Tissue and Cell 48 (2016) 8188

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Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

The role of curcumin in streptozotocin-induced hepatic damage and

the trans-differentiation of hepatic stellate cells
Hesham N. Mustafa
Anatomy Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

a r t i c l e

i n f o

Article history:
Received 5 December 2015
Received in revised form 19 January 2016
Accepted 7 February 2016
Available online 9 February 2016
Keywords:
Curcumin
Hepatic stellate cells
Streptozotocin
Lipid peroxidation
Trans-differentiation

a b s t r a c t
Diabetic patients frequently suffer from non-alcoholic steatohepatitis. The current study aimed to investigate the role of curcumin and the response of hepatic stellate cells in streptozotocin (STZ)-induced
hepatic damage. Sixty male rats were divided into three groups. The normal control injected with a
citrate buffer vehicle and the diabetic control group which was injected intraperitoneally (IP) with a
single-dose of streptozotocin (50 mg/kg body weight) and a diabetic group was treated with an oral
dose of curcumin at 80 mg/kg body weight daily for 60 days. Curcumin effectively counteracts oxidative
stress-mediated hepatic damage and improves biochemical parameters. Alpha-smooth muscle actin (SMA) was signicantly reduced, and insulin antibodies showed strong positive immunoreactivity with
curcumin administration. These results optimistically demonstrate the potential use of curcumin, which
is attributed to its antiradical/antioxidant activities and its potential -cell regenerative properties. Also,
it has the capability to encourage the trans-differentiation of hepatic stellate cells into insulin-producing
cells for a period of time. In addition, as it is an anti-brotic mediator that inhibits hepatic stellate
cell activation and the transition to myobroblast-like cells, this suggests the possibility of considering
curcumins novel therapeutic effects in reducing hepatic dysfunction in diabetic patients.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Diabetic patients frequently suffer from a hepatic impairment
recognized as non-alcoholic steatohepatitis which is associated
with severe complications such as deposition of glycogen, steatosis,
cirrhosis, brosis and occasionally hepatic cancer (Bugianesi et al.,
2007).
Streptozotocin (STZ) is an N-nitroso-N-methylurea derivative
of 2-deoxy-d-glucose which is a diabetogenic agent that damages
the islet -cells in the pancreas selectively to produce insulindependent diabetes mellitus (IDDM) (Yang et al., 2010).
Alpha-smooth muscle actin (-SMA) is an indicator for the
recognition of myobroblast-like cells plus hepatic stellate cells
(HSCs) which are also known as Ito cells (Clement et al., 2010).
In diabetes, the glucose accessibility is increased and this leads
to an accelerated formation of advanced glycation end products
(AGEs). AGEs interact with the receptor for AGEs (RAGEs) and
consequently increase oxidative stress and cellular growth. This

Corresponding author at: King Abdulaziz University, Faculty of Medicine,

Anatomy Department, Ground Floor Building No. 8, P.O. Box: 80205, Jeddah 21589,
Saudi Arabia. Tel.: +966 566 764 762.
E-mail address: hesham977@hotmail.com
http://dx.doi.org/10.1016/j.tice.2016.02.003
0040-8166/ 2016 Elsevier Ltd. All rights reserved.

leads to an increased proliferation of HSCs, which is noticed during

hepatic brogenesis that is accompanied by the up-regulation of
RAGEs.
Oxidative stress plays a crucial role in the chronic complications
of the diabetic liver, where it is associated with an overproduction of oxygen free radicals and lipid peroxidation (Saravanan and
Ponmurugan, 2011).
The use of herbal medicine in defending against STZ-induced
liver damage looks promising. Curcumin has been gaining attention because of its health benets, such as its anti-inammatory,
antioxidant, and immune modulatory effects. Curcumin is the chief
curcuminoid of Curcuma Longa, which is a member of the Zingiberaceae family (Kumar et al., 2015).
The polyphenol curcumin improves diabetes-induced dysfunction by decreasing the level of glucose, inhibiting protein-kinase
C, and lowering superoxide production (Rungseesantivanon et al.,
2010). It reverses insulin resistance, hyperglycemia, and hyperlipidemia by inhibiting the pro-inammatory transcription factors,
signal transducers and stimulating anti-inammatory signaling
pathways (Tang and Chen, 2010).
This study aimed to investigate the probable inuence of hepatic
stellate cells in the protective capability of curcumin in STZ-induced
liver damage. It was also intended to provide an important support
in understanding the mechanism of curcumin treatment for hepatic
dysfunction.

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H.N. Mustafa / Tissue and Cell 48 (2016) 8188

2. Materials and methods

2.8. Serum parameters

2.1. Ethical approval

ALT, AST, and ALP, which were increased following hepatocyte

injury, were assessed according to the protocol detailed in the
manuals of the diagnostic kits (Randox Laboratories Ltd., Crumlin, County Antrim, UK). Serum albumin, total protein, and total
bilirubin were determined by spectrophotometer using the corresponding colorimetric kits supplied by Bio Diagnostic Company
(Cairo, Egypt) (Lee et al., 2012).

This study was conducted after receiving the approval of the

Medical Research Ethics Committee, Faculty of Medicine, King
Abdulaziz University, Jeddah, Saudi Arabia.
2.2. Chemicals and Reagents
Streptozotocin (STZ) and curcumin were purchased from
SigmaAldrich Chemicals (St. Louis, MO, USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline
phosphatase (ALP) kits were purchased from Randox Laboratories
Ltd. (Crumlin, County Antrim, UK). Serum albumin, total protein,
and total bilirubin colorimetric kits were supplied by the Bio Diagnostic Company (Cairo, Egypt).
2.3. Animals
Sixty male adult albino Wistar rats, weighing 190 20 g, that
were used were obtained from the animal house. Animals were
housed in a (24 C 3 C) temperature-controlled room with
4070% humidity and 12/12 h light/dark cycle. Rats were fed a
standard diet and tap water ad libitum throughout the experiment.
The experimental procedures were performed in accordance with
the international guidelines for the care and the use of animals in
a laboratory.
2.4. Induction of diabetes
Fasted rats (12 h) received a single intraperitoneal (IP) injection
of freshly prepared STZ (50 mg/kg body weight) dissolved in 0.1 M
citrate buffer (pH 4.5). STZ-injected animals were given a 5% glucose solution for 24 h to overcome drug-induced hypoglycemia. On
the third day after STZ injection, glucose levels were estimated by
obtaining blood samples from the cut tip of the tail using Diagnostic Accu-Chek test strips (Roche Diagnostics, Mannheim, Germany).
Blood glucose levels of 250 mg/dl or more were considered diabetic.
2.5. Experimental design
The animals were distributed into 3 groups (20/group). Group
1 (normal control) was injected IP with a citrate buffer vehicle.
Group 2 (diabetic control) received a single IP injection of STZ
(50 mg/kg body weight). Group 3 received a single IP injection of
STZ (50 mg/kg body weight) and, on the third day after the STZ
injection, curcumin was given orally with a dosage of 80 mg/kg
body weight and continued daily for 60 days (Zhang et al., 2013). At
the end of the experiment, blood samples were collected from the
retro-orbital sinus in heparinized capillary tubes for serum analysis. Animals from all groups were sacriced, and the livers were
processed for histological studies.
2.6. Plasma glucose estimation
Plasma glucose was measured using an enzymatic colorimetric method with commercially available kits (Randox Laboratories,
Ltd., Antrim, UK).
2.7. Plasma insulin estimation
Plasma insulin was determined using an insulin enzyme-linked
immunosorbent assay (ELISA) kit (code no. AKRIN-010T; Shibayagi
Co., Ltd., Gunma, Japan).

2.9. Histological examination

Livers were washed with a phosphate buffer solution and then
xed in 10% neutral buffered formalin. Tissues were dehydrated
through a graded series of alcohol, cleared in xylene, and embedded in parafn wax. Tissues were then cut into sections of 35 m
in thickness using a microtome and stained with hematoxylin and
eosin (H&E) for histopathological evaluation and with periodic
Acid-Schiff (PAS) for observation of glycogen. For each specimen,
at least three to ve slides were examined using an Olympus
BX53 microscope equipped with a DP73 camera (Olympus, Tokyo,
Japan).
2.10. Histopathological evaluation
The sections were analyzed for hydropic swelling, parenchymatous degeneration, microvesicular vacuole, macrovesicular
vacuole, focal necrosis, inammatory inltrations, brosis, and
sinusoids hyperemia. At the end of the analyses, the ndings were
presented in a table which showed the degree of degeneration
(Guven et al., 2006). Score levels of 0, +1, +2, +3 were equivalent to no, mild, moderate, and severe, respectively. The scores
represented values obtained from the tissue sections of six animals from each group with ve elds/section (Mustafa et al.,
2015).
2.11. Immunohistochemical examination
The standard peroxidase immunohistochemistry technique was
applied to slides of parafn-embedded tissue. Sections were
de-waxed in xylene, rehydrated, and pretreated with 3% of hydrogen peroxide solution to block endogenous peroxidase activity.
Microwave-assisted antigen retrieval was performed for 20 min.
Slides were then incubated overnight at 4 C with the primary
antibody against -SMA (a mouse monoclonal antibody [Dako,
Carpinteria, California, USA] with a dilution of 1:50; cellular site was
cytoplasmic) as a marker of activated HSCs with varying degrees
of intensity in smooth muscles and myobroblasts. They were
similarly incubated with the primary antibody against insulin (a
mouse monoclonal antibody [Dako, Carpinteria, California, USA]
with a dilution of 1:100; cellular site was cytoplasmic). The sections
were incubated with biotinylated IgG and then with streptavidinperoxidase conjugate (Zymed Corp, San Francisco, CA, USA).
Sections were then washed with phosphate-buffered saline (PBS)
and incubated with 3, 3 -diaminobenzidine tetrachloride (DAB)
substrate chromogen solution (1 drop of DAB chromogen/1 mL of
substrate buffer) for 5 min to detect immunoreactivity. All sections
were counter-stained with Mayers hematoxylin. Negative control
sections were prepared by omitting the primary antibody. Positive
control standard laboratory slides were used for all stains to prove
the success of the technique. All slides were examined under light
microscopy, and the presence of labeled cells was documented.
Absence of staining was recognized as a negative result (), while
the presence of brown staining was recognized as positive result
(+) (Mustafa et al., 2015).

H.N. Mustafa / Tissue and Cell 48 (2016) 8188

83

Table 1
Body and liver weight of different groups.
Normal control (n = 20)
Liver weight (g)

11.14 1.54

Body weight (g)

187.65 6.11

STZ-diabetic control (n = 20)

STZ-diabetes + curcumin (n = 20)

9.91 1.04
P* < 0.01

10.83 0.85
*P > 0.05
**P < 0.05
180.47 3.89
*P < 0.001
**P < 0.001

163.94 4.9
*P < 0.001

Values are means SD (n = 20 each group). ANOVA followed by Tukeys post hoc test.
*P: compared to normal control. **P: compared to diabetic control.

Fig. 1. Body and liver weight of different groups. The mean is given in columns, and error bars represent the standard deviation (SD).

2.12. Quantitative morphometric measurements

Ten non-overlapping elds for each animal were selected indiscriminately and analyzed. The measurements were done by using
Image-Pro Plus v6.0 software (Media Cybernetics, Silver Spring,
MD, USA) and the NIH ImageJ (v1.50) program (http://rsb.info.
nih.gov/ij/) with an Olympus BX53 microscope (Olympus, Tokyo,
Japan). The area percentages of -SMA immunopositive cells and
the insulin-positive cells number were evaluated (Hussein et al.,
2015).
2.13. Statistical analysis
Quantitative data were expressed as the mean SD of different
parameters for the treated groups. The data were analyzed using
one-way analysis of variance (ANOVA) followed by Tukeys post hoc
test. The statistical analysis was performed using IBM SPSS version
23. The values were considered signicant when p < 0.05.

albumin and total protein in the diabetic control group, while

curcumin treatment showed a signicant increase in these parameters compared to those of the diabetic control group. Conversely,
the total bilirubin was increased in the diabetic control group
and decreased signicantly with curcumin treatment (Table 2 and
Fig. 3).
3.3. Histopathological ndings
The normal control group showed normal cytoarchitecture. The
diabetic control group showed parenchymatous degeneration and
loss of architecture. With curcumin treatment, hepatic changes
were reduced (Table 3).
PAS-stained sections of the normal control group showed PASpositive red granules in the cytoplasm of the hepatocytes and
more granules around the central vein (Fig. 4A), while those
of the diabetic control group showed few PAS-positive granules
(Fig. 4B). With curcumin treatment, mild PAS-positive granules
were observed (Fig. 4C and Table 3).

3. Results
3.4. Immunohistochemistry-stained sections
3.1. Effect on liver and body weight
At the end of 60 days, curcumin treated specimens showed a
signicant increase in the body and liver weight as compared to
the diabetic control (Table 1 and Fig. 1).
3.2. Biochemical parameters
The mean glucose level of the diabetic control group was significantly increased while curcumin treatment showed a signicant
improvement in glucose level (Table 2 and Fig. 2). The mean insulin
level of the diabetic control group was signicantly decreased while
curcumin treatment showed a signicant improvement in insulin
level (Table 2 and Fig. 2).
Plasma ALT, AST, and ALP levels were elevated in the diabetic
control group in comparison to those of the normal control group,
while with curcumin treatment all of these parameters are reduced
signicantly. Furthermore, there was a signicant decrease in the

In the normal control group, -SMA staining was observed only

in the media of the blood vessels (portal veins, hepatic artery
branches, and central veins) (Fig. 5A). While the diabetic control
group showed positive -SMA immunoreactivity in the media of
the portal vessels and the periportal area and along the perisinusoidal spaces (Fig. 5B and C). In the curcumin treatment group,
mild immunoreactivity was observed (Fig. 5D). The area percentage of -SMA positive cells was signicantly higher in the diabetic
control group, while in the curcumin treatment group it was
reduced signicantly (Table 4 and Fig. 7).
Insulin-immunostained sections for both the normal control
(Fig. 6A) and diabetic control (Fig. 6B) groups showed no or minimal insulin immune positive cells. The curcumin treatment group
showed many insulin positive immune cells in the portal area
around the bile ductule (Fig. 6C). The number of insulin positive
cells was signicantly higher in the curcumin treated group (Table 4
and Fig. 7).

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H.N. Mustafa / Tissue and Cell 48 (2016) 8188

Fig. 2. Plasma glucose and insulin of different groups. The mean is given in columns, and error bars represent the standard deviation (SD).

Table 2
Biochemical parameters of the different groups.
Parameters

Normal control

STZ-diabetic control

STZ-diabetes + curcumin

Plasma glucose (mg/dl)

87.14 4.28

280.12 57.24
*P < 0.001

Plasma insulin (mIU/L)

5.17 0.73

0.54 0.11
*P < 0.001

ALT (U/l)

65.4 1.2

119.6 2.30
*P < 0.001

AST (U/l)

139.13 3.82

321.35 2.80
*P < 0.001

ALP (U/l)

68.10 2.30

85.2 4.60
*P < 0.001

Albumin (g/dl)

4.14 0.60

1.3 0.87
*P < 0.001

Total protein (g/dL)

5.94 0.89

2.03 0.78
*P < 0.001

Total bilirubin (mg/dL)

0.24 0.20

0.78 0.07
*P < 0.001

160.12 63.27
*P < 0.001
**P < 0.001
4.06 0.45
*P < 0.001
**P < 0.001
76.8 4.60
*P < 0.001
**P < 0.001
204.68 1.96
*P < 0.001
**P < 0.001
79.5 8.10
*P < 0.001
**P < 0.01
3.81 0.40
*P > 0.05
**P < 0.001
4.62 0.13
*P < 0.001
**P < 0.001
0.36 0.06
*P < 0.05
*P < 0.001

Values are means SD (n = 20 each group). ANOVA followed by Tukeys post hoc test.
*P: compared to normal control. **P: compared to diabetic control.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; mg/dl, milligrams per deciliter; mIU/L, milli-international units per liter; U/l,
units per liter; g/dl, grams per deciliter.

4. Discussion
In the current study, the diabetic control group revealed a reduction in body weight, which is attributed to the muscle destruction or
catabolism of structural protein and fat (Salahuddin and Jalalpure,
2010). Curcumin administration improves the body weight, and
this indicates the effect of curcumin on the degradation of structural protein, control of muscle wasting, and increase of metabolic
processes in the body.
The current data showed a signicant decrease in the plasma
insulin levels, which coincides with other ndings (Abdel Aziz et al.,
2013). Improved insulin level supports the hypothesis that curcumin causes -cells neogenesis and protection (Meghana et al.,
2007), and this was evidently proven by this study. Furthermore,
it is proposed that controlling hyperglycemia encourages further
regeneration of -cells (Guz et al., 2001). This may be attributed
to the suggestion that dying -cells are regenerated continuously
(Montanya et al., 2000).
Hepatocellular injury is credited to reactive oxygen species
(ROS) and lipid peroxidation that causes direct damage to hepatocytes by disrupting the membranes, protein, and DNA (Asmah
Rahmat, 2015). The injured hepatocytes release aldehyde end products, which causes further hepatocellular damage and emergence of
brosis (Novo and Parola, 2012). Also, lipid peroxidation causes an
inammatory reaction that involves the occurrence of cytokines,

mainly tumor necrosis factor (TNF-), interleukin-1 (IL-1), and

consensus interferon (IFN-c) (Sugano et al., 2006). Meanwhile, curcumin treatment alleviates the hepatocellular injuries.
In the present study, the diabetic control group revealed a congestion of the portal triad with inammation and notable brosis
near the central vein. This is attributed to tissue degradations that
are caused by depletion of the endogenous antioxidant enzyme
stores as superoxide dismutase (SOD) and catalase (CAT) (Chang
and Chuang, 2010) and promotion of de novo generation of free
radicals (Maritim et al., 2003). Consequently, the higher level of
antioxidants that exists in curcumin could boost the quenching of
some free radicals inside hepatocytes, which defend the hepatic
tissue against oxidative stress damage (Afrin et al., 2015), and this
is supported by the current ndings.
In liver impairment, hepatic stellate cells (Ito cells)
undergo trans-differentiation from lipid storing pericytes to
myobroblast-like cells that lead to an increased collagen and
extracellular matrix that is the crucial event in hepatic brogenesis
(She et al., 2005). Curcumin treatment in the current study caused
a reduction of -SMA positive cells, which is the marker of Ito
cell activation and in turn, indicates inhibition of Ito cells and the
improvement of hepatic brosis (Erenoglu et al., 2011).
Curcumin suppresses the AGEs mediated by the stimulation of RAGEs gene expression. This leads to inhibition of the
RAGE activated pathways which, in turn, prevents oxidative

H.N. Mustafa / Tissue and Cell 48 (2016) 8188

85

Fig. 3. Biochemical parameters of different groups. The mean is given in columns, and error bars represent the standard deviation (SD).

Table 3
Histopathological ndings of the liver.

Hydropic swelling
Parenchymatous degeneration
Microvesicular vacuole
Macrovesicular vacuole
Focal necrosis
Inammatory inltrations
Fibrosis
Sinusoids hyperemia
PAS

Normal
control

STZ-diabetic
control

STZdiabetes +
curcumin

0
0
0
0
0
0
0
0
+3

+2
+2
+0.5
+0.5
+2
+1
+1
+2
+1

+1
+1
+1
+1
+0.5
+1
+0.5
+1
+2

N = 20; Scale: No (0), mild (+1), moderate (+2), severe (+3).

stress, inammation, and hepatic stellate cell activation, a hallmark of non-alcoholic steatohepatitis and hepatic brogenesis
associated with type 2 diabetes mellitus (Stefanska, 2012). Moreover, AGE-encouraged production of reactive oxygen species and
lipid peroxides was attenuated after treatment with curcumin
(Stefanska, 2012).
In the current ndings, the trans-differentiation of Ito cells into
myobroblast-like cells is attributed to intercellular connections
between Ito cells and activated Kupffer cells, damaged hepatocytes,
and inammatory cytokines (Hellerbrand, 2013). Other factors that
cause this trans-differentiation include the platelet-derived growth
factor (PDGF), epidermal growth factor (EGF), and transforming
growth factor 1 (TGF-1) that are released after activation of
Kupffer cells (Nosseir et al., 2012).

In hyperglycemic conditions, the diabetic control group in the

current study showed minimal insulin immune reactions of hepatic
stellate cells. This indicates that diabetes encourages the transition of hepatic stellate cells to insulin-producing cells but it cannot
maintain their production. Furthermore, it was found that this
trans-differentiation occurs early and lasts for two weeks in the
diabetic liver (Kim et al., 2007). This transition is attributed to the
fact that both the liver and the pancreas originate from the upper
primitive foregut endoderm appendages (Lammert et al., 2003) and
the late separation of the pancreas and liver during organogenesis
might leave pluripotent cells, which are able to give rise to both
pancreatic and hepatic lineage (Meivar-Levy and Ferber, 2003).
In supporting this hypothesis, a previous study has discussed the
existence of extra-pancreatic insulin-producing cells in the liver in
the hyperglycemic condition. However, the number and production of these insulin-producing cells are not enough to improve the
hyperglycemic condition (Kojima et al., 2004). The current results
support the hypothesis that the hypoglycemic effect of curcumin is
attributed to preserving functional insulin-producing cells. In the
curcumin group in the current study, insulin immune positive cells
were increased which supports the suggestion that curcumin may
maintain insulin production by trans-differentiated hepatic stellate
cells for a period after STZ injection.
These results optimistically demonstrate the potential use
of curcumin for the treatment of STZ-induced liver damage
because of its antiradical/antioxidant activities, potential -cell
regenerative properties, and capability to encourage the transdifferentiation of hepatic stellate cells into insulin-producing cells
for a period of time. In addition, as it is an anti-brotic mediator
that inhibits hepatic stellate cell activation and the transition to

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H.N. Mustafa / Tissue and Cell 48 (2016) 8188

Fig. 4. (A) Normal control group showed PAS-positive reaction (distribution of liver glycogen) (arrow); (B) diabetic control group showed few PAS-positive reaction (arrow);
(C) curcumin treatment group showed mild PAS-positive reaction (arrow) (PAS, scale bar 20 m).

Fig. 5. (A) Normal control group showed minimal immunostaining (arrow); (B) diabetic control group showed positive immunostaining (activated HSCs) in the media of the
portal blood vessels and scattered in the periportal area (arrow); (C) the previous group with positive immunostained cells in the perisinusoidal spaces (arrow); (D) curcumin
treatment group showed mild immunostaining (arrow) (-SMA, scale bar 20 m).

H.N. Mustafa / Tissue and Cell 48 (2016) 8188

87

Table 4
Mean SD of the area percentage of -smooth muscle actin positive cells and the number of insulin-positive cells.
Normal control

STZ-diabetic control

STZ-diabetes + curcumin

Area % of -SMA

0.43 0.28

10.20 0.62
*P < 0.001

Number of insulin positive cells

0.00

0.28 0.01
*P > 0.05

1.06 0.87
*P < 0.01
**P < 0.001
5.10 3.50
*P < 0.001
**P < 0.001

Values are means SD (n = 20 each group). ANOVA followed by Tukeys post hoc test.
*P: compared to normal control. **P: compared to diabetic control.

Fig. 6. (A) Normal control group showed a negative immune reaction; (B) diabetic control group showed a minimal immune reaction; (C) the curcumin treatment group
showed strong positive immunoreactivity (insulin-producing cells) (arrows) (insulin immune reaction, scale bar 20 m).

Fig. 7. Area % of -SMA and number of insulin positive cells. The mean is given in columns, and error bars represent the standard deviation (SD).

myobroblast-like cells, consequently the way is now open to

consider curcumins novel therapeutic effects in reducing hepatic
dysfunction in diabetic patients.

Conicts of interest

5. Conclusion

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