats, hypothesizing the restoring effect of TQ on islets angiogenesis and vascularization in this model, and in turn represent as an additional mechanism to regenerate the destructed β-cells. In agreement, the recent advancement in
the field of pancreatic islets vascularization and transplantation indicated that VEGF system is essential for the
proliferation of pancreatic islet endocrine endothelial
cells to give functionally active insulin-producing β-cells
[6, 40, 41]. Furthermore, native pancreatic islets are
densely vascularized structures, in which CD31 and
Fig. 7. Histopathological (H&E) findings of the resected livers and
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T
VEGF play a critical role in their vascularization and provide enough oxygenated blood for β-cells to maintain
their normal mass and function to produce insulin of demand [41]. Also, studies on pancreatic tissues of both human diabetic patients [6] and experimental animals with
induced DM including those with STZ model [42, 43],
have confirmed the vital role of CD31 and VEGF on islet
vascularization, oxygenation, nourishment, survival, and
insulin productivity [40–43].
Next, TQ is known to have strong anti-inflammatory,
immunomodulatory, and antioxidant effects [13, 14, 18].
Such properties of TQ were also observed here, whereby
its administration to STZ-diabetic rats had significantly
decreased their elevated pancreatic levels of the inflammatory cytokine, IL-1β, while increasing the lowered levels of the anti-inflammatory cytokine, IL-10. In a similar
line, therapy with TQ resulted in a significant decrease of
the end product of lipid peroxidation, TBARSs, while increasing the levels of the GSH and SOD in the pancreatic
tissues of STZ-diabetic rats. TQ reduced IL-1β and
TBARS, and upregulated IL-10, GSH, and SOD in different disease modalities [13, 14, 18]. The pivotal diabetogenic role of IL-1β, through inducing β-cell dysfunction,
DNA fragmentation and apoptotic death, and induction
of peripheral insulin resistance, is now decisively known
[44, 45]. β-cells overexpressing IL-1β have been obviously detected in pancreatic tissues of patients with type II
DM but not in nondiabetic control subjects [44], and IL1β-antagonism therapy has been tested clinically in patients with type II DM [45, 46]. On the contrary, increasing IL-10 levels in the pancreatic tissues has shown to subdue pancreatic islet inflammation (insulitis) and improve
diabetes-associated hyperglycemia and insulinopenia
[47]. Likewise, there is now a compelling body of evidence
that strengthens the axial pathogenic role of lipid peroxidation and oxidative stress that occur secondary to longterm hyperglycemia in organizing and aggravating insulitis, β-cell dysfunction, insulin resistance, and diabetic
organ complications. On the contrary, suppression of endogenous lipid peroxidation and replenishment of antioxidant status are essential therapeutic demands in DM
besides adequate glycemic control [7, 8, 12].
Lastly, both hepato-renal protective and blood lipid
lowering effects mediated by TQ therapy were clearly detected in STZ-diabetic rats. Progressive renal tubulointerstitial and glomerular damage with ultimate diabetic
nephropathy is a highly prevalent complication in uncontrolled diabetic patients, accounting for approximately
50% of worldwide end-stage renal disease [48], and there
are several mechanisms that implicate the development
of liver injury in diabetic patients [49]. Diabetic hyperglycemia inducing hypoxia, inflammation, and lipid peroxidation, and likewise, insulin deficiency leading to increased blood levels of amino acids, have been hypothesized as key factors in hepato-renal cellular and tissue
damage among diabetic patients [48–49]. Thus, treatments display antioxidant and anti-inflammatory effects
and normalized hyperglycemic status could eventually
delay or prevent the development of hepato-renal injury
in diabetic patients [50, 51]. Similarly, DM is closely associated with the dysregulation of lipid metabolism with
subsequent development of diabetic dyslipidemia and
cardiovascular disease (CVD), raising the importance of
blood lipids lowering therapy to hide CVD and its comorbidities in diabetic patients [52, 53]. The utilization of
standard cholesterol lowering (statin)-based medications
for the treatment of hyperlipidemia of diabetic patients
has been significantly declined due to their common
dose-dependent diabetogenic property including inducing new-onset DM and increasing the risks of presented
diabetes [54, 55]. Moreover, statin-evoked suppression of
prenyl intermediates during cholesterol biosynthesis has
been revealed to be positively linked to the induction of
atherosclerosis and heart failure, suggesting the importance of utilizing natural medicines instead of statins for
the management of hyperlipidemia in diabetic patients
[53, 54]. Towards this goal, several natural medications
with different lipid lowering mechanisms have been studied extensively for the prevention of hyperlipidemia and
CVD in DM; however, none have been recommended to
be effective in improving long-term outcomes [53]. Thus,
based on our present findings and previously published
data [13, 14, 17, 21], TQ appears to be the most promising
alternative anti-dyslipidemic agent to manage hyperlipidemia in diabetic patients.
Antidiabetic Role of TQ in STZ-Diabetic
Rats, and Its Underlying Mechanisms
Pharmacology 2018;101:9–21
DOI: 10.1159/000480018
Data of the present study demonstrated the potential
ameliorative effects of TQ therapy on STZ-induced diabetes in rats. Therapy with TQ was associated with reduction of pancreatic inflammation and lipid peroxidation, exhibited potent regenerative effects on insulin producing β-cells, and as a novelty, successfully repressed
apoptosis of β-cells and enhanced islet revascularization
in this diabetic model. These anti-diabetic findings in
turn raise the value of using TQ as an add-on supplementary therapy to improve the treatment efficacy for
DM and its complications. However, further studies are
19
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Conclusions
still required to support these findings, elucidate additional molecular and other explanatory mechanisms,
and realize the possibility of their clinical translation and
importance.
Grant Support and Acknowledgments
Disclosure Statement
The authors declare that they have no conflict of interest.
Author’s Contributions
This research work was financially supported by a
grant (43509032) from the Institute of Scientific Research
(ISRRIH) and Deanship of Scientific Research, Umm AlQura University, Holy Makkah, The Kingdom of Saudi
Arabia.
A.G.E. conceived and designed the study and the entire experiment; A.G.E., O.A.K., A.A., and M.H.M. performed the experiments and laboratory processes, and contributed in acquisition,
analysis, and interpretation of data for the work; and A.G.E. wrote
and submitted the manuscript. All authors approved the manuscript to be published.
20
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