TNF-a suppression by glutathione preconditioning attenuates hepatic ischemia reperfusion injury in young and aged rats

ORIGINAL RESEARCH PAPER

TNF-a suppression by glutathione preconditioning attenuateshepatic ischemia reperfusion injury in young and aged rats

Arumugam Suyavaran • Chitteti Ramamurthy •

Ramachandran Mareeswaran • Ariraman Subastri •

Polaki Lokeswara Rao • Chinnasamy Thirunavukkarasu

Received: 5 September 2014 / Accepted: 7 November 2014

� Springer Basel 2014

Abstract

Background and aim Hepatic ischemia reperfusion (I/R)

stimulates Kupffer cells and initiates injury through tumor

necrosis factor-a (TNF-a) upregulation. Aim of this study

was to compare the variable effects of reduced glutathione

(GSH) pre-treatment on I/R liver injury in young and aged

rats.

Methods Wistar male rats were sorted into young (groups

I–III) and aged (groups IV–VI). All groups except sham

(groups I and IV) were subjected to 90-min ischemia and

2-h reperfusion. The treatment groups received 200 mg/kg

bwt (groups III and VI) of GSH, 30 min prior to I/R.

Variable effects of GSH were studied by transaminase

activities, thiobarbituric acid-reactive substances (TBARS),

GSH level, GSH/oxidized GSH (GSSG) ratio, TNF-a level,

apoptotic markers and confirmed by histopathological

observations.

Results Our findings revealed that I/R inflicted more liver

damage in aged rats than young rats. The GSH treatment

prior to surgery significantly lowered the serum transami-

nase activities, hepatic TBARS level and effectively

restored the GSH/GSSG ratio in both young and aged rats

more remarkably in the mitochondria. Western analysis

depicted that the GSH treatment effectively suppressed

TNF-a expression and apoptotic markers in both young and

aged rats. These findings were further confirmed by ter-

minal deoxynucleotide transferase dUTP nick end labeling

assay and histopathological observations of liver sections

of young and aged rats.

Conclusion Restoration of GSH/GSSG ratio through

GSH pre-conditioning inhibits TNF-a and apoptosis in

hepatic I/R injury. Hence, GSH pre-conditioning may be

utilized in both young and aged individuals during liver

transplantation/surgery for better post-operative outcomes.

Keywords Ischemia/reperfusion � TNF-a � Glutathione �Ageing � GSH/GSSG ratio

Introduction

The hepatic inflow from portal triad is occluded during

transection procedures to prevent severe blood loss and

reduce the morbidity [1]. Pringle maneuver is the procedure

by which the portal triad is clamped either partially or fully

until surgical intervention and then released to re-establish

the blood flow [2, 3]. The occlusion of blood flow during this

procedure leads to ischemia of the hepatic parenchyma

which leads to accumulation of toxic metabolites further

worsening the hypoxic condition. The ischemia reperfusion

(I/R) injury occurs due to oxidative stress, during restoration

of blood supply to hypoxic organ. Such sudden release of

blood supply, also results in reperfusion of toxic metabolites

and inflammatory substances into the parenchyma [4]. Ear-

lier studies have shown that I/R-induced oxidative damage is

associated with increased rate of acute liver graft failure and

chronic liver dysfunction after liver transplantation [4]. The

I/R is unavoidable in hepatic surgical procedures including

liver transplantation. Hence, therapeutic strategies to curtail

I/R injury are being considered for the better clinical care of

hepatic disorders.

The reperfusion injury has been largely attributed to

activation of Kupffer cells (KC) and generation of reactive

oxygen species (ROS) [5, 6]. It has been shown earlier that,

A. Suyavaran � C. Ramamurthy � R. Mareeswaran � A. Subastri �P. Lokeswara Rao � C. Thirunavukkarasu (&)

Department of Biochemistry and Molecular Biology,

Pondicherry University, Puducherry 605014, India

e-mail: [email protected]

Inflamm. Res.

DOI 10.1007/s00011-014-0785-6 Inflammation Research

123

upon reperfusion-induced activation KC releases inflam-

matory mediators such as tumor necrosis factor-a (TNF-a),

interleukins and chemokines into sinusoidal space, which

further leads to ROS generation mediating proinflamma-

tory gene activation resulting in further damage to hepatic

parenchyma [7–10].

Aged liver does not show significant morphological

changes when compared to young liver. However, reduced

blood flow and bile flow have been shown to be associated

with liver ageing [11, 12]. The extent to which aged liver

can withstand oxidative insult is a mystery, since the

enzymatic and non-enzymatic antioxidant machinery in

aged is not as efficient as in young [13, 14]. Reports have

shown that ROS accumulation in aged liver mitochondria

affects its DNA and membrane integrity leading to apop-

tosis and organ dysfunction [15].

Reduced glutathione (GSH) an endogenous antioxidant

which protects the liver from various intrinsic oxidants

formed during cellular metabolism and also external oxi-

dant sources by neutralizing them and maintaining the

cellular homeostasis [16, 17]. The GSH from GSH trans-

porters of hepatocytes has been shown to act against KC-

generated ROS, thereby protecting the hepatic infrastruc-

ture [18, 19]. The endogenous GSH is usually depleted

during pre-operative starvation which makes the hepatic

parenchyma vulnerable to I/R injury and the risk is higher

in aged liver [20]. Mitochondrial GSH pool acts as the

primary defense against peroxide-induced stress and pro-

tects against oxidative damage [21]. We suspect that

depletion of liver mitochondrial GSH pool with ageing

may lead to defective detoxification of mitochondrial ROS

paving way to cumulative oxidative damage and increased

susceptibility to hepatic I/R damage.

The earlier studies on GSH pre-conditioning show its

effectiveness in reduction of hepatic I/R injury and improved

survival rates [22, 23]. Though these studies elucidate the

potential protective effect of GSH administration against

hepatic I/R injury, there is a greater lacuna in the under-

standing of variable age-dependent response of GSH pre-

conditioning in I/R injury. Hence, we devised the present

study emphasizing the protective effect of GSH pre-treat-

ment against I/R injury in aged rat liver compared with that of

young rat. GSH was preferred to other antioxidants, since it is

an intrinsic antioxidant and its mode of action and metabo-

lism have been extensively studied [19, 21–23].

Materials and methods

Animals

Male Wistar (approved by the Institutional Animal Ethics

Committee, Pondicherry University, Puducherry, India)

young rats of age 6 weeks weighing 140 ± 20 g and aged

rats of age 24 months weighing 300 ± 30 g at the time of

surgery were used for the study. Animals were maintained

in central animal facilities of Pondicherry University. They

were allowed free access to food and water ad libitum until

8 h before surgery. Animals were treated and experimented

as per the guidelines of the Committee for the Purpose of

Control and Supervision of Experiments on Animals,

Government of India.

Experimental design

The animals were divided into six groups (n = 6):

Group I: Young sham (without surgical procedure).

Group II: Young I/R—subjected to saline pre-treatment

via intraperitoneal (i.p.), 30 min before I/R surgery (90-

min ischemia and 2-h reperfusion).

Group III: Young rats pre-treated with GSH (200 mg/kg

bwt, i.p.) 30 min before I/R surgery.

Group IV: Aged sham (without surgical procedure).

Group V: Aged I/R—subjected to saline pre-treatment

(i.p.) 30 min before I/R surgery.

Group VI: Aged rats pre-treated with GSH (200 mg/kg

bwt, i.p.) 30 min before I/R surgery.

The rats were anesthetized by ketamine/xylazine

(100 mg/kg bwt/10 mg/kg bwt, i.p) and then subjected to

midline laparotomy [24]. Hepatic I/R was achieved by

subjecting the rats to hepatic ischemia by partial hepatic

occlusion (covers 70 % of hepatic parenchyma) for 90 min

and released for 2 h for reperfusion. The hepatic pedicle

was identified and the branch left of porta-hepatis which

supplies median and left lobe were clamped with micro-

vascular clamp for 90 min (ischemia) and released exactly

for 2 h (reperfusion). This method prevented congestion of

mesenteric venous drain by permitting decompression of

portal supply through right and caudate lobes [25]. About

2 ml of blood was collected for serum analysis from vena

cava caudalis before killing the rats. Liver tissues were

washed with 0.9 % saline and divided into three portions.

First portion was wrapped in aluminum foil and stored at

-80 �C, for biochemical assays. Second portion was fixed

in neutral buffered formalin for histopathological studies

and the third portion was saved for western blot analysis.

The 2-h reperfusion model was chosen for the analysis

of reperfusion injury in young and aged rats because KC

activation and the release of proinflammatory cytokines are

initiated at this early stage, which later progresses to induce

hepatic fibrosis [26]. The molecular changes during the

early phase of reperfusion are critical since they determine

the ultimate fate of the cells. We assume that the effective

suppression of these changes in ischemia reperfusion may

yield better post-operative outcome.

A. Suyavaran et al.

123

Serum analysis

Hemolysis-free serum from rats was subjected to ALT and

AST analysis using commercially available kit (Cayman

chemical company, MI, USA). The GSH and oxidized

GSH (GSSG) in serum were estimated by HT glutathione

assay kit from Trevigen (Trevigen, Inc, MD, USA).

Hepatic GSH, GSSG and TBARS levels

Tissues were homogenized with 0.25 M sucrose–phosphate

buffer solution (50 mM, pH 7.4) at 4 �C to obtain 10 %

homogenate (1 g tissue in 10 ml of ice cold buffer) in a

motorized homogenizer. Mitochondrial fraction was sepa-

rated by differential centrifugation method [27]. Lipid

peroxidation level in mitochondrial and cytosol fractions

was estimated in terms of the thiobarbituric acid-reactive

substances (TBARS) using malondialdehyde as standard

[28] and GSH and GSSG were estimated by using com-

mercially available kit (Trevigen, Inc, MD, USA).

Western blot analysis

The expression levels of active caspase-3, N-terminal cleavage

fragment of poly (ADP-ribose) polymerases-1 (PARP-1) and

TNF-a were analyzed by Western blot. The samples (30 lg of

protein) were subjected to 12 % SDS-PAGE and then trans-

ferred onto a polyvinylidene fluoride membrane. The

membranes were blocked with 1 % BSA in TBST (Tris-buf-

fered saline, 0.01 % Tween 20) for 2 h at RT and then incubated

with appropriate primary antibodies for 2 h at RT, washed

(3 9 15 min each) with TBST and incubated with appropriate

secondary antibody. The membranes were subjected to

enhanced chemiluminescence reaction and densitometric anal-

yses of the blots were done by Image J—image analysis

software (NIH, Bethesda, USA).b-Actin expression was used as

an internal control to confirm equal protein loading. The anti-

bodies for caspase 3, PARP-1, TNF-a and b–Actin were

purchased from Santacruz biotechnology Inc, USA.

Terminal deoxynucleotide transferase dUTP nick end

labeling (TUNEL) assay

The extent of DNA fragmentation in hepatocytes was ana-

lyzed by TUNEL staining using TACS 2 TdT-Fluor in situ

apoptosis detection kit (Trevigen Inc, MD, USA). Minimum

of ten different fields were observed for each slide.

Assessment of serum and liver TNF-a level

The TNF-a level was quantified by using InvitrogenTM

ELISA kit (KRC3011). Values are expressed as pg/ml in

serum and pg/mg protein in liver tissue.

Histopathological assessment

Liver tissues fixed in neutral buffered formalin were

dehydrated in ascending series of alcohol, cleared in xylene

and embedded in paraffin. The sections (4 lm thickness)

were cut using Leica RM2125 rotatory microtome (LeicaTM,

Germany) and fixed onto gelatin–formaldehyde coated

slides. The slides were deparaffinized in xylene and stained

using hematoxylin and eosin (H&E). They were pictured in

109 objective field using an Olympus CX40 microscope

with ProgresTM image capture setup.

Statistical analysis

Statistical analyses was performed using one-way analysis

of variance (ANOVA) followed by Tukey’s multiple test.

Differences were considered to be significant at P B 0.05

against control. Data were presented as mean ± SD

(standard deviation).

Results

Alteration in liver marker enzyme activities

The ALT and AST activities were significantly (P B 0.05)

elevated in I/R (group II) rats when compared with sham

(group I) rats. The transaminase activities in GSH-treated

group (group III) were significantly lowered when com-

pared with I/R group (group II) (P B 0.05). The similar

trend was observed in aged rats subjected to I/R and pre-

treated with GSH. However, the ALT and AST activities

were elevated in aged I/R (group V) (P B 0.05) when

compared with young I/R (group II) rats (Fig. 1a, b).

GSH, GSSG and its ratio in serum and liver tissue

The serum GSH level was significantly (P B 0.05) lower in

I/R groups (groups II and V) compared with sham groups

(group I and IV). However, GSH level was significantly

(P B 0.05) decreased in aged I/R rats (group V) when

compared with young I/R (group II) rats. Recovery

(P B 0.05) in serum GSH level was observed in GSH-

treated groups both in young and aged rats (groups III and

VI). No significant changes were observed in GSSG level

in all groups (Fig. 2a, b).

In general, the mitochondrial GSH level in liver tissue

was about tenfold higher than that of cytosol fraction. The

liver tissue cytosol and mitochondrial GSH level of I/R

groups (groups II and V) were significantly (P B 0.05)

lower than respective sham groups (groups I and IV).

Significantly (P B 0.05) elevated level of GSH was

observed in cytosol and mitochondria of all GSH-treated

TNF-a suppression by glutathione preconditioning

123

groups (both young and aged rats) when compared with

that of respective I/R rats (groups II and VI) (Table 1).

The significant (P B 0.05) decrease in serum and liver

(cytosol and mitochondria) GSH/GSSG ratio was observed

in I/R groups (groups II and V) when compared with group

I and IV (control), though the reduction in mitochondrial

GSH/GSSG ratio was more remarkable in these groups

(groups II and IV). In groups (III and VI) treated with GSH,

both cytosol and mitochondrial GSH/GSSG ratio was

restored and was similar to that of sham control (groups I

and IV) (Fig. 2a, b; Table 1).

Lipid peroxidation in liver

The level of TBARS in cytosol and mitochondria was sig-

nificantly (P B 0.05) elevated in rats subjected to I/R (groups

II and VI) when compared with that of respective sham groups

(groups I and V). In general, TBARS level of aged I/R rats was

significantly (P B 0.05) higher when compared with that of

young I/R rats and more evidently in the mitochondrial frac-

tions than in cytosol. Pre-treatment with GSH (groups III and

V) suppressed the TBARS level significantly (P B 0.05) in

cytosol and mitochondria when compared with respective I/R

rats (groups II and VI) (Fig. 2c).

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Fig. 1 a Effect of glutathione pre-treatment on serum AST activity of

young and aged rats subjected to I/R. b Effect of glutathione pre-

treatment on serum ALT activity of young and aged rats subjected to

I/R. Values are shown as mean ± SD (n = 6). Groups were treated as

mentioned in ‘‘Materials and methods’’. Statistical significance was

evaluated by one-way ANOVA followed by Tukey’s test. *P B 0.05

vs. group I; #P B 0.05 vs. group IV; @P B 0.05 vs. group II;$P B 0.05 vs. group V

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Fig. 2 a Effect of GSH treatment on serum GSH level in young and

aged rats. b Effect of GSH treatment on GSH/GSSG ratio of serum in

young and aged rats (GSH reduced glutathione; GSSG oxidized

glutathione). c Effect of GSH treatment on hepatic I/R-induced lipid

peroxidation denoted as TBARS (nmol/mg of protein) in liver cytosol

and mitochondrial fractions. Values are represented as mean ± SD;

*significance level of P B 0.05 vs. group I; @significance level of

P B 0.05 vs. group II; #significance level of P B 0.05 vs. group IV;$significance level of P B 0.05 vs. group V

A. Suyavaran et al.

123

Alterations in inflammatory and apoptotic markers

Western blot was performed to compute the extent of

apoptotic and inflammatory liver damage. The young and

aged I/R groups (groups II and V) showed (P B 0.001 vs.

group I; P B 0.01 vs. group IV) higher expression levels of

active caspase-3, N-terminal cleavage fragment of PARP-1

and TNF-a compared with that of respective sham groups.

The young and aged rats pre-treated with GSH (groups III

and VI) showed significant decrease in expression of cas-

pase-3, PARP-1 and TNF-a when compared with those of

young and aged I/R group rats (groups II and V). The

expression levels of TNF-a and PARP-1 were significantly

(P B 0.01) increased in aged I/R group (group VI) when

compared with those of young I/R group (group II) rats,

while difference in caspase-3 level between these groups

was insignificant (Fig. 3a–d).

Attenuation of serum and liver TNF-a level by GSH

The inflammatory progression was further confirmed by

measuring TNF-a level in serum and liver. The serum and

liver TNF-a levels of young and aged I/R group (groups II

and VI) rats were significantly elevated (P B 0.05—serum;

P B 0.01—liver) when compared with those of respective

sham groups (groups I and IV). The rise in serum TNF-alevel in aged I/R group rats (group V) was about twofold

higher (P B 0.05) than in young I/R group (group II). The

groups treated with GSH prior to I/R surgery (groups III

and VI) showed significant reduction in TNF-a level in

serum and liver compared with respective I/R groups. The

liver TNF-a level of aged I/R (group V) rats was signifi-

cantly (P B 0.01) higher than that of young I/R (group II)

rats. The reduction in liver TNF-a level of aged rats upon

GSH pre-treatment was slightly lower when compared with

that of young rats (Fig. 4a, b).

TUNEL staining

The extent of DNA damage was analyzed by TUNEL

staining. The TUNEL-stained sections of sham group

(groups I and IV) showed very few apoptotic cells. The

liver sections from rats subjected to I/R injury (groups II

and V) showed significantly (P B 0.01) higher number of

apoptotic cells as evidenced by deep fluorescent green-

stained nuclei compared with those of corresponding sham

group (groups I and IV) rats (Fig. 5a, b). Reduction

(P B 0.01) in number of apoptotic nuclei was observed in

groups treated with GSH prior to I/R surgery (groups III

and VI). The reduction of apoptotic nuclei in aged rats was

generally lower than young rats upon GSH treatment, but it

was not statistically significant.

Histopathological assessment

The histopathological changes were observed by H&E

staining. The stained liver sections of young and aged sham

group rats (groups I and IV) exhibited normal morphology.

The liver sections in I/R group rats (groups II and V)

demonstrated epithelial dystrophy of portal vein branch

with venous hemorrhage, sinusoidal congestion and hepa-

tocytes ballooning with nuclear pyknosis and areas of focal

necrosis. In liver sections of aged I/R rats, the hepatocyte

cytoplasm was lightly stained and there were patches of

hepatocyte apoptosis with neutrophil intrusion. Liver sec-

tions of young and aged rats pre-treated with GSH (groups

III and IV) showed reduction in above morphological

changes (Fig. 6).

Discussion

In recent years, hepatic disorders have increased expo-

nentially at global scale. In critical cases, such as

hepatocellular carcinoma, colorectal metastases to liver,

portopulmonary hypertension, primary sclerosing cholan-

gitis, etc., treatment involves surgical intervention or

transplantation [29, 30]. During surgical procedures, the

hepatic ischemia reperfusion injury results due to clamping

of portal vein. The hypoxic state of hepatocytes during

ischemia leads to impaired mitochondrial respiration.

Table 1 Levels of GSH, GSSG and GSH/GSSG ratio in liver of

control and experimental rats

Groups GSH (nmol/mg) GSSG (nmol/mg) GSH/GSSG

Cytosol

Group I 2.6 ± 0.36 0.8 ± 0.07 2.9 ± 0.19

Group II 0.7 ± 0.12* 0.7 ± 0.12 0.9 ± 0.15*

Group III 4.9 ± 1.76*, @ 1.5 ± 0.17*, @ 3.1 ± 0.58*, @

Group IV 1.1 ± 0.05 0.5 ± 0.03 2.3 ± 0.27

Group V 0.6 ± 0.03 0.7 ± 0.14 0.8 ± 0.35#

Group VI 4.3 ± 0.85#, $ 1.4 ± 0.26#, $ 2.9 ± 0.72#, $

Mitochondrial

Group I 16.5 ± 1.57 0.5 ± 0.03 33.0 ± 4.78

Group II 8.05 ± 1.35* 0.9 ± 0.07 14.1 ± 1.75*

Group III 18.1 ± 2.76*, @ 0.5 ± 0.15 36.2 ± 2.15@

Group IV 14.5 ± 1.03 0.6 ± 0.12 24.1 ± 2.48

Group V 7.8 ± 1.09# 0.8 ± 0.08 9.8 ± 0.93#

Group VI 16.2 ± 1.42#, $ 0.6 ± 0.17 27.2 ± 1.73$

Groups were treated as mentioned in ‘‘Materials and methods’’; val-

ues are represented as mean ± SEM

GSH reduced glutathione, GSSG oxidized glutathione* P B 0.05 vs. group I; @ P B 0.05 vs. group II; # P B 0.05 vs.

group IV; $ P B 0.05 vs. group V

TNF-a suppression by glutathione preconditioning

123

Reduction in cellular energy reserve initiates the break-

down of metabolic pathways and impaired membrane ion

transport which in turn leads to ionic imbalance thereby

activating the KC, which are further aggravated during

reperfusion [31, 32]. The activated KC release the

inflammatory mediators which in turn stimulate the

sequestration of neutrophils giving rise to the release of

ROS at the sinusoidal interfaces further aggravating the

injury [33, 34]. Since oxidative stress is the major con-

tributor for I/R-induced liver injury, therapeutic strategies

employing antioxidants has gained interest.

Though there are earlier studies regarding the effect

of pre-treatment of antioxidants on the hepatic I/R injury

in adult rats, only meager information is available on the

comparison of the liver of young and aged rats. There

has been no adequate insight into the differences

between molecular as well as biochemical changes that

occur in young and aged rat liver during I/R injury.

Since, aged rat liver has reduced antioxidant defense

system it may be more susceptible to I/R injury, and

hence a detailed comparison on the differential outcome

of antioxidant pre-treatment in young and aged rats

becomes essential.

In the current investigation, young and aged male Wistar

rats were subjected to 90-min ischemia and 2-h reperfu-

sion. Schauer et al. [23] found that 90-min hepatic ischemia

induces more liver injury than 60 min hepatic ischemia and

no further changes occur after 120 min of ischemia. Later,

other studies have employed 90 min as optimum period to

induce hepatic ischemia [35–37]. Previous studies have

shown that two distinct phases exist in hepatic reperfusion

injury. The early phase (during the first 2 h) during which

the KC are activated leading to ROS release and expression

of TNF-a and interleukin-1, while the late phase (12 h after

reperfusion) is characterized by activation of neutrophils

and progressive hepatic damage leading to fibrosis in later

stages [38]. Reperfusion-induced stress stimulus triggers

nuclear factor j-B (NFjB), which transcriptionally acti-

vates TNF-a. Takahashi et al. [39] observed biphasic

activation of NFjB level during 1–3 h (early phase) and

12 h (late phase) reperfusion of liver grafts transplanted in

rats, which depicts the occurrence of multiple peaks in

inflammatory signal.

Though multiple peaks of inflammatory mediators are

observed during hepatic I/R injury, the observations from

earlier studies suggest that intervention during the initial

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Fig. 3 a Representation of western blot images of active caspase 3,

PARP1, and TNF-a. b-Actin level has been shown as internal loading

control. 30 lg of protein was loaded into each well and western

blotting was performed as mentioned in ‘‘Materials and methods’’.

b Densitometry analysis of caspases-3; c densitometry analysis of

PARP 1 and d densitometry analysis of TNF-a. Values represent

relative density ratio against b-actin. Images were processed using

Image-J software (NIH, USA). Data are shown as mean ± SD

(n = 6). *P B 0.001 vs. group I; #P B 0.01 vs. group IV; @P B 0.05

vs. group II; $P B 0.05 vs. group V

A. Suyavaran et al.

123

phase of reperfusion injury (during first 2 h) result in sig-

nificant recovery of hepatic tissue rather than intervention

in later phases [40]. Moreover, the changes that occur

during the initial phase are stronger and determine the

ultimate fate of the organ. Hence, the current study focused

on the changes that occur during the 90-min ischemia and

2-h reperfusion (initial phase)-induced injury in rat liver, in

order to determine the effect of GSH pre-treatment in

young and aged rats.

I/R injury in liver involves complex network of path-

ways and mediators. Several pathways such as, KC-

induced NFjB-mediated proinflammatory pathway, nitric

oxide synthase (NOS) pathway and heme oxygenase (HO)

system are activated in parallel during the I/R stimulus

[41]. The proinflammatory pathway leads to deleterious

changes in liver post I/R injury. On the other hand, the

NOS pathway and HO system counteract the I/R-mediated

injury and proved as beneficial for the improvement of

liver function following ischemic stimulus [42, 43].

Kaizu et al. had shown that inducible nitric oxide syn-

thase (iNOS) gene therapy reduced I/R injury in rat liver.

They have further shown that increased production of NO

induces heme oxygenase-1 (HO-1) which in turn counter-

acts the inflammatory response [44]. Involvement of such

multitude of factors makes it difficult to treat I/R injury by

targeting a single intervening factor. Since ROS are central

mediators of I/R injury, better clinical outcome can be

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Fig. 4 TNF-a level in serum and liver of young and aged rats.

Statistical significance was evaluated by one-way ANOVA followed

by Tukey’s test. Values represent mean ± SD (n = 6). a Graph

represents serum TNF-a level (pg/ml of serum), *P B 0.05 vs. group

I; �P B 0.05 vs. group V; @P B 0.05 vs. group II; $P B 0.05 vs.

group VI. b Graph represents liver TNF-a level (pg/mg of protein),*P B 0.01 vs. group I; �P B 0.01 vs. group V; @P B 0.05 vs. group

II; $P B 0.05 vs. group VI

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Fig. 5 a Terminal deoxynucleotide transferase dUTP nick end

labeling (TUNEL)-stained liver sections of young and aged rats.

b Graphic representation of apoptotic grading in TUNEL-stained

sections; a minimum of 10 fields were chosen per slide and the

number of apoptotic nuclei under the high power (940) was counted

and values were represented as mean ± SD (n = 6). *P B 0.01 vs.

group I; �P B 0.01 vs. group IV; P B 0.01 vs. group II; $P B 0.01 vs.

group V

TNF-a suppression by glutathione preconditioning

123

achieved through blocking ROS-mediated injury by

employing antioxidant treatment prior to surgery.

In the present study, we have compared the effect of

GSH treatment prior to surgery on hepatic recovery from

early phases of I/R-induced injury in young and aged rats.

The outcomes were analyzed by various biochemical

markers and histopathological analysis. The elevation of

serum transaminases is a reliable indicator for the extent of

hepatic damage [23]. The elevation of ALT and AST

activities in serum of young and aged I/R group rats por-

trays the extent of liver I/R injury. The elevation of AST

and ALT suggests the increased susceptibility of aged rat

liver to I/R injury compared with the young and the

restoration of transaminase activities explains the reduction

in liver damage in both age groups with GSH pre-

treatment.

The reduced serum and hepatic GSH level in I/R groups

(groups II and V) and their resurgence in GSH pre-treated

groups reveals the effective absorption and restoration of

GSH/GSSG ratio upon extraneous treatment in young and

aged rats alike. Lang et al. [45] had reported that GSH level

in young individuals was 17 % higher on average than that

of elderly subjects. The significant decline in mitochondrial

GSH/GSSG ratio in I/R groups substantiates the oxidative

damage at mitochondrial level. The observed decline in

GSH/GSSG ratios of mitochondrial fraction of aged I/R

Fig. 6 Hematoxylin and eosin-

stained images of paraffin-

embedded rat liver sections

(910). a Young sham rats,

b young rats subjected to I/R

injury without prior GSH

treatment, c young rats pre-

treated with GSH (200 mg/kg

bwt) before surgery, d aged

sham rats, e aged rats subjected

to I/R injury without GSH

treatment, f aged rats pre-treated

with GSH (200 mg/kg bwt)

before surgery; (n = 6) (PV

portal vein). the arrows indicate

vascular endothelial damage

and hypertrophy, while the

asterisk indicates focal necrosis

and pyknotic hepatocyte

nucleus

A. Suyavaran et al.

123

groups (group VI) and its recovery with GSH treatment in

aged groups are comparable to the similar outcome in

young rats, thereby suggesting the mitochondrial GSH

replenishing efficiency of extraneous GSH administration

prior to hepatic surgery across age barrier.

Lipid peroxidation is the key mechanism by which ROS

execute the oxidative damage in liver [46–48]. Moreover,

mitochondrial lipid peroxidation due to oxidative injury

has been described as the major culprit in the hepatic I/R

injury [49]. Ageing has been shown to be associated with

increased hepatic lipid peroxidation in aged subjects [50].

In our study, TBARS level was elevated in the aged rats

subjected to hepatic I/R, but were subdued in the rats

treated with GSH prior to surgery.

In the current study, we have observed significantly high

expression of TNF-a, active caspase-3 and PARP-1 (N-

terminal fragment) in rats subjected to hepatic I/R injury

without GSH treatment. TNF-a has been observed to play a

major role in the onset of I/R-mediated oxidative hepatic

injury. Previous studies have elucidated the mechanism of

TNF-a-induced activation of apoptosis and its role in

hepatic injury [51–53]. Circulating neutrophils have been

shown to elicit TNF-a-induced caspase-3 activation [54].

PARP-1 cleavage fragments are a signature of apoptotic

progression [55]. Our observation of correlated increase in

TNF-a, caspase-3 and PARP-1 confirms a positive loop of

apoptotic signaling involved in hepatic I/R injury. The rise

in serum and liver tissue TNF-a level upon hepatic I/R

injury and its reduction in GSH pre-treated rats, as shown

by ELISA further supports the inflammatory process aug-

mented by I/R liver injury.

The increase in TUNEL-positive nuclei in young and

aged rats of I/R group elucidates the DNA damage inflicted

by I/R-mediated oxidative damage. Earlier studies have

reported similar increase in TUNEL-positive cells upon

reperfusion followed by ischemia [56, 60]. The effective-

ness of GSH pre-treatment prior to surgery in reducing

oxidative DNA damage is demonstrated by reduction in

number of TUNEL-positive cells in GSH-treated young

and aged groups.

Liver sections of aged I/R groups showed vascular

endothelial degradation and patches of hepatocyte necrosis

with neutrophil intrusion, while hepatocytes ballooning and

pyknotic nucleus was observed in young rats subjected to

hepatic I/R injury. The liver damage in I/R injury has been

proven earlier to be extensively due to the triggering of

inflammatory mediators released by KC stimulation and

the resulting release of inflammatory mediators like TNF-

a, IL1-a and IL6 [57, 58]. The elevated expression of TNF-

a has been shown to be the culprit for P-selectin upregu-

lation and neutrophil recruitment [59]. Suppression of

TNF-a-mediated apoptotic signaling by GSH pre-treatment

can be explained by attenuation of KC simulation by

lowering the oxidative stress stimulus.

Conclusions

We have observed that the administration of GSH prior to

hepatic I/R surgery protects both the young and aged rats

from I/R-induced oxidative damage. The pre-treatment

with GSH significantly reduced the apoptosis and TNF-aby restoration of GSH/GSSG ratio at mitochondrial level,

in young and aged rats. These findings suggest that GSH

supplementation prior to surgery would be an efficient

therapeutic strategy and can be used synergistically with

other treatments to yield better post-operative outcomes,

thus irrespective of age factor.

Acknowledgments The authors duly acknowledge the funding

support from Indian Council of Medical Research (ICMR Ref: 52/13/

2007) and Department of Science and Technology (NO.SR/FT/LS-63/

2011 & DST-FIST), New Delhi, India. Author A. Suyavaran

acknowledges UGC, New Delhi, India, for financial support in the

form of Junior Research Fellowship [CSIR-UGC-JRF; S. No. F.17-

115/98 (SA-I)].

References

1. Carden DL, Granger DN. Pathophysiology of ischemia–reperfu-

sion injury. J Pathol. 2000;190:255–66.

2. Smyrniotis VE, Kostopanagiotou GG, Contis JC, Farantos CI,

Voros DC, Kannas DC, et al. Selective hepatic vascular exclusion

vs. Pringle maneuver in major liver resections: prospective study.

World J Surg. 2003;27:765–9.

3. Man K, Fan ST, Ng IO, Lo CM, Liu CL, Wong J. Prospective

evaluation of Pringle maneuver in hepatectomy for liver tumors

by a randomized study. Ann Surg. 1997;226:704–11.

4. Kupiec-Weglinski JW, Busuttil RW. Ischemia and reperfusion in

liver transplantation. Transpl Proc. 2005;37:1653–6.

5. Rymsa B, Wang JF, de Groot H. O2.- release by activated Kupffer

cells upon hypoxia–reoxygenation. Am J Physiol.

1991;261:G602–7.

6. Tomiyama K, Ikeda A, Ueki S, Nakao A, Stolz DB, Koike Y,

et al. Inhibition of Kupffer cell-mediated early proinflammatory

response with carbon monoxide in transplant-induced hepatic

ischemia/reperfusion injury in rats. Hepatology.

2008;48:1608–20.

7. Essani NA, McGuire GM, Manning AM, Jaeschke H, et al.

Endotoxin-induced activation of the nuclear transcription factor

kappa-B and expression of E-selectin messenger RNA in hepa-

tocytes, Kupffer cells and endothelial cells in vivo. J Immunol.

1996;156:2956–63.

8. Hansford RG, Hogue BA, Mildaziene V. Dependence of H2O2

formation by rat heart mitochondria on substrate availability and

donor age. J Bioenerg Biomembr. 1997;29:89–95.

9. Miquel J, Economos CA, Fleming J, Johnson JE Jr. Mitochon-

drial role in cell aging. Exp Gerontol. 1980;15:575–91.

10. Kim HG, Hong SM, Kim SJ, Paark HJ, Lee YY, Moon JS, et al.

Age related changes in the activity of antioxidant and redox

enzymes in rats. Mol Cells. 2003;16:278–84.

TNF-a suppression by glutathione preconditioning

123

11. Le Couteur DG, McLean AJ. The aging liver. Drug clearance and

an oxygen diffusion barrier hypothesis. Clin Pharmacokinet.

1998;34:359–73.

12. Strolin BM, Whomsley R, Baltes EL. Differences in absorption,

distribution, metabolism and excretion of xenobiotics between

the paediatric and adult populations. Expert Opin Drug Metab

Toxicol. 2005;1:447–71.

13. Mallikarjuna K, Shanmugham KR, Nishanth K, Mc Wu, Hou

CW, Kuo CH, et al. Alcohol-induced deterioration in primary

antioxidant and glutathione family enzymes reversed by exercise

training in the liver of old rats. Alcohol. 2010;44:523–9.

14. Castro MR, Suarez E, Kraiselburd E, Isidro A, Paz J, Ferder L,

et al. Aging increases mitochondrial DNA damage and oxidative

stress in liver of rhesus monkeys. Exp Gerontol. 2012;47:29–37.

15. Sastre J, Pallardo FV, Pla R, Pellin A, Juan G, O’Connor JE, et al.

Aging of the liver: age-associated mitochondrial damage in intact

hepatocytes. Hepatology. 1996;24:1199–205.

16. Kosower NS, Kosower EM. The glutathione status of cells. Int

Rev Cytol. 1978;54:109–60.

17. Han D, Hanawa N, Saberi B, Kaplowitz N. Mechanisms of liver

injury. III. Role of glutathione redox status in liver injury. Am J

Physiol Gastrointest Liver Physiol. 2006;291:G1–7.

18. Brass CA, Roberts TG. Hepatic free radical production after cold

storage: Kupffer cell-dependent and independent mechanism in

rats. Gasteroenterology. 1995;108:1167–75.

19. Fernandez-Checa JC, Yi JR, Ruiz CG, Ookhtens M, Kaplowitz N.

Plasma membrane and mitochondrial transport of hepatic reduced

glutathione. Semin Liver Dis. 1996;16:147–58.

20. Selzner M, Selzner N, Jochum W, Graf R, Clavien PA. Increased

ischemic injury in old mouse liver: an ATP-dependent mecha-

nism. Liv Transpl. 2007;13:382–90.

21. Fernandez-Checa JC, Kaplowitz N. Hepatic mitochondrial glu-

tathione: transport and role in disease and toxicity. Toxicol Appl

Pharmacol. 2005;204:263–73.

22. Xue F, Wang G, Pang Z, Liu C, Liang T. Protective effect of

glutathione against liver warm ischemia–reperfusion injury in rats

is associated with regulation of P-selectin and neutrophil infil-

tration. Anat Rec. 2008;291:1016–22.

23. Schauer RJ, Gerbes AL, Vonier D, Meissner H, Michl P, Leiderer

R, et al. Glutathione protects the rat liver against reperfusion

injury after prolonged warm ischemia. Ann Surg.

2004;239:220–31.

24. Day YJ, Marshall MA, Huang L, McDuffie MJ, Okusa MD,

Linden J. Protection from ischemic liver injury by activation of

A–A adenosine receptors during reperfusion: inhibition of che-

mokine induction. Am J Physiol Gastrointest Liver Physiol.

2004;286:G285–93.

25. Camargo CA Jr, Madden JF, Gao W, Selvan RS, Clavien PA.

Interleukin-6 protects liver against warm ischemia/reperfusion

injury and promotes hepatocyte proliferation in the rodent.

Hepatology. 1997;26:1513–20.

26. Ozkan E, Yardimci S, DUlundu E, et al. Protective potential of

montelukast against hepatic ischemia/reperfusion injury in rats.

J Surg Res. 2010;159:588–94.

27. Sahoo A, Chainy GB. Alterations in the activities of cerebral

antioxidant enzymes of rat are related to aging. Int J Dev Neu-

rosci. 1997;15:939–48.

28. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal

tissues by thiobarbituric acid reaction. Anal Biochem.

1979;95:351–8.

29. Anaya DA, Becker NS, Abraham NS. Global graying, colorectal

cancer and liver metastasis: new implications for surgical man-

agement. Crit Rev Oncol Hematol. 2011;77:100–8.

30. Goldberg DS, Fallon MB. Model for end-stage liver disease-

based organ allocation: managing the exceptions to the rules. Clin

Gastroenterol Hepatol. 2013;11:452–3.

31. Clavien PA, Harvey PR, Strasberg SM. Preservation and reper-

fusion injuries in liver allografts. An overview and synthesis of

current studies. Transplantation. 1992;53:957–78.

32. Rosser BG, Gores GJ. Liver cell necrosis: cellular mechanisms

and clinical implications. Gastroenterology. 1995;108:252–75.

33. Jaeschke H, Bautista AP, Spolarics Z, Spitzer JJ. Superoxide

generation by Kupffer cells and priming of neutrophils during

reperfusion after hepatic ischemia. Free Radic Res Commun.

1991;15:277–84.

34. Jaeschke H, Farhood A. Neutrophil and Kupffer cell-induced

oxidant stress and ischemia–reperfusion injury in rat liver. Am J

Physiol. 1991;260:G355–62.

35. Xu SQ, Li YH, Hu SH, Chen K, Dong LY. Effect of Wy14643 on

hepatic ischemia reperfusion injury in rats. World J Gastroen-

terol. 2008;14:6936–42.

36. Wu C, Wang P, Rao J, Wang Z, Zhang C, Lu L, Zhang F.

Triptolide alleviates hepatic ischemia/reperfusion injury by

attenuating oxidative stress and inhibiting NF-jB activity in

mice. J Surg Res. 2011;166:e205–13.

37. Lin CM, Lee JF, Chiang LL, Chen CF, Wang D, Su CL. The

protective effect of curcumin on ischemia–reperfusion induced

liver injury. Transplant Proc. 2012;44:974–7.

38. Glantzounis GK, Salacinski HJ, Yang W, Davidson BR, Seifalian

AM. The contemporary role of antioxidant therapy in attenuating

the ischemia–reperfusion injury: a review. Liver Transpl.

2005;11:1031–47.

39. Takahashi Y, Ganster RW, Gambotto A, Shao L, Kaizu T, Wu T,

et al. Role of NF-kappaB on liver cold ischemia–reperfusion

injury. Am J Physiol Gastrointest Liv Physiol.

2002;283:G1175–84.

40. Crockett ET, Galligan JJ, Uhal BD, Harkema J, Roth R, Pandya

K. Protection of early phase hepatic ischemia–reperfusion injury

by cholinergic agonists. BMC Clin Pathol. 2006;6:3.

41. Vardanian AJ, Busuttil RW, Kupiec-Weglinski JW. Molecular

mediators of liver ischemia and reperfusion injury: a brief review.

Mol Med. 2008;14:337–45.

42. Hines IN, Harada H, Flores S, Gao B, McCord JM, Grisham MB.

Endothelial nitric oxide synthase protects the post-ischemic liver:

potential interactions with superoxide. Biomed Pharmacother.

2005;59:183–9.

43. Tsuchihashi S, Zhai Y, Bo Q, Busuttil RW, Kupiec Weglinski

JW. Heme-oxygenase-1 mediated cytoprotection against liver

ischemia and reperfusion injury: inhibition of type-1 interferon

signaling. Transplantation. 2007;83:1628–34.

44. Kaizu T, Ikeda A, Nakao A, Takahashi Y, Tsung A, Kohmoto J,

et al. Donor graft adenoviral iNOS gene transfer ameliorates rat

liver transplant preservation injury and improves survival.

Hepatology. 2006;43:464–73.

45. Lang CA, Naryshkin S, Schneider DL, Mills BJ, Lindeman RD.

Low blood glutathione in healthy aging adults. J Lab Clin Med.

1992;120:720–5.

46. Glantzounis GK, Salacinski HJ, Yang W, Davidson BR, Seifalian

AM. The contemporary role of antioxidant therapy in attenuating

liver ischemia–reperfusion injury: a review. Liver Transpl.

2005;11:1031–47.

47. Zhang W, Wang M, Xie HY, Zhou L, Meng XQ, Shi J, et al. Role

of reactive oxygen species in mediating hepatic ischemia–reper-

fusion injury and its therapeutic application in liver

transplantation. Transplant Proc. 2007;39:1332–7.

48. Knight TR, Fariss MW, Farhood A, Jaeschke H. Role of lipid

peroxidation as a mechanism of liver injury after acetaminophen

overdose in mice. Toxicol Sci. 2003;76:229–36.

49. Mukhopadhyay P, Horvath B, Zsengeller Z, Batkai S, Cao Z,

Kechrid M, et al. Mitochondrial reactive oxygen species gener-

ation triggers inflammatory response and tissue injury associated

with hepatic ischemia–reperfusion: therapeutic potential of

A. Suyavaran et al.

123

mitochondrially targeted antioxidants. Free Radic Biol Med.

2012;53:1123–38.

50. Farahmand SK, Samini F, Samini M, Samarghandian S. Safarnal

ameliorates antioxidant enzymes and suppresses lipid peroxida-

tion and nitric oxide formation in aged male rat liver.

Biogerontology. 2013;14:63–71.

51. Okajima K, Harada N, Kushimoto S, Uchiba M. Role of micro-

thrombus formation in the development of ischemia/reperfusion-

induced liver injury in rats. Thromb Haemost. 2002;88:473–80.

52. Birk AV, Liu S, Soong Y, Mills W, Singh P, Warren JD, et al.

The mitochondrial-targeted compound SS-31 re-energizes

ischemic mitochondria by interacting with cardiolipin. J Am Soc

Nephrol. 2013;24:1250–61.

53. Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa

JC. Mitochondrial glutathione, a key survival antioxidant. Anti-

oxid Redox Signal. 2009;11:2685–700.

54. Geering B, Gurzeler U, Federzoni E, Kaufmann T, Simon HU. A

novel TNFR1-triggered apoptosis pathway mediated by class IA

PI2Ks in neutrophils. Blood. 2011;117:5953–62.

55. Chaitanya GV, Alexander JS, Babu PP. PARP-1 cleavage frag-

ments: signatures of cell-death proteases in neurodegeneration.

Cell Commun Signal. 2010;8:31. doi:10.1186/1478-811X-8-31.

56. Schmitt-Graff A, Kruger S, Bochard F, Gabbiani G, Denk H.

Modulation of alpha smooth muscle actin and desmin expression

in perisinusoidal cells of normal and diseased human livers. Am J

Pathol. 1991;138:1233–42.

57. Kim do Y, Chung SI, Ro SW, Paik YH, Lee JI, Jung MK, et al.

Combined effects of an antioxidant and Caspase inhibitor on the

reversal of hepatic fibrosis in rats. Apoptosis. 2013;18:1481–91.

58. Menger MD, Richter S, Yamauchi J. Vollmar. Role of micro-

circulation in hepatic ischemia/reperfusion injury. Hepato

Gastroenterol. 1999;46:1452–7.

59. Peralta C, Fernandez L, Panes J, Prats N, Sans M, Pique JM, et al.

Preconditioning protects against systemic disorders associated

with hepatic ischemia–reperfusion through blockade of tumor

necrosis facto-induced P-selectin up-regulation in the rat. Hepa-

tology. 2001;33:100–13.

60. Serbetci K, Uysal O, Erkasap N, Koken T, Baydemir C, Erkasap

S. Anti-apoptotic and antioxidant effect of leptin on CCl4 induced

acute liver injury in rats. Mol Biol Rep. 2012;39:1173–80.

TNF-a suppression by glutathione preconditioning

123