1 effect of-glycyrrhizin

Effect of glycyrrhizin on lipopolysaccharide/D-galactosamine-

induced acute hepatitis in albino rats: a histological and

immunohistochemical studyNashwa Fathy El-Tahawy, Aza Husein Ali, Saadia Ragab Saiedand Zahraa Abdel-Wahab

Department of Histology, Faculty of Medicine,El-Minia University, Egypt

Correspondence to Nashwa Fathy El-Tahawy,Department of Histology, Faculty of Medicine,El-Minia University, EgyptTel: + 145435777; + 86 2342813;e-mail: [email protected]

Received 24 January 2011Accepted 23 April 2011

The Egyptian Journal of Histology

2011, 34:518–52747 (1284 -2011)

Background

Acute liver diseases constitute a global concern. Medical treatments for these

diseases have limited efficacy. Lipopolysaccharide (LPS) and D-galactosamine

(D-GaIN) cause hepatic failure in rodents. Glycyrrhizin (GL) was reported to treat

increased serum aminotransferase activity in chronic hepatitis. However, its role in

acute hepatitis remains unclear.

Aim of the study

To investigate the protective and curative effect of GL in an animal model of acute

hepatitis.

Materials and methods

Thirty adult male albino rats were divided into five groups: group I = control group,

group II = LPS/D-GaIN-induced hepatitis model, group III = treated with GL 1=2 h

before LPS/D-GaIN injection, groups IV = treated 1=2 h after LPS/D-GaIN, and group

V = treated 4 h after LPS/D-GaIN. Serum ALT and AST levels were assayed. Animals

were killed by decapitation. Livers were processed for histological and

immunohistochemical studies. The results were statistically analyzed.

Results

This study revealed hepatocellular degeneration, and many hepatocytes exhibited

apoptosis-like features after LPS/D-GaIN administration. Pretreatment with GL

significantly improved this microscopic picture, whereas posttreatment with GL also

reduced the effects of LPS/D-GaIN, but this reduction decreased with the time of

administration. There was a significant increase in caspase-3-immunolabeled

hepatocytes and in tumor necrosis factor a-immunolabeled Kupffer cells in group II

compared with the control, whereas a significant decrease was observed in groups III

and IV, and to a lesser extent in group V compared with group II (all P < 0.05). Serum

levels of ALT and AST showed a significant increase in group II compared with the

control, whereas a significant decrease was observed in groups III and IV, and to a

lesser extent in group V (all P < 0.05), which was in harmony with the histological

results.

Conclusion

This study provides evidence for the protective and curative effect of GL against LPS/

D-GaIN-induced hepatotoxicity in rats. The anti-inflammatory and antiapoptotic effects

of GL evidently provide a new insight in treating acute hepatitis.

Keywords:

acute hepatitis, glycyrrhizin, lipopolysaccharide, rats

Egypt J Histol 34:518–527�c 2011 The Egyptian Journal of Histology1110-0559

IntroductionAcute liver diseases constitute a global concern, and the

medical treatments for these diseases are often difficult

to handle and have limited efficacy. Developing

therapeutically effective agents from natural products

may reduce the risk of toxicity when the drug is used

clinically [1]. Lipopolysaccharide (LPS)/D-galactosamine

(D-GaIN)-induced liver injury has been widely used to

study the mechanisms of human hepatitis and to examine

hepatic protection of compounds [2]. LPS is a toxic

component of the cell walls of gram-negative bacteria and

is widely present in the digestive tracts of humans and

animals [3]. LPS produces fulminated liver injury

characterized by widespread death of hepatocytes [4].

Under stimulation by LPS, liver macrophages secrete

various proinflammatory cytokines including the tumor

necrosis factor a (TNF-a), which is a terminal mediator

for apoptosis, subsequently leading to hepatic necrosis

[2,5]. D-GaIN is an amino sugar selectively metabolized

518 Original article

1110-0559 �c 2011 The Egyptian Journal of Histology DOI: 10.1097/01.EHX.0000399701.81302.e1

Copyright © The Egyptian Journal of Histology. Unauthorized reproduction of this article is prohibited.

by hepatocytes. D-GaIN is known for inducing the features

of acute hepatitis in rats by depletion of the uridine

triphosphate pool and thereby inhibition of macromolecule

(RNA, protein, and glycogen) synthesis in the liver [6].

The combination of LPS and D-GAlN specifically causes

hepatic failure in rodents [7]. The hepatic lesion in LPS/

D-GaIN induced acute hepatitis model resembles that of

human hepatitis as the upregulation of the TNF-a level

and hepatic apoptosis has been reported as a pathogenic

symptom in human hepatitis [8].

Glycyrrhizin (GL) is an aqueous extract of licorice root

(Glycyrrhiza glabra), which is a time-honored herbal

medicine in the major world herbal traditions [9]. GL

was reported to treat increased serum aminotransferase

activity in chronic hepatitis [10,11]. However, its role in

acute hepatitis and the nature of this protective

mechanism remain unclear.

The aim of this study was to investigate the protective

and curative effect of GL in LPS/D-GaIN induced acute

hepatitis model and to shed light on the possible

mechanisms of this effect.

Materials and methodsThirty adult, 8–10-week-old, male albino rats, weighing

150–200 g, were given food and water ad libitum for 1 week

before use. LPS (Escherichia coli, O55 : B5), N-acetyl

D-GAlN, and GL were purchased from Sigma (Sigma-

Aldrich, Egypt). LPS was given in a dose of 50 mg/kg body

weight, and D-GaIN in a dose of 300 mg/kg body weight

[8]. GL was given in a dose of 100 mg/kg body weight

[12,13]. These reagents were dissolved in sterile

pyrogen-free 0.9% sodium chloride and were injected

once in all by an intravenous injection in the tail vein.

Control animals were intravenously injected with an

equal volume of pyrogen-free 0.9% sodium chloride. Rats

were randomly assigned into five equal groups: group

I = injected with physiological saline (control), group

II = injected with LPS/D-GaIN (hepatitis model), group

III = injected with GL 1=2 h before LPS/D-GaIN injection,

group IV = injected with GL 1=2 h after LPS/D-GaIN

injection, and group V = injected with GL 4 h after LPS/

D-GaIN injection. Animals were killed by decapitation 8 h

after injection. Blood samples were collected by cardiac

punctures. Livers were rapidly removed and fixed in 10%

buffered formalin, followed by paraffin embedding.

Serum levels of aspartate transaminase (AST) and alanine

transaminase (ALT) were assayed by the method of

Reitman and Frankel [14].

Seven-micrometer sections were stained with hemato-

xylin and eosin (H&E) for histological examination using

a light microscope. Other sections were used for immuno-

histochemical staining for the following antibodies: (a)

anticleaved caspase-3 [15,16] and (b) anti-TNF-a [17].

In brief, sections were deparaffinized and rehydrated; to

retrieve antigen, sections were incubated with 0.1%

trypsin and 0.1% CaCl2 2H2O in Tris buffer (50 mmol/l)

at pH 7.4 at 371C for 120 min. Sections were soaked in

absolute methanol containing 0.3% hydrogen peroxide for

30 min at room temperature, to eliminate endogenous

peroxidase activity. The sections were then incubated

with 1.5% nonimmunized goat serum for 30 min at room

temperature, then incubated with the diluted primary

antibodies (1 : 500) for cleaved caspase-3 and TNF-a (5–

10 mg/ml) for 30 min at room temperature, and washed

three times with phosphate-buffered saline for 30 min.

Thereafter, the sections were incubated with biotinylated

goat antimouse immunoglobulin serum for 60 min.

After being washed with phosphate-buffered saline, the

sections were incubated with avidin/biotin peroxidase

complex (Vector, Burlingame, California, USA). Sites of

peroxidase binding were detected using chromogenic

3,30-diaminobenzidine tetra hydrochloride substrate.

Tissue sections were counterstained with hematoxylin.

Image capture

Tissue sections were examined and images were digitally

captured using a hardware consisting of a high-resolution

color digital camera mounted on an Olympus microscope

(Olympus, Japan), connected to a computer, and then

analyzed using Adobe Photoshop.

Morphometry

Caspase-3-immunolabeled cells and TNF-a-immuno-

labeled cells were counted in 10 adjacent nonover-

lapping fields of the tissue sections of each rat. The

total number of hepatocytes was also assessed by

counting their all nuclei in the same fields. The ratio

between numbers of caspase-3-immunolabeled hepato-

cytes to the total number of hepatocytes was calculated

in each experimental group [15]. The percentage range

was calculated for each group.

Data handling and statistics

Analysis of the data was carried out using SPSS version 13

(SPSS Inc., Chicago, Illinois, USA). The following

statistical tests were used:

(1) Mean and standard deviation to describe quantitative

data.

(2) Student’s t-test was used to compare between two

groups as regards parametric data.

For all tests, a probability (P value) of less than 0.05 was

considered significant.

ResultsLaboratory findings

Group II showed a significant increase in serum ALT and

AST levels compared with the control group. GL

pretreatment tended to normalize the serum ALT and

AST levels. Although GL 1=2 h posttreatment significantly

decreased the serum levels of ALT and AST, their levels

were higher than the control. Group V also showed a

significant decrease in ALT and AST serum levels

compared with group II, but the levels are still higher

when compared with groups III and IV (Table 1).

Effect of glycyrrhizin El-Tahawy et al. 519


Histological and immunohistochemical results

Hematoxylin and eosin-stained sections

Tissue sections of the control group presented a normal

lobular architecture (Fig. 1a). The hepatocytes radiated

from the central vein forming anastomozing fenestrated

plates of liver cells, separated from each other by irregular

vascular spaces, hepatic sinusoids. The hepatocytes

appeared polyhedral with acidophilic cytoplasm and large,

central, rounded nuclei. The nuclei of hepatocytes were

vesicular with prominent nucleoli. Hepatocytes may be

binucleated (Fig. 1b). Portal tracts contained many

structures including branches of portal vein with large

diameter and thin wall, branches of hepatic artery with

small diameter and thick wall, and branches of bile duct

(inset of Fig. 1b).

Marked morphological changes appeared in group II; it

exhibited disrupted lobular architecture with dilated

blood sinusoids in some areas (Fig. 2a). There was

marked hepatic injury, especially in the pericentral

region, characterized by hepatocellular degeneration.

The periportal injury was considerably weak, but the

portal vein as well as the central vein seemed to be

congested (inset of Fig. 2a). Many hepatocytes showed

densely stained acidophilic cytoplasm with condensed

(pyknotic) or fragmented nuclei (karyorrhectic), which

resembled apoptosis-like features. Some hepatocytes

appeared to be isolated in halo due to shrinkage, with

small dense nuclei, and intense acidophilic cytoplasm

(Fig. 2b). Furthermore, some degenerated hepatocytes

were observed with ill-defined cell boundaries, condensed

pyknotic nucleus, and vacuolated cytoplasm (Fig. 2c).

In group III, livers preserved the general architecture and

lacked evidence of major morphological injury. Hepato-

cyte degenerations appeared to be remarkably reduced.

Sections contained scarce degenerated cells with densely

stained acidophilic cytoplasm and condensed nuclei;

most hepatocytes retained their normal basophilic

granules (Fig. 3).

In group IV, livers also preserved the general architecture

and lacked evidence of major morphological injury.

Hepatocytes retained their normal basophilic granules;

only few dispersed degenerated cells with densely

stained acidophilic cytoplasm and condensed nuclei

were found, especially around the central vein (Fig. 4).

Although group V had preserved the general architecture,

few hepatocytes showed densely stained acidophilic cyto-

plasm; condensed nuclei appeared in the pericentral area

(Fig. 5).

Table 1. Serum ALT and AST levels in control and experimental groups

Serum ALT level (IU/l) Serum AST level (IU/l)

Group Mean ± SD P value Mean ± SD P value

Control 10.8 ± 3.7 82.17 ± 3.5LPS/D-GaIN (hepatitis model) 100.3 ± 2.3 0.000a 259.4 ± 17.938 0.000a

GL pretreatment 15.0 ± 1.5 0.000b 110.80 ± 14.703 0.000b

GL 1/2 h posttreatment 33.0 ± 3.7 0.000b 120.20 ± 9.859 0.000b

GL 4 h posttreatment 60.0 ± 2.8 0.000b 181.80 ± 16.300 0.000b

ALT, alanine transaminase; AST, aspartate transaminase; D-GaIN, D-galactosamine; GL, glycyrrhizin; LPS, lipopolysaccharide; SD, standard deviation.P < 0.05 is significant.aLPS/D-GaIN group vs. control.bGL treated groups vs. LPS/D-GaIN group.

Figure 1.

Photomicrographs of rat liver tissues of the control group showing (a)normal lobular architecture. (b) The polyhedral hepatocytes radiatedfrom the central vein in plates (arrows) separated by sinusoids (S), withacidophilic cytoplasm and vesicular nuclei (blue arrow). Hepatocytesmay be binucleated (red arrow). Portal tracts contained branches ofportal vein (PV), hepatic artery (HA), and bile duct (BD) (inset).

H&E a: �10, b: inset �40.

520 The Egyptian Journal of Histology


Immunolabeled sections

Liver sections of control (1) group displayed normal

lobular architecture with no detectable immunolabeling

for activated caspase-3 (apoptotic marker) (Fig. 6). Group

II showed obvious high immunolabeling for activated

caspase-3, especially around the central vein (Fig. 7).

Pretreatment of rats with GL (group III) completely

abolished the positive immunolabeling for activated

caspase-3 (Fig. 8). Group IV showed minimal immuno-

labeled cells around the central vein (Fig. 9). Caspase-3-

immunolabeled cells were decreased in group V and

were mainly localized in the pericentral area. The

immunolabeling was mainly confined to hepatocytes.

Figure 2.

Photomicrographs of rat liver tissues of group II showing (a) disruptedlobular architecture with dilated blood sinusoids (arrows) andhepatocellular degeneration. The inset showing degeneration (circle)around central vein (CV) being more than around portal vein (PV) andboth veins showing congestion. (b) Clustered degenerated cellsaround a CV, with densely stained acidophilic cytoplasm and darkcondensed (black arrows) or fragmented nuclei (blue arrow). Observethe shrunken apoptotic cells present in a halo (green arrow). (c)Degenerated cells with pyknotic nucleus and vacuolated cytoplasm(circle).

H&E a and inset: �10, b: �40, c: �100.

Figure 3.

Photomicrograph of rat liver tissues of group III showing retainedlobular architecture with scarce hepatocytes with densely stainedacidophilic cytoplasm and dark, condensed nuclei (black arrow) atareas around a central vein (CV). Observe that most hepatocytesretained their normal basophilic granules (blue arrow).

H&E �40.

Figure 4.

Photomicrograph of rat liver tissues of group IV showing retainedlobular architecture. Few hepatocytes with densely stained acidophiliccytoplasm and small, dark, condensed nuclei (black arrows) at areasaround a central vein (CV). Observe that most hepatocytes retainedtheir normal basophilic granules (blue arrows).

H&E �40.



Staining of some centrilobular hepatocytes was observed,

but some scattered individual positive cells were also

present (Fig. 10). The expression in the hepatocytes

showed a pattern of heterogeneity; most hepatocytes had

shown both cytoplasmic and nuclear expressions, whereas

others showed either cytoplasmic or nuclear expression only.

Examination of wide fields of control group (I) showed

that few TNF-a-immunolabeled cells were barely detec-

ted along the blood sinusoids (Fig. 11). High immuno-

labeling for TNF-a was markedly observed in the liver

sections of group II (Fig. 12). The immunolabeling

was mainly cytoplasmic, and most of the positive Kupffer

cells appeared large, elongated, and branched (inset of

Fig. 12). Groups III, IV, and V (Figs 13–15) showed a

decrease in the level of TNF-a expression when

compared with the extensive immunolabeling seen in

group II. The immunoreactivity for TNF-a was mainly

restricted to macrophages, Kupffer cells, and endothelial

cells.

Morphometric results

There was a significant increase in caspase-3-

immunolabeled hepatocytes and TNF-a-immunolabeled

Kupffer cells in group II compared with the control,

whereas a significant decrease was observed in groups

III and IV, and to a lesser extent in group V compared

with group II (all P < 0.05) (Tables 2 and 3, respectively).

Figure 5.

Photomicrograph of rat liver tissues of group V showing fewhepatocytes with densely stained acidophilic cytoplasm and dark,condensed nuclei (arrows). CV, central vein.

H&E �40.

Figure 6.

Rat liver tissues labeled for activated caspase-3 in the control groupshowing no detectable immunolabeling. CV, central vein.

Immunohistochemistry counterstained with H �40.

Figure 7.

Rat liver tissues labeled for activated caspase-3 in group II showingextensive positive reaction of hepatocytes clustered (circle) aroundcentral vein (CV) or scattered cells (blue arrows). Immunolabeling inmost positive cells was both cytoplasmic and nuclear (black arrows),whereas some cells showed cytoplasmic (green arrows) or nuclear (redarrows) immunolabeling.


Figure 8.

Rat liver tissues labeled for activated caspase-3 in group III showingnegative reaction. CV, central vein; PV, portal vein.




Figure 9.

Rat liver tissues labeled for activated caspase-3 in group IV showingfew positive cells (arrow) around central vein (CV).


Figure 10.

Rat liver tissues labeled for activated caspase-3 in group V showingmoderate positive reaction (arrows) around a central vein (CV).


Figure 11.

Figure 12.

Rat liver tissues labeled for tumor necrosis factor a in group II showingextensive immunolabeling of Kupffer (black arrow) and endothelial (bluearrow) cells. Observe the infrequent hepatocytes with positive reaction(red arrow). Kupffer cells appeared large, elongated, irregular, andbranched (inset). CV, central vein.

Immunohistochemistry counterstained with H �40, inset: �100.

Figure 13.

Rat liver tissues labeled for tumor necrosis factor a in group III showingfewer immunolabeled Kupffer (black arrow) and endothelial (blue arrow)cells around a central vein (CV).


Figure 11.

Rat liver tissues labeled for tumor necrosis factor a in the control groupshowing very few endothelial (arrow) cells with faint positive reaction.CV, central vein.




DiscussionConsiderable efforts have been made to clarify the

mechanisms for the development of acute hepatitis and

various measures have been taken for its treatment;

however, the prognosis of acute hepatitis is still quite

poor and there is no completely effective treatment for

the disease [18]. In many liver disorders, inflammation

and apoptosis are important pathogenic components,

finally leading to acute liver failure [19]. This study was

carried out with regard to the structural changes in the rat

liver after LPS/D-GaIN administration and with regard to

the evaluation of the efficacy of a natural hepato-

protective agent, GL, for treatment of these changes.

This study showed that liver injury by LPS/D-GaIN had

marked hepatocellular degeneration. Many hepatocytes

appeared with hypereosinophilic, shrunken cytoplasm,

and pyknotic nuclei, and few cells had fragmented nuclei

(apoptosis-like features). These morphological findings

were in accordance with the results of Wilhelm et al.[18]. Hase et al. [20] confirmed their results with

electrophoresis of total hepatic DNA that showed a

typical apoptotic ladder pattern fragmented at the

nucleosome unit. Mignon et al. [21] suggested that LPS

toxicity actually resulted exclusively from severe

apoptotic liver injury, and not from the systemic

inflammatory response, as it was suggested previously.

Apoptosis induced by LPS/D-GaIN resulted from a

combination of two pathways: the extrinsic apoptotic

pathway by activation of Kupffer cells, which secrete

TNF-a, and the intrinsic pathway by generation of

oxidative stress [22]. Hence, the downregulation

of these pathways could interpret the reducing effect of

GL on apoptosis. Histological sections of the liver

specimens of LPS/D-GaIN group also exhibited the

emergence of some degenerated hepatocytes with ill-

defined cell boundaries and vacuolated cytoplasm; these

findings were in accordance with Le-Minh et al. [23]. The

concentration of the previous changes in the pericentral

area (zone three) could be explained by the fact that the

cells in zone three are especially rich in enzymes involved

in drug metabolism [24]. Injection of GL 1=2 h before

LPS/D-GaIN administration showed a marked reduction

in the previous histopathological changes. Similar results

were obtained when GL was injected 1=2 h after LPS/

D-GaIN administration. These results were in agreement

with Ikeda et al. [13] who reported the reducing effect of

Figure 14.

Rat liver tissues labeled for tumor necrosis factor a in group IV showingfewer immunolabeled Kupffer (black arrow) and endothelial (blue arrow)cells around central vein (CV), and infrequent immunolabeled hepatocytes(red arrow).


Figure 15.

Rat liver tissues labeled for tumor necrosis factor a, in group V, showingincreased immunolabeled Kupffer cells (black arrows) around central vein(CV). Observe the infrequent hepatocytes with positive reaction (red arrow).

Immunohistochemistry counterstained with H�40.

Table 2. Active caspase-3 immunolabeled hepatocytes of

different experimental groups (n = 6 animals per group)

Caspase positivehepatocytes

Group Range (%) Mean ± SD P value

Control 0–0.33 0.17 ± 0.4LPS/D-GaIN (hepatitis model) 9–21.2 36.16 ± 5.6 0.002a

GL pretreatment 0–0.9 1 ± 0.37 0.002b

GL 1/2 h posttreatment 2–3 5 ± 3.65 0.003b

GL 4 h posttreatment 4.8–9.6 20.3 ± 2.07 0.056b

D-GaIN, D-galactosamine; GL, glycyrrhizin; LPS, lipopolysaccharide;SD, standard deviation.P < 0.05 is significant.aLPS/D-GaIN group vs. control.bGL treated groups vs. LPS/D-GAIN group.

Table 3. Tumor necrosis factor a immunolabeled Kupffer cells of

different experimental groups (n = 6 animals per group)

Group Mean ± SD P value

Control 0.5 ± 0.3LPS/D-GaIN (hepatitis model) 27 ± 3.8 0.001a

GL pretreatment 2.8 ± 0.94 0.000b

GL 1/2 h posttreatment 5 ± 1.03 0.004b

GL 4 h posttreatment 13.16 ± 1.19 0.021b

D-GaIN, D-galactosamine; GL, glycyrrhizin; LPS, lipopolysaccharide;SD, standard deviation.P < 0.05 is significant.aLPS/D-GaIN group vs. control.bGL treated groups vs. LPS/D-GaIN group.



GL on apoptotic changes evidenced by the quantitative

determination of DNA fragmentation of liver tissues.

In contrast, Hase et al. [20] reported that oral GL

pretreatment did not inhibit hepatic DNA fragmentation.

The discrepancy between our results and Hase results

may be due to the difference in route of administration,

as glycyrrhetic acid is absorbed through the intestines

with only a minimal absorption of GL [25]. A relative

decrease in the histopathological changes was observed

when GL was injected 4 h after LPS/D-GaIN

administration. This was in contrast to Ikeda et al. [13]

who reported that the protective effect of GL (evidenced

by its lowering effect on ALT serum level only) appeared

on GL administration 10–60 min after LPS/D-GaIN

administration.

Caspase-3 activation is a hallmark of almost all apoptotic

systems [26]. The expression of activated caspase-3 and

the release of cytochrome C into the cytoplasm of liver

cells were both enhanced by LPS, indicating the

involvement of activated caspase-3 in the intrinsic

pathway of apoptosis induced by LPS [27]. In this

study, it was found that LPS/D-GaIN administration

markedly increased the expression of active caspase-3

in rat liver tissues. In terms of expression, these results

coincided with Erik and Poh-Gek [28], who found

numerous hepatocytes positively stained for cleaved

caspase-3 in liver sections of mice treated with LPS/

D-GaIN for 6 h; however in terms of distribution, they

found that, in contrast, the positive cells were scattered

throughout the parenchyma and not concentrated around

the central vein as it was observed in this study. This

variation in distribution may be due to the short duration

of intoxication in their study. The labeling distribution

pattern for activated caspase-3 in rat liver sections of

LPS/D-GaIN also paralleled the findings obtained in this

study by hematoxylin and eosin, in which increasing

numbers of positive cells (apoptotic cells) were observed

in the pericentral area. In this study, GL pretreatment

abolished the immunolabeling for activated caspase-3

caused by LPS/D-GaIN injection; this was in agreement

with Tang et al. [29]. While post-treatment with GL also

reduced reactivity for caspase-3 but with lesser extent

with time delaying.

TNF-a is an important member of cytokines, which is a

key mediator of hepatic apoptosis and necrosis in LPS/D-

GAlN-induced liver failure [30]. Plasma TNF-a level is

also known to be elevated in patients with acute alcoholic

hepatitis [31] and chronic hepatitis caused by hepatitis

B virus infection [32]. Therefore, TNF-a plays a role in

the pathogenesis of not only endotoxin-induced experi-

mental liver injury but also in many human liver diseases.

Kim et al. [33] reported that the serum TNF-a level

significantly increased after the LPS/D-GaIN treatment,

matching the high immunolabeling for TNF-a in the liver

sections of LPS/D-GAlN group compared with controls

in this study. Similar to the results of Mohammed et al.[34], the positive cells for TNF-a in these results were

confined to blood sinusoids and the expression was

mainly restricted to Kupffer cells. Endothelial cells

secrete proinflammatory cytokines [35] and this can

explain the positive immunolabeling for TNF-a, observed

in some endothelial cells. Treatment with GL showed

marked attenuation of TNF-a immunolabeling in liver

sections. The study of Lee et al. [36] also established the

lowering effect of GL on the increased level of circulating

TNF-a in CCl4-induced liver injury, as an analog of the

liver damage caused by various hepatotoxins in humans.

The suppressive effect of GL on TNF-a may be

considered as one of the major mechanisms of its

hepatoprotective role in this model. The relation

between TNF-a, apoptosis, and caspase-3 was explained

by Zang et al. [30] that TNF-a combined with TNF-areceptor on the membrane of liver cells through a series

of signal transmission activating caspase-3 and then

inducing apoptosis.

Serum ALT and AST are the most sensitive markers used

in the diagnosis of hepatic damage. They are cytoplasmic

enzymes released into circulation after cellular damage in

acute hepatoxicity [37]. In accordance to Ito et al. [38],

these results exhibited an increase in AST and ALT

activities after LPS/D-GAlN exposure compared with

controls. The histological observations in this study

strongly support the release of aminotransferases by the

damaged hepatocytes. Although apoptotic cells generally

do not release cell contents, the high ALT values

indicated that some of the apoptotic hepatocytes were

undergoing secondary necrosis [39]. This may be caused

by neutrophil cytotoxicity [40]. There was no increase in

the serum levels of ALT or AST after GL pretreatment.

GL posttreatment significantly decreased the levels of

these markers, which vary by the time of administration.

The light microscopic pictures, in all groups, were in

harmony with results of aminotransferase level.

ConclusionIn conclusion, the results of this study provide evidence

for the protective and curative effect of GL against LPS-

induced acute hepatotoxicity in rats. GL pretreatment is

more effective than posttreatment. The effect of

GL decreased with delaying the time of administration

in the acute LPS/D-GAlN-induced hepatitis model. The

anti-inflammatory and antiapoptotic effects of GL were

evident and these results attributed GL hepatoprotective

effect mainly to the suppression of TNF-a and active

caspase-3.

These results should thus provide a new insight in treating

patients with acute hepatitis and paves the future for a

new rationale in the treatment of liver diseases. However,

further experimental studies are still needed for more

details on GL as a drug for acute hepatitis.

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