Advances in Bioscience and Biotechnology, 2013, 4, 41-47 ABB
http://dx.doi.org/10.4236/abb.2013.411A2006 Published Online November 2013 (http://www.scirp.org/journal/abb/)
Melatonin modulates the expression of Bcl-2 family
proteins in liver after thermal injury in rats
Ganka Bekyarova1*, Maria Tzaneva2, Minka Hristova1
1Division of Pathophysiology, Medical University of Varna, Varna, Bulgaria
2Department of General and Clinical Pathology, Medical University of Varna, Varna, Bulgaria
Received 26 September 2013; revised 28 October 2013; accepted 9 November 2013
Copyright © 2013 Ganka Bekyarova et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Melatonin, the principal secretory product of the
pineal gland, functions as a potent antioxidant and
free radical scavenger. Additionally, the antiapoptotic
effect of melatonin has been observed both in vivo
and in vitro. The aim of this study was to investigate
the protective effects of melatonin against burn-in-
duced injury in rat liver and whether these changes
were associated with oxidative stress and changes in
the expression of apoptosis related genes Bcl-2 and
Bax. Melatonin (10 mg/kg, i.p.) was applied immedi-
ately after 30% of total body surface area (TBSA)
burns of male Wistar rats. Malondialdehyde (MDA)
as marker of oxidative stress and tumor necrosis fac-
tor (TNF-α) as inflammatory marker were assayed by
biochemical methods. The hepatic apoptosis related
genes Bcl-2 and Bax using light immunоchistochem-
istry were investigated, too. Hepatic TNF-α and MDA
levels were increased significantly following severe
burn. Thermal trauma increased the Bax expression
without any changes of anti-apoptotic Bcl-2 protein in
sinusoidal endothelial cells (SECs) of burn-treated
animals compared with the control group animals as
well as elevated ratio Bax/Bcl-2 suggesting the sus-
ceptibility of these cells to apoptosis. Melatonin sig-
nificantly decreased the MDA and TNF-α levels in the
liver tissue. It decreased also expression of Bax, in-
creased expression of Bcl-2 and reduced Bax/Bcl-2
ratio. In conclusion, experimental data show that me-
latonin modulates the expression of Bcl-2 family pro-
teins by increasing anti-apoptotic Bcl-2, inhibits apop-
tosis and restricts the burn-induced damage.
Keywords: Melatonin; Liver; Bcl-2; Bax; Tumor
Necrosis Factor; Lipid Peroxidation; Thermal Skin
Injury
1. INTRODUCTION
Severe burn is still a serious clinical problem in emer-
gency medicine causing damages to organs distant from
burn wound. Clinical studies have shown that liver dys-
function and damage are essential for survival in cri-
tically ill and severely burned patients [1,2]. Various cel-
lular and molecular interactions such as neutrophil and
macrophage activation, mitochondrial dysfunction oxy-
gen radical, cytokine overproduction, depletion of glu-
tathione, may be involved in the processes [3-6]. These
complex mechanisms cause liver dysfunction, alteration
of hepatic parenchyma and eventually cell death (apopto-
sis).
The increased apoptosis in liver and other organs is a
result of “systemic apoptotic response” after burns [7].
Pathophysiology of burn-induced liver injury includes
many mechanisms and is not entirely defined yet. The
possible mechanisms for increased apoptosis are a de-
creased perfusion and elevation of pro-inflammatory
cytokines early post burn. Oxidative stress is recognized
as an important mechanism of apoptosis of hepatocytes
in liver injury [8,9]. The increased ROS production re-
sults in an imbalance between anti-apoptotic Bcl-2 and
pro-apoptotic Bax proteins which contributes to the sus-
ceptibility of the cell to apoptosis. Bax and Bcl-2 pro-
teins are among the most commonly used markers for
apoptosis [10]. Literature data about the role of free ra-
dicals in liver apoptosis after burns are scarce.
Melatonin (N-acetyl-5-methoxytryptamine) is an in-
doleamine endogenously produced in the pineal gland
and extrapineal tissues, liver including [11-13]. Mela-
tonin possesses a wide variety of biologic effects such as
sedative, anxiolytic, antinociceptive. Melatonin plays a
direct role in mitochondrial homeostasis by scavenging
reactive oxygen species (ROS) and reactive nitrogen
species (RNS), improving mitochondrial respiration,
increasing the level of ATP synthesis, and reducing the
*Corresponding author.

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47 42
harmful decrease in the mitochondrial membrane poten-
tial that can trigger the apoptotic cascade [14,15]. Mela-
tonin is widely used to prevent oxidative stress induced
by ischemia/reperfusion (I/R) in the liver [9,16,17] as
well as other organs such as gastric mucosa [10].
The aim of this experimental study was to investigate
the protective effects of melatonin against burn-induced
injury and whether these changes were associated with
oxidative stress and changes in the expression of apop-
tosis-related proteins Bcl-2 and Bax in rat liver.
2. MATERIAL AND METHODS
2.1. Animals
The experimental procedure was approved by the Home
Office for Care and Use of Laboratory Animals and per-
formed with a strong consideration for ethics of animal
experimentation. Age-matched male rats weighing be-
tween 220 and 250 g fasted for 12 h were allowed free
access to water before injury. Animals were housed in a
20˚C and offered rat chow and water ad libitum. They
were kept in dark: light cycles (DL = 12:12 h) in indi-
vidual wire-bottomed cages. Thus, lights were turned off
at 8:00 p.m. and turned on at 8:00 a.m. for achieving
satisfactory photoperiod.
2.2. Thermal Injury and Melatonin Treatment
After light ether inhalation, general anesthesia was per-
formed using thiopental (30 mg/kg i.p.). In order to ac-
complish 30% of third degree burn hot boiling water
(90˚C) was applied on the back of the animals during a
period of 10 sec. For those rats which were subjected to
burn injury 4 mL of physiological saline was applied
intraperitoneally (i.p) for immediate resuscitation fol-
lowing burn injury. No animals died within the first 24 h
of post-burn period. Twenty-four male Wistar rats were
divided into three equal (n = 8) groups: 1. control—non-
burned, non-treated group (C); 2. burned, nontreated (B),
3. burned rats, melatonin-treated (B+Mel). All the ani-
mals were given buprenorphine (0.3 mg/kg i.p. b.w.)
twice daily for pain control post burn. They were re-
anesthetized with thiopental and sacrificed 24 h after
burns as liver was sampled.
2.3. Biochemical Analysis
Livers was gently separated from the underlying tissue
and homogenized in 1:5 w/v 50 mM phosphate buffer
(pH 7.4) containing 0.1 mM EDTA at 4000 rpm for 10
min. The homogenate was centrifuged at 800 × g rpm for
15 min to discard the sediment and supernatant was fro-
zen until analysis. All the manipulations were performed
at 4˚C - 8˚C. Analysis was carried out immediately after
thawing of the samples. For apoptosis and immunohis-
tochemical analysis tissue samples were fixed in 10%
neutral buffered formalin and embedded in paraffin.
Membrane lipid peroxidation was assayed by MDA
measured by its thiobarbituric acid (TBA) reactivity of
liver homogenate using the method of Porter et al. (1976)
[18]. Results were expressed as nmol MDA/g tissue.
They were determined using the extinction co-efficient
of MDA-TBA complex at 532 nm = 1.56 × 10−5
cm−1·M−1 solution.
Hepatic tumor necrosis factor (TNF)-α level was quan-
tified using enzyme-linked immunosorbent assay (ELISA)
kit (Diaclone Gen-Probe, France) specific for the previ-
ously mentioned rat cytokines according to the manu-
facturer’s instructions and guidelines. Hepatic TNF-α
level was expressed as pg/mL.
2.4. Immunohistochemical Examination for
Bcl-2 and Bax in Liver
Immunohistochemical staining was performed with rab-
bit anti-rat Bcl-2 or Bax on paraffin sections according to
the manufacturer’s instructions (Santa Cruz).
Morphometric method was used to assess quantita-
tively enzyme contents of Bax and Bcl-2. Enzyme con-
tent was determined as: strong, score 3; moderate, score
2; weak, score1, and lacking, score 0 on the basis of the
occurrence of immunodeposits (Tzaneva, 2001) [19]. Bax
and Bcl-2 contents of the liver cells were defined as the
enzyme content of each cell was multiplied by their
scoring factors and divided by total number of cells.
Morphometric investigation was performed on 50 cells
from each sample.
2.5. Histopathological Examinations
Tissue specimens were fixed in 10% buffered formalin
(pH 7.2), dehydrated in ascending series of ethyl alcohol
(70% - 100%), cleared in methyl benzoate and embedded
in paraffin wax. Tissue sections of 5 μm were stained
with hematoxylin and eosin (H&E) and examined using
light microscope (Olympus BH-2, Tokyo, Japan). Apop-
totic SECs were examined by light microscopy at a mag-
nification of 400× (high power field). Approximately 500
SECs were examined in each high power field. The
apoptotic index was defined as the number of stained
cells per high power field.
Statistics
Our data were log-transformed to satisfy the assumptions
required to perform parametric tests and, therefore, pre-
sented as geometric mean and 95% confidence intervals
of the mean. Orthogonal contrasts in ANOVA were used
to statistically analyze the difference between any two
specified groups.

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47 43
3. RESULTS
3.1. Changes in the Hepatic Malondialdehyde
and Hepatic TNF Levels
The hepatic MDA level was found to be higher by 48%
(p < 0.05) in the burned group than that of the control
group (Figure 1). Treatment with melatonin significantly
reduced the elevation in mucosal MDA level, maintain-
ing the level close to the control values.
The plasma TNF-α level increased by 79% (p < 0.001)
in the burned group when compared to those of the con-
trol one. When the rats were treated with melatonin, the
concentration of hepatic TNF-α decreased by 21% sig-
nificantly, reaching the control values at the 24th hour
after burns (Figure 2).
3.2. Changes in the Expression on
Apoptosis-Related Bcl-2 in Liver
The expression of Bcl-2 was detected in sinusoidal en-
dothelial cells (SECs) in the control group (Figure 3).
The mean Bcl-2 content in the cells was 1.36 ± 0.48
(Figure 3). In the burned group, Bcl-2 expression re-
mained low (1.14 ± 0.69) and did not show significant
changes compared to controls. The expression Bcl-2 was
Figure 1. Effect of melatonin on burn-induced hepatic MDA
level. Results are given as mean ± SEM; *p < 0.05 compared
with control group; •p < 0.05 compared with burned group.
Figure 2. Effect of melatonin on burn-induced hepatic TNF-
alpha level. Results are given as mean ± SEM; ***p < 0.001
compared with Control group; •••p < 0.001 compared with
burned group.
increased in sinusoidal endothelial cells of the burned
group treated with melatonin (Figure 3). It was moderate
to strong in sinusoidal endothelial cells and their mean
content (2.22 ± 0.74) was significantly higher 63% (p <
0.001) than this of the controls.
3.3. Changes in the Expression on
Apoptosis-Related Bax in Liver
The data showed that Вax expression was detected
mainly in the SECs in the control group (Figure 4). The
intensity of the immune reaction ranged from mild to
moderate (1.46 ± 0.29) Bax expression in SECs and iso-
lated hepatocytes was moderate to strong (2.34 ± 0.59) in
the burned goup and the number of positive cells was
significantly higher by 60% (p < 0.001) compared to
controls. The expression Bax was reduced by 62% (p <
0.01) in the burned group treated with melatonin main-
taining the level close to the control values.
3.4. Changes in the Ratio Bax/Bcl-2 in Liver
The results presented in Table 1 showed that the ratio
BAX/Bcl-2 was significantly higher by 91% (p < 0.01)
in the burned group compared to controls. Treatment
with melatonin significantly reduced the BAX/Bcl-2
ratio after burns maintaining the level close to the control
values.
4. DISCUSSION
Burn trauma initiates systemic inflammatory response
where the generation of proinflammatory cytokines and
free radicals causes tissue injury to skin and to remote
organs liver including [4-6,20]. A significant increase of
proinflammatory cytokine TNF-α and MDA as a marker
of lipid peroxidation in liver has been reported in our
animal models of burns along with evidence of histopa-
thological changes such as degenerative changes and
hepatic apoptotic bodies (Figure 5) occurred in rats after
burns. The present findings showed that administration
of antioxidant melatonin can block this progress by at-
tenuating oxidative stress, inflammatory response and
apoptosis of SECs and hepatocytes.
Table 1. Changes in the expression of Bax, Bcl-2 in liver tissue
of rats after burns and ratio of Bax/Bcl-2.
Group Bcl-2 Bax Bax/Bcl-2
C group 1.36 ± 0.048 1.46 ± 0.25 1.07
B group 1.14 ± 0.69 2.34 ± 0.59*** 2.05*
B+MEL group2.22 ± 0.74+++ 1.46 ± 0.29+ 1.23+
Results are given as mean ± SEM; ***p < 0.001 compared with Control (C)
group; +++p < 0.001 compared with burned (B) group. *p < 0.05 compared
ith Control (C) group; +p < 0.05 compared with burned (B) group. w

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47 44
Figure 3. Immunohistochemistry detection for Bcl-2 in liver after burns. It is localized in sinusoidal endo-
thelial cells (SECs) in the control group (A). In the burned group, Bcl-2 expression remained low (B) The
expression Bcl-2 of the burned group treated with melatonin was moderate to strong in SECs (C).
Figure 4. Immunohistochemistry detection for Bax in liver after burns. Original magnification, 200×. It is
localized in sinusoidal endothelial cells (SECs) of the liver in the control group. (A) The staining intensity
of Bax positive cells was mild to moderate. In the burned group induction of Bax positive cells was princi-
pally in SECs and isolated hepatocytes around the central vein (B). It was moderate to strong in the indi-
vidually cells. The expression BAX was reduced in the burned group treated with melatonin maintaining the
level close to the control values (C).
Figure 5. Effect of melatonin on burn-induced histomorphological changes in liver in rats (A) control group,
(B) burned group, (C) burned, treated with melatonin group. Original magnification 400×. H & E staining,
original magnification. Arrows indicate apoptotic cells.
Oxidative stress is recognized as an important mecha-
nism of apoptosis [21,22]. The involvement of ROS is
suggested in apoptotic cell death of hepatocytes [8,23]
and endothelial cells [23].
Mitochondria are an important source and target of
ROS and RNS even when their production is growing at
a reduced antioxidant protection. ROS and RNS activate
lipid peroxidation damage and mitochondrial membranes,
protein and DNA. During the ROS-induced lipid peroxi-
dation and damage of mtDNA of the cell (internal signals)
pro-apoptotic Bax which underwent a conformational
change (externalization of phosphatidylserine (ps) trans-
locates from the cytosol to the mitochondrial membrane
where anti-apototic proteins Bcl-2 are localized [11]. The
Bcl-2 prevents the cell death whereas Bax forming het-
erodimers with Bcl-2 appears to accelerate the cell death
signal [24]. The increased ROS production results in an
imbalance between anti-and pro-apoptotic Bcl-2 family
proteins, membrane permeability transition, release of
cytochome C and further production of ROS, which con-
tributes to the susceptibility of the cell to apoptosis [25].
In the current study, we observed a significant increase
in Bax protein levels in SECs at the periphery of the si-
nusoidal space in liver. Burn-induced lipid peroxidation
was found to be accompanied by elevation of pro-apop-
totic Bax protein without changes of anti-apoptotic Bcl-2
protein of burn-treated animals compared with the con-
trol group animals. This was confirmed by the ratio Bax/

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47 45
Bcl-2 which was higher in the burned group compared
with the control group and the large number of Bax pos-
sitive stained SECs and hepatocytes together with few
Bcl-2 possitive stained SEC and hepatocytes (Table 1)
indicating the susceptibility of these cells to apoptosis.
Our results were consistent with privious reports about
elevation of Bax protein levels and reduction of the
anti-apoptotic protein Bcl-2 and its translocation to mi-
tochondria causing apoptosis in the gut after experiment-
tal thermal trauma [26]. Similar findings for apoptotic
proteins Вax and Bcl-2 in SECs and hepatocytes are
demonstrated in cold I/R model [27].
The expression of proapototic BAX protein can also
be increased and under the action of external death
signals [28,29]. The increased Bax proapoptotic protein
expression may associate with translocation of proapop-
totic Bid protein to mitochondria by the Fas/TNF-R1
pathway [30]. There is strong evidence that burn induces
Fas/FasL [31] representing one possible pathway for
burn induction of hepatic apoptosis and dysfunction.
Bid-mediated mitochondria pathway seems to be critical
in certain types of cells, such as hepatocytes [32]. Thus,
Bid connects the death receptor pathway and the mito-
chondria pathway and is responsible for cytochrome C
release and the downstream caspase activities after Fas/
TNF-R1 activation. Overexpression of pro-inflamema-
tory cytokines and decreased level of Bcl-2 protein were
reported in liver after burns [26]. Jeschke et al. (2001)
investigating molecular mechanisms of burn-induced
apoptosis and dysfunction found out that burn injury leads
to endoplasmic reticulum (ER) stress, mitochondrial da-
mage with release of cytochome C and apoptotic cell
death. Other authors suggest that Bid can induce release
of cytochrome C by mechanism independent of Bax and
increasing permeability of the mitochondrial membrane
[16].
Treatment of the animals with melatonin (2 × 10 mg/kg)
significantly prevented those changes resulting in a sig-
nificantly lower ratio Bax/Bcl-2 compared with burned
untreated rats. Melatonin is able to shift the balance to-
wards a cell-protective state. Indeed many studies dem-
onstrate that antiapoptotic effect involves the down re-
gulation of BAX or up regulation of Bcl-2 [11]. Mela-
tonin increased expression of Bcl-2, decreased expres-
sion of Bax in SECs and hepatocytes, indicating de-
creased susceptibility of these cells to apoptosis and he-
patic damage in burned rats. Therefore, anti-apoptotic
effects of melatonin are related with decrease of ROS
production in mitochondria and activate of anti-apoptotic
and redox-sensitive system Bcl-2/Bax.
The present study provides morphological and mo-
lecular biological evidence for the protective role of the
melatonin in ameliorating oxidative stress, hepatic apop-
tosis, and inflammation in experimental thermal trauma.
Melatonin may protect liver from oxidative stress by
scavenging the free radicals in mitochondria inhibiting
lipid peroxidation and production of proinflammatory
cytokine TNF-α. Thus, melatonin modulates the expres-
sion of Bcl-2 family proteins by increasing anti-apoptotic
Bcl-2 in SECs and by inhibiting apoptosis restricts the
damage of the liver.
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ABBREVIATIONS
SECs—Sinusoidal Endothelial Cells,
ROS—Reactive Oxygen Species,
RNS—Reactive Nitrogen Species,
MDA—Malondialdehyde,
TNF-α—Tumor Necrosis Factor,
Bcl-2—Anti-Apoptotic Protein,
Bax—Pro-Apoptotic Protein,
I/R—Ischemia/Reperfusion,
TBSA—Total Body Surface Area.