Protective effect of Quercetin on hepatic tissue injury in
Streptozotocin- induced Diabetic Rats
Ola A. El Gohary MD and Abeer A. Shoman MD,
Departments of Physiology faculty of medicine Banha Univeristy
Abstract:
The negative impact of diabetes on the liver is well recognized. This study was designed to
evaluate the hepatoprotective properties of Quercetin in streptozotocin-induced diabetes in
rats. Male Wistar rats were made diabetic with a single injection of STZ (40 mg/kg i.p.). Rats
were randomly divided into four groups 8 animals each: Group 1, healthy control rats; Group
2 non-diabetic rats treated with 15 mg/kg b.w./day i.p. injection of Quercetin; Group 3,
diabetic rats; Group 4, diabetic rats treated with Quercetin (15 mg/kg b.w./day, i.p.) for 8
weeks. Finally, serum ALT, AST, ALP and albumin levels as well as liver MDA contents and
activities of GSH-Px, were measured to assess hepatic injury. Liver tissues of Rat in whole
groups were removed then prepared for Apoptosis analysis. Liver MDA content and serum
ALT, AST, ALP and bilirubin levels in Groups 3 were found to be significantly increased as
compared to Group 1 (P<0.001) and these parameters in Group 4 were significantly
decreased as compared to Group 3 (P<0.001). Liver GSH-Px contents and serum albumin
level in Group 3 was significantly decreased as compared to Group 1 (P<0.001) and were
found to be significantly increased in Group 4 as compared to Groups 3 (P<0.001).
Histopathological examination revealed that diabetes increased apoptotic index in liver tissue
while the treatment of diabetic rats with Quercetin was shown to have anti-apoptosis effect.
This study showed that Quercetin have hepatoprotective effects in experimentally induced
diabetic rats.
Keywords: Apoptosis, Diabetic, Quercetin, Streptozotocin, Liver, Rat
Introduction
Diabetes mellitus is one of the most common endocrine metabolic disorders.
It is a chronic disease that characterized by hyperglycemia (1). Hyperglycemic
in long time have side effect in other tissues especially in liver. Liver
dysfunctional has seen indirectly or directly, the liver is a major target of
insulin action. The onset of diabetes is accompanied by development of major
biochemical and functional abnormalities in the liver, including alterations in
carbohydrate, lipid, and protein metabolism, and changes in antioxidant status
(2,3,4,5). On the other hand, it was established that hyperglycemia increases
mitochondrial reactive oxygen species (ROS) production, which could
represent a key event in the development of diabetes complications (6,7). The
initial cellular response to high glucose challenge is the generation of ROS,
which rapidly induces apoptotic cell death (8). The balance of ROS and
antioxidant is a major mechanism in preventing damage by oxidative stress.
However, although it may not be possible to completely reverse diabetic
complications, antioxidants could be useful in preventing or attenuating the
adverse effects of chronic hyperglycemia (9). Therefore, the dietary
supplement of antioxidants such as vitamins, flavonoids has been used to
prevent the occurrence of many chronic diseases (10,11) . Flavonoids are a
large group of natural polyphenolic substances widely distributed in the plant
kingdom (12). They are important constituents of the nonenergetic part of the
human diet and are thought to promote optimal health, via their antioxidant
effects in protecting cellular components against ROS (13). Quercetin
1
(3,5,7,3′4′-pentahydroxy flavon) is one of the most widely distributed
flavonoids, present in fruit, vegetables, tea olive oil and many other dietary
sources (14). It is a strong antioxidant and it has been shown to reduce
oxidative stress (15,16). It has been demonstrated that quercetin exhibits its
therapeutic potential against many diseases, including ischemic heart
diseases, atherosclerosis, liver fibrosis, renal injury, and chronic biliary
obstruction (17,18,19).
Because liver is subjected to ROS-mediated injury in diabetes (20), our
experiments were performed to investigate the potential protective effects of
quercetin treatment on liver oxidative stress.
Material and Methods
Animals :
This study was conducted on 32 adult Wistar albino male rats 6-8 weeks old,
weighing between 170 and 200 g. Animals were housed in the animal
laboratory at the medical research center at Benha faculty of medicine. They
were housed at room temperature (25°C) and 12h/12h light/ dark cycle . All
Rats were fed a standard diet and water.
Groups of the experiment:
The animals were randomly divided into 4 groups each consisted of 8 rats as
follow:
Group (I) : Control group injected with citrate buffer daily, intraperitoneal (IP).
Group (II) : Quercetin (QR) group that received 15 mg/kg QR (IP).
Group (III) : Diabetic group that received 40 mg/kg streptozotocin (IP).
Group (IV):Treatment group received 40mg/kg (IP) STZ plus15mg/kg QR (IP).
Induction and diagnosis of diabetes mellitus:
Diabetes was induced by intraperitoneal (ip) injection of a single dose of STZ
(40 mg/kg in freshly prepared citrate buffer pH 4.5). The animals were allowed
to drink 5% glucose solution overnight to overcome drug induced
hypoglycemia. Control rats were injected by the buffer alone (21).
Diabetes was verified 72 hours later by measuring blood glucose levels (after
an overnight fasting) with the use of glucose oxidase reagent strips. Rats
having blood glucose level of ≥ 250 mg/dl were considered to be diabetic.
Quercetin administration:
Quercetin (QR) treatment was initiated 5 days after the administration of
streptozotocin. Quercetin (QR) injections of 15mg/kg intraperitoneal (IP) (22)
were continued daily to the end of the study (for 8 weeks) (23).
Chemicals used:
*Streptzotocin drug:
2
It was purchased from Sigma- Aldrich Company (USA). It is presented in
powder form, purity more than 99% to be dissolved in freshly prepared
sodium citrate buffer pH 4.5.
*Sodium citrate buffer pH 4.5:
Preparation of 0.1MCitrate Buffer:
Weigh accurately citric acid 10.5 gm and sodium citrate 14.7 gm. Mix it with
500 ml water. Make up volume to 1000 ml with distilled water.
Adjust pH 4.5 by sodium hydroxide (24).
*Quercetin drug:
Quercetin powder was obtained from Sigma Chemical Company (St. Louis,
MO, USA). It was dissolved and diluted with 20% glycerol in 0.9% normal
saline, mixed vigorously and stored in a dark bottle at 4ºC. The quercetin
solution was freshly prepared each week.(22)
Procedure of the experiments:
At the end of the treatment period, the animals were anesthetized with diethyl
ether. The animals were fixed on operating table and the blood samples were
taken as follow:
Blood sample collection
A craniocaudal incision of about 2 cm is made, parallel and with slightly to the
left of the sternum through the skin and pectoral muscles to expose the ribs. A
blunt curved forceps is then binged between the 5th and 6th ribs, through the
intercostals muscles. The gap is widened so that the rapidly beating heart
becomes visible, then the blood sample were taken from the right ventricle.
Biochemical assessment
Plasma activities of Alanine Transaminase (ALT), Aspartate Transaminase
(AST) , alkaline phosphatase (ALP) and concentration of glucose, albumin
and total bilirubin were determined by a standard automated technique using
Hitachi Analyzer Model 911 and adequate kits from Roche Company
(Switzerland). (25)
Tissue preparation
A midline laparotomy was performed to remove the liver.The liver was
dissected and fixed in 10% formalin solution at room temperature. Slices of
liver tissue were processed for histopathological & immunohistochemical
studies.
Analysis of apoptosis
3
Measurement of antioxidant activity
The rat's Liver were removed immediately and washed in normal saline and
homogenate 10% prepared in 1.15% w/v of potassium chloride. The
homogenate was centrifuged in 7000 x g for 10 minutes at 4˚C and
supernatant were used for measurement of oxidative stress by determination
of malondialdehyde (MDA) as well as estimation of antioxidant enzymes such
as glutathione peroxidase (GSH-Px). (26) Tissue MDA levels were
determined by the thiobarbituric acid (TBA) method and expressed as nmol
MDA formed/mL. Plasma MDA concentrations were determined with
spectrophotometer (27). Glutathione peroxidase (GSH-Px) activity was
measured by NADPH oxidation, using a coupled reaction system consisting of
glutathione, glutathione reductase, and cumene hydroperoxide . One unit of
enzyme activity is defined as the amount of enzyme that transforms 1 μmol of
NADPH to NADP per minute. Results are expressed as units/mg protein (28).
Statistical analysis :
All data were expressed as mean  S.D; data were evaluated by the one way
analysis of variance. Difference between groups were compared by Student's
t-test with P  0.05 selected as the level of statistical significance.
Results
Results of the effect of daily treatment of Quercetin at a dose of 15mg/kg for 8
weeks on blood glucose levels of experimental rats are presented in Table 1.
The Quercetin treatment produced hypoglycemic effect in both normal and
diabetic rats after 8 weeks of administration,but this hypoglycemic effect is
significant in diabetic group (P<0.001). Table 1 shows the effects of Quercetin
treatment on the serum levels of markers of liver injury (ALT, AST, ALP and
bilirubin) in diabetic rats. ALT, AST, ALP and bilirubin serum contents in
Groups 3 was found to be significantly increased as compared to Group 1
(P<0.001) and these parameters in Group 4 were significantly decreased as
compared to Group 3 (P<0.001). The albumin serum level in Group 3 was
significantly decreased as compared to Groups 1 (P<0.001) and this
parameter was significantly increased in Group 4 as compared to Group 3
(P<0.001).
Table 1: Comparison of the effect of Quercetin on blood glucose levels and
serum markers of liver tissue injury among the experimental groups
(mean ± SD)
4
Grou
Groups
Biochemical parameters
G
Blood
glucose
level
(mg/dL)
ALT
(U/L)
AST
(U/L)
ALP
(U/L)
Total serum
bilirubin
(Mg/dl)
Albumin
(g/dl)
Control group
rats
119±2.49
86±1.55
123±1.83
156±1.45
0.83±0.01
4.37±0.02
Nondiabetic
rats+
Quercetin
(QR) treatment
113±3.12
86±1.28
124±1.04
157±1.41
0.84±0.02
4.36±0.02
Diabetic rats
304±5.99 a
181±1.30 a
272±1.67 a
245±2.03 a
1.26±0.1 a
3.14±0.01 a
Diabetic rats+
Quercetin
(QR) treatment
127±2.06 b
87±1.60 b
135 ±1.58 b
181±1.48 b
0.88±0.01 b
4.35±0.02 b
*p<0.001 ; acompared to Group 1, bcompared to Group 3.
Table 2 shows the effects of Quercetin treatment on antioxidative activity in
liver tissue of diabetic rats. MDA contents of the liver tissue in Groups 3 was
found to be significantly increased as compared to Group 1 (P<0.001) and
liver MDA level in Group 4 were significantly decreased as compared to
Group 3 (P<0.001). The GSH-Px contents of the liver in Group 3 were
significantly decreased as compared to Groups 1 (P<0.001) and GSH-Px
activity were increased in Group 4 as compared to Group 3 (P<0.001).
Table 2: Comparison of the effect of Quercetin treatment on liver MDA and
antioxidant enzymes activities (GSH-Px) among the experimental groups
(mean ± SD)
Groups
Biochemical parameters
MDA (nmol/g protein)
GSH-Px (U/mg protein)
Control group rats
3.24±0.08
22.76±0.04
Nondiabetic
rats+ Quercetin (QR) treatment
3.23±0.01
22.44±0.22
Diabetic rats
5.37±0.03 a
17.51±0.14 a
Diabetic rats+
Quercetin (QR) treatment
4.35±0.02 b
20.65±0.13 b
5
*p<0.001; acompared to Group 1, bcompared to Group 3.
Pathologically, liver histological structure was normal in healthy control group
(Fig. 1, A). In Group 2 also there were no pathological changes so that
hepatic lobular structure seemed quite normal (Fig. 1, B). In group 3, Diabetic
rats showed (Fig. 1, C). Finally in group 4, Quercetin treatment of diabetic rats
prevented the pathologic changes in the liver (Fig. 1, D).
B
Fig 1: Microscopic appearance from liver tissues of the experimental rats
(strept – avidin – biotin) x200 . (A) Healthy control rat liver showing weak BCLX expression in cytoplasm of hepatocytes. (B) non-diabetic+ Quercetin treated
rat liver shows weak BCL-X expression in cytoplasm of hepatocytes. (C) Diabetic
rat liver showing strong BCL-X expression (arrows) in cytoplasm of hepatocytes.
(D) Quercetin treatment of diabetic rats prevented the pathologic changes in the
liver.
DISCUSSION
Worldwide studies have been done to make use of herbal medicine in
different fields of medicine. Base on ancient Persians traditional books Use of
herbal medicine has positive effect on treatment of different diseases
especially on diabetes mellitus (29). Quercetin as an important and main
flavonoids found in human mail (30) has an useful effect in human health
involves prevention of diabetes induced cataract, reduced blood vessels
fragility, anti microbial, anti viral, anti allergy, and anti inflammatory effects
and prevention of platelet aggregation (30,31,32,33). In both type 1 and type 2
diabetes mellitus the late diabetic pathological complications are mostly due
to excessive elevated production of reactive oxygen species over the capacity
6
of their removal by internal enzymatic and non-enzymatic mechanisms (34).
Therefore, additional numerous dietary artificial or natural antioxidants may be
of great importance in such cases (35). Various natural products have long
been used in traditional medical systems for treating diabetes (36). Most of
them contain a wide scale of antioxidants with a potent scavenging activity for
reactive oxygen species. Therefore, it might be assumed that these products
or isolated natural compounds could play a very important role in adjuvant
therapy. In current study, Intraperitoneal injection of Quercetin caused
significant reductions in blood glucose levels of healthy normal rats. Quercetin
also caused significant hypoglycemic effect in diabetic rats. This results
coincides with results of Mahesh and Menom (37) or Coskum et al. (38), who
found a hypoglycemic effect of quercetin when given to streptozotocindiabetic rats. It has been shown that, hypoglycemic effect of Quercetin is
mediated through stimulation of synthesis and/or release of insulin (23). In the
current study, significant decline in serum albumin level and elevations in
markers of liver injury (ALT, AST, ALP, and bilirubin) reflects the hepatocytes
injury in experimental diabetes. These results are consistent with the findings
reported by Ramesh et al (39). The data of our study also revealed that daily
treatment with Quercetin markedly improves biochemical parameters of rats
with streptozotocin induced diabetes.
Liver function tests (LFTs) are
commonly used in clinical practice to screen for liver disease, monitor the
progression of known disease, and monitor the effects of potentially
hepatotoxic drugs. The most common LFTs include the serum
aminotransferases,
alkaline
phosphatase,
bilirubin,
and albumin.
Hepatocellular damage causes release of these enzymes into circulation.
Increase in serum levels of AST shows hepatic injuries similar to viral
hepatitis, infarction, and muscular damages. ALT, which mediates conversion
of alanine to pyruvate and glutamate, is specific for liver and is a suitable
indicator of hepatic injuries. Increased levels of these enzymes are an
indicator of cellular infiltration and functional disturbance of liver cell
membranes (40). In addition, ALP is membrane bound and its alteration is
likely to affect the membrane permeability and produce derangement in the
transport of metabolites (41). On the other hand, bilirubin and albumin values
are associated with the function of hepatic cells (42). Return of the above
enzymes to normal serum values following Quercetin treatment may be due to
prevention of intracellular enzyme leakage resulting from cell membrane
stability or cellular regeneration (43). Effective control of bilirubin and albumin
shows early improvement of functional and secretory mechanism of hepatic
cells. In this study, histopathological evaluation of liver tissues showed liver
tissue damage and apoptosis induced by diabetes mellitus of the livers in
diabetic rats. With Quercetin treatment in diabetic rats no considerable
pathological changes were observed demonstrating the protective effect of
Quercetin against hepatic complications of diabetes. In this study, significant
reduction of antioxidant enzymes (GSH-Px) activity as well as significant
increase in MDA reflects oxidative stress of the liver in experimental diabetes.
These results are in line with the findings reported by Khaki et al. (22)
Increased oxidative stress in the tissues of streptozotocin diabetic rats was
similarly reported. This was said to be a contributory factor in the development
of the complications of diabetes (44,45).The data of our study also revealed
that daily treatment of Quercetin markedly improves antioxidant status of liver
7
tissue of rats with streptozotocin-induced diabetes as GSH-Px significantly
increased and MDA level markedly decreased. This indicates that in the
presence of Quercetin ,there is an improvement in the oxidative stress. This
finding is completely in agreement with those of Dias et al (23) who
demonstrated antioxidant activity of Quercetin in streptozotocin induced
diabetic mice. Liver is one of the most important organs that maintains blood
glucose levels within normal limits thus enhancement of blood glucose leads
to imbalance of oxidation-reduction reactions in hepatocytes, so that,
hyperglycemia through increasing in advanced glycation end products (AGEs)
facilities free radicals production through disturbance in ROS production (46).
Therefore, it reveals that diabetic hepatic damage is not controllable only by
inhibition of hyperglycemia (47). In other words, in early stages of diabetes,
tissues injuries are in association with hyperglycemia but its progress is not
related to hyperglycemia. Therefore, monitoring of blood glucose levels solely
is not sufficient in retarding diabetes complications. Thus, a suitable drug
must have both antioxidant and blood glucose decreasing properties (48).
One of the Quercetin anti oxidant mechanism is removal of free radical such
as xanthine super oxide and xanthine oxidase (49). Therefore suggested,
increased use of herbal medicine, fruit, vegetables, onion, tea and black
burgundy grape which are full of flavonoids and Quercetin can decrease side
effects of diabetes mellitus on liver tissue in diabetic patient complicated with
hepatic diseases.
CONCLUSION
We observed that Quercetin improved serum biomarkers of liver tissue injury
and histopathologic properties of this organ. It is presumed that Quercetin
prevents diabetic complications and ameliorates diabetic hepatopathy through
its antioxidant potential.
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