Melatonin modulates the expression of Bcl-2 family proteins in liver after thermal injury in rats

doi:10.4236/abb.2013.411A2006

Paper Menu >>

Journal Menu >>

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

Email: *[email protected]

Received 26 September 2013; revised 28 October 2013; accepted 9 November 2013

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.

OPEN ACCESS

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47

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

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/

OPEN ACCESS

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.

REFERENCES

[1] Lausevic, Z., Lausevic, M., Trbojevic-Stankovic, B., Krs-

tic, S. and Stojimirovic, B. (2008) Predicting multiple

organ failure in patients with severe trauma. Canadian

Journal of Surgery, 51, 97-102.

[2] Jeschke, M.G. (2009) The hepatic response to thermal

injury: Is the liver important for postburn outcomes. Mo-

lecular Medicine, 15, 337-351.

http://dx.doi.org/10.2119/molmed.2009.00005

[3] Nishiura, T., Nishimura, T., de Serres, S., Godfrey, V.,

Bradham, C.A., Nakagawa, T., Brenner, D.A. and Meyer,

A.A. (2000) Gene expression and cytokine and enzyme

activation in the liver after a burn injury. Journal of Burn

Care & Rehabilitation, 21, 135-141.

http://dx.doi.org/10.1097/00004630-200021020-00009

[4] Chen, X.L., Xia, Z.F., Yu, Y.X., Wei, D., Wang, C.R.

and Ben, D.F. (2005) p38 mitogen-activated protein ki-

nase inhibition attenuates burn-induced liver injury in rats.

Burns, 31, 320-330.

http://dx.doi.org/10.1016/j.burns.2004.10.015

[5] Gauglitz, G.G., Song, J., Herndon, D.N., Finnerty, C.C.,

Boehning, D., Barral, J.M. and Jeschke, M.G. (2008)

Characterization of the inflammatory response during

acute and post-acute phases after severe burn. Shock, 30,

503-507.

http://dx.doi.org/10.1097/SHK.0b013e31816e3373

[6] Agay, D., Andriollo-Sanchez, M., Claeyssen, R., Touvard,

L., Denis, J., Roussel, A.M. and Chancerelle, Y. (2008)

Interleukin-6, TNF-alpha and interleukin-1 beta levels in

blood and tissue in severely burned rats. European Cyto-

kine Network, 19, 1-7.

[7] Gravante, G., Delogu, D. and Sconocchia, G. (2007) “Sys-

temic apoptotic response” after thermal burns. Apoptosis,

12, 259-270.

http://dx.doi.org/10.1007/s10495-006-0621-8

[8] Kurose, I., Higuchi, H., Miura, S., Saito, H., Watanabe,

N., Hokari, R., Hirokawa, M., Takaishi, M., Zeki, S.,

Nakamura, T., Ebinuma, H., Kato, S. and Ishii, H. (1997)

Oxidative stress-mediated apoptosis of hepatocytes exposed

to acute ethanol intoxication.Hepatology, 25, 368-378.

http://dx.doi.org/10.1002/hep.510250219

[9] Guha, M., Maity, P., Choubey, V., Mitra, K., Reiter, R.J.

and Bandyopadhyay, U. (2007) Melatonin inhibits free

radical-mediated mitochondrial-dependent hepatocyte apop-

tosis andliver damage induced during malarial infection.

Journal of Pineal Research, 43, 72-81.

http://dx .do i.org/1 0.1111 /j.1600-079X.2007.00488.x

[10] Maity, P., Bindu, S., Dey, S., Goyal, M., Alam, A., Pal,

C., Reiter, R. and Bandyopadhyay, U. (2009) Melatonin

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) **-**

OPEN ACCESS

reduces indomethacin-induced gastric mucosal cell apo-

ptosis by preventing mitochondrial oxidative stress and

the activation of mitochondrial pathway of apoptosis.

Journal of Pineal Research, 46, 314-323.

http://dx .do i.org/1 0.1111 /j.1600-079X.2009.00663.x

[11] Reiter, R.J., Tan, D.X., Manchester, L.C. and Qi, W.

(2001) Biochemical reactivity of melatonin with reactive

oxygen and nitrogen species: A review of the evidence.

Cell Biochemistry and Biophysics, 34, 237-256.

http://dx.doi.org/10.1385/CBB:34:2:237

[12] Reiter, R.J. (2000) Melatonin. Lowering the high price of

free radicals. News in Physiological Sciences, 15, 246-

250.

[13] Rodriguez, C., Mayo, J.C., Sainz, R.M., Antolнn, I., Her-

rera, F., Martнn, V. and Reiter, R.J. (2004) Regulation of

antioxidant enzymes: A significant role for melatonin.

Journal of Pineal Research, 36, 1-9.

http://dx.doi.org/10.1046/j.1600-079X.2003.00092.x

[14] López, A., García, J.A., Escames, G., Venegas, C., Ortiz,

F., López, L.C. and Acuña-Castroviejo, D. (2009) Mela-

tonin protects the mitochondria from oxidative damage

reducing oxygen consumption, membrane potential, and

superoxide anion production. Journal of Pineal Research,

46, 188-198.

http://dx .do i.org/1 0.1111 /j.1600-079X.2008.00647.x

[15] Acuña Castroviejo, D., López, L.C., Escames, G., López,

A., García, J.A. and Reiter, R.J. (2011) Melatonin-mi-

tochondria interplay in health and disease. Current Topics

in Medicinal Chemistry, 11, 221-240.

[16] Kim, S.H. and Lee, S.M. (2011) Cytoprotective effects of

melatonin against necrosis and apoptosis induced by

ischemia/reperfusion injury in rat liver. Journal of Pineal

Research, 44, 165-171.

http://dx .do i.org/1 0.1111 /j.1600-079X.2007.00504.x

[17] Tuñón, M.J., San Miguel, B., Crespo, I., Jorquera, F.,

Santamaría, E., Alvarez, M., Prieto, J. and González-

Gallego, J. (2011) Melatonin attenuates apoptotic liver

damage in fulminant hepatic failure induced by the rabbit

hemorrhagic disease virus. Journal of Pineal Research,

50, 38-48.

http://dx .do i.org/1 0.1111 /j.1600-079X.2010.00807.x

[18] Porter, N.A., Nixon, J.R. and Isaac, R. (1976) Cyclic pero-

xidase and thiobarbituric assay. Biochimica et Biophysica

Acta, 441, 596-599.

[19] Tzaneva, M. (2001) Electron microscopic immunohis-

tochemical investigation of chromogranin A in endocrine

cells in human oxyntic gastric mucosa. Acta Histochemi-

ca, 103, 179-194.

http://dx.doi.org/10.1078/0065-1281-00585

[20] Jeschke, M.G., Einspanier, R., Klein, D. and Jauch, K.W.

(2002) Insulin attenuates the systemic inflammatory res-

ponse to thermal trauma. Molecular Medicine, 8, 443-

450.

[21] Sarafian, T.A. and Bredesen, D.E. (1994) Is apoptosis

mediated by reactive oxygen species? Free Radical

Research, 21, 1-8.

http://dx.doi.org/10.3109/10715769409056549

[22] Singh, R. and Czaja, M.J. (2007) Regulation of hepato-

cyte apoptosis by oxidative stress. Journal of Gastroen-

terology and Hepatology, 22, S45-S48.

http://dx .do i.org/1 0.1111 /j.1440-1746.2006.04646.x

[23] Rauen, U., Polzar, B., Stephan, H., Mannherz, H.G. and

de Groot, H. (1999) Cold-induced apoptosis in cultured

hepatocytes and liver endothelial cells: Mediation by

reactive oxygen species. FASEB Journal, 13, 155-168.

[24] Radogna, F., Diederich, M. and Ghibelli, L. (2010) Mela-

tonin: A pleiotropic molecule regulating inflammation.

Biochemical Pharmacology, 80, 1844-1852.

http://dx.doi.org/10.1016/j.bcp.2010.07.041

[25] Lucken-Ardjomande, S. and Martinou, J.C. (2005) Regu-

lation of Bcl-2 proteins and of the permeability of the

outer mitochondrial membrane. Comptes Rendus Biolo-

gies, 328, 616-631.

http://dx.doi.org/10.1016/j.crvi.2005.05.002

[26] Klein, D., Schubert, T., Horch, R.E., Jauch, K.W. and

Jeschke, M.G. (2004) Insulin treatment improves hepatic

morphology and function through modulation of hepatic

signals after severe trauma. Annals of Surgery, 240, 340-

349.

http://dx.doi.org/10.1097/01.sla.0000133353.57674.cd

[27] Yue, L.H., Zhao, Y.L., Chen, J. and Lu, D.R. (2010)

Effect of fusion protein TAT and heme oxygenase-1 on

liver sinusoidal endothelial cells apoptosis during pre-

servation injury. Chinese Medical Journal, 123, 68-73.

[28] Ding, W.X. and Yin, X.M. (2004) Dissection of the

multiple mechanisms of TNF-alpha-induced apoptosis in

liver injury. Journal of Cellular and Molecular Medicine,

8, 445-454.

http://dx .do i.org/1 0.1111 /j.1582-4934.2004.tb00469.x

[29] Shuh, M., Bohorquez, H., Loss Jr., G.E. and Cohen, A.J.

(2013) Tumor necrosis factor-α: Life and death of he-

patocytes during liver ischemia/reperfusion injury. Ochs-

ner Journal, 13, 119-130.

[30] Yin, XM. (2000) Bid, a critical mediator for apoptosis

induced by the activation of Fas/TNF-R1 death receptors

in hepatocytes. Journal of Molecular Medicine, 78, 203-

211. http://dx.doi.org/10.1007/s001090000099

[31] Jeschke, M.G., Micak, R.P., Finnerty, C.C. and Herndon,

D.N. (2007) Changes in liver function and size after a

severe thermal injury. Shock, 28, 172-177.

http://dx.doi.org/10.1097/shk.0b013e318047b9e2

[32] Chen, X., Ding, W.X., Ni, H.M., Gao, W., Shi, Y.H., Gam-

botto, A.A., Fan, J., Beg, A.A. and Yin, X.M. (2007)

Bid-independent mitochondrial activation in tumor necro-

sis factor alpha-induced apoptosis and liver injury. Mole-

cular and Cellular Biology, 27, 541-553.

http://dx.doi.org/10.1128/MCB.01166-06

G. Bekyarova et al. / Advances in Bioscience and Biotechnology 4 (2013) 41-47 47

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.