Effect of Selected Natural Products, Thioproline and
Pegasys® on Hepatic Platelet Activating Factor (PAF) in
CCl4-induced Hepatic Fibrosis In Rats
Farid A. Badria1 and Hala A. Attia2*
1
Department of Pharmacognosy, 2Department of Biochemistry, Faculty of
Pharmacy, Mansoura University 35516, Mansoura, Egypt.
*
To whom correspondence should be addressed.
Abstract
This study aimed to estimate hepatic levels of platelet activating factor
(PAF) in liver fibrosis induced by CCl4 in rats. A group of selected natural
products; boswellic acids, curcumin and glycyrrhizin (preparation named OMNI ®
; a drug under clinical trials for treatment of hepatitis C virus), Mirazid ® (a
commercially available schistomicidal drug), Thioproline (a commercially
available hepatoprotective agent) and Pegasys® (peg interferon alpha-2a; a
commercially available therapy for treatment of hepatitis C virus) were examined
for their effect on hepatic PAF after CCl4 intoxication. For this purpose, 54 rats
were used in the study. Rats were divided into 6 groups each comprised 9 rats.
Group 1 was treated only with CCl4, groups 2 to 5 were treated with OMNI®,
Mirazid®, Thioproline and Pegasys®, respectively, whereas the sixth group was
the normal control group (with no treatment, except an injection of the vehicle).
Liver damage was induced in all groups except normal control group (groups 1 to
5) by i.p. injection of 40% CCl4 in corn oil (0.375 ml/kg) 3 times a week for 3
weeks. One week after CCl4 intoxication, all tested drugs were injected i.p. daily
for 3 weeks. Hepatic PAF concentration was estimated by HPTLC (high
performance thin layer chromatography), while, levels of serum transaminases
(ALT, AST), hepatic hydroxyproline (as markers of liver fibrosis), serum
malondialdehyde and catalase (as markers of oxidative stress) were estimated
spectrophotometrically. The hepatic PAF levels were significantly higher in CCl 4
group (24.24±2.01 pmol equiv./mg) as compared to normal control (8.81±1.09 pmol
®
®
equiv./mg) (p<0.001). Treatment with OMNI , Mirazid , Thioproline and
Pegasys® reduced hepatic PAF significantly to be 11.84±0.22, 14.5±1.00,
13.17±0.54 and 14.26±1.09 pmol equiv./mg, respectively. This study may add
further rational to the anti-fibrotic activity of the tested drugs via reduction of
hepatic PAF.
Key words: platelet activating factor (PAF), OMNI®, Mirazid®, Thioproline,
Pegasys® and liver fibrosis.
-1-
Introduction
Platelet
activating
factor
(PAF:1-0-alkyl-2-acetyl-sn-glycero-3phosphocholine) is a potent pro-inflammatory phospholipid which exerts diverse
biological activities including platelet secretion and aggregation, hypotension,
bronchoconstriction, increased vascular permeability and stimulated synthesis of
mediators such as eicosanoids (1-6). PAF acts as a mediator of inflammation,
allergic reactions and shock and as a specific membrane bound adhesion molecule
(6-8). The PAF content of the intact liver is elevated by various types of injury
including ischemia reperfusion (9), endotoxin exposure in vivo (10) and obstructive
jaundice (11). Plasma and liver tissue PAF levels are increased in experimental
obstructive jaundice and activation of this mediator contributes to the ongoing
liver injury (12).
PAF has been suspected to play an important role in liver
pathophysiology. The cultured Kupffer and endothelial cells produce and release
PAF in order to facilitate communication between hepatic sinusoidal and
parenchymal cells (12). Moreover, PAF is an important mediator of hepatic injury.
Inappropriate production of PAF leads to pathologic inflammation, and PAF is
clearly involved in liver damage as PAF receptor blockers reduce hepatic damage
after ischemia reperfusion. (13).
Villamediana et al. (14) found that, in the cirrhotic rats, PAF levels
were elevated in systemic blood but made no measurements of the hepatic PAF
system or altered hepatic responses to PAF. Marathe et al. (15) reported increased
hepatic PAF two hours after CCl4 administration but did not determine if the PAF
content of liver was elevated during chronic injury. Therefore, a thorough study
was needed to estimate the level of hepatic PAF after intoxication for prolonged
time.
The present study aimed to estimate hepatic PAF levels 3 weeks after
CCl4-induced liver damage in rats. Different agents with different postulated
mechanisms of action on liver injury were selected; OMNI® (a drug under clinical
trials for antiviral therapy), Mirazid® (Schistomicidal agent), Thioproline (a well
known antioxidant agent) and Pegasys® (long acting interferon). This study, in
addition, aimed to test the influence of these drugs on hepatic PAF levels after
CCl4 intoxication and subsequently the possible contribution of this effect to their
known hepatoprotective action.
OMNI®, a drug under clinical trials for treatment of hepatitis C virus
(HCV), is composed of boswellic acids, curcumin and glycyrrhizin. Boswellic acids
are the effective compounds isolated from the gum-resin of Boswellia serrata and
Boswellia carterii that have been used for the treatment of rheumatoid arthritis
and inflammatory bowel diseases (16) and may have cytostatic and apoptotic
effects towards a variety of malignant cells (17). Curcumin is a natural compound
-2-
present in the rhizome of plant Curcuma longa Linn, it has anti-inflammatory,
anti-carcinogenic, anti-thrombotic, antispasmodic and potent antioxidant effects
(18). Glycyrrhizin, a major component of licorice root, is an anti-inflammatory
and antioxidant compound isolated from Glycyrrhiza glabra (19). It has inhibitory
effect on PAF production in human neutrophils in a dose dependent manner (20)
and it was shown to significantly suppress the production of PAF from the rat
peritoneal exudate cells (21). The anti-inflammatory and anti-allergic effects of
glycyrrhizin were suggested to be due to the suppression of PAF production (21).
Mirazid® is a commercially available drug used as a safe and effective
natural product against Schistosoma mansoni infection (22). It is a special
formulation of myrrh; the oleo-gum resin obtained from the stems of Commiphora
molmol, and probably other species of Bursearacae (23). Myrrh is useful for
treatment of sore throat, bleeding gums, chronic pharyngitis and amenorrhea (24)
and is used widely in Somalia for treatment of diarrhea and stomach complains
(25). Moreover, myrrh is approved by the Food and Drug Adminstation (FDA) for
food use (21 CFR 172.510).
Thioproline (thiazolidine-4-carboxylic acid, timonacic acid or
Hepargen ) is a cyclic sulfur containing amino acid and is a condensation product
of cysteine and formaldehyde (26). Animal studies confirmed the anti-toxic effects
of Thioproline, particularly on the liver (27). Thioproline has been clinically used,
mainly in the treatment of liver diseases and related gastrointestinal disturbances
(27).
Pegasys® (peg interferon alpha-2a) is a long acting interferon used in
antiviral therapy in humans, mainly for viral hepatitis B and C. An anti-fibrotic
effect of interferon has been postulated even in the absence of antiviral response,
suggesting that, interferon directly inhibits fibrogenesis (28-30). Moreover, its
effect on the reduction of fibrosis has been discovered in different experimental rat
models of hepatic fibrosis (31-33).
®
Materials and Methods
A) Chemicals:
ALT and AST kits were purchased from bioMerieux Co.; Thioproline
powder, chloramine-T and perchloric acid were obtained from Aldrich Co.; pdimethylamino-benzaldehyde and authentic PAF (1-O-palmityl-2-acetyl-snglycero-3-phosphocholine) were purchased from Sigma Chemical Co.
B) Animals:
Fifty four Wistar rats, weighing 180-250 gm, were used in this study.
They were fed with a standard laboratory diet and tap water ad libitum and
housed in individual cages. Houses were kept at 255oC, 555% humidity, and a
12 h light/dark cycle. Rats were divided into 6 groups each comprised 9 rats.
Based on the treatment protocol and dosage regimen described previously by the
author (34), animals were treated as follow: Group 1 was treated only with 40%
-3-
CCl4 in corn oil ( 0.375 ml/kg), groups 2 to 5 were treated with OMNI®, Mirazid®,
Thioproline and Pegasys®, respectively, one week after CCl4 intoxication, whereas
the 6th group was the normal control group, which only received an injection of
corn oil (0.375 ml/kg). Liver damage was induced in all groups except normal
control group (groups 1 to 5) by i.p. injection of CCl4 3 times a week for 3 weeks.
One week after CCl4 intoxication, drugs were injected i.p. daily for 3 weeks in the
following doses; OMNI® (600 mg/kg), Mirazid® (500 mg/kg), Thioproline (100
mg/kg) and Pegasys® (0.5 μg/rat).
C) Assessment of liver fibrosis:
i- Estimation of liver enzymes (ALT and AST):
At the and of the 3 weeks of study, blood was withdrawn from the eye
ball under ether anesthesia. The blood was allowed to coagulate then centrifuged
at 3000 rpm for 10 min. to separate the serum. Serum levels of ALT and AST were
measured immediately according to the method of Reitman and Frankel (35).
ii- Estimation of hepatic hydroxyproline:
The content of hepatic hydroxyproline was measured to estimate the
anti-fibrotic property of the injected drugs by using the modified method of
Woessner (36). Briefly, at room temperature, liver tissues of the right lobe were
dehydrated by 95% alcohol for 5-6 hrs, and defatted by acetone for two days. The
obtained defatted tissues were dried at 110oC and ground into powder. Forty mg
of liver tissue powder were hydrolysed in 3 ml of 6 M HCl at 110o for 6 hrs. The
hydrolysates were filtered and diluted to 50 ml with distilled water, and the
solution was neutralized to pH 6 by 6 M NaOH. Two ml of the solution and 1 ml of
0.05 M chloramine-T were placed in a glass tube, shaken vigorously and left at
room temperature for 20 min., followed by addition of 1 ml 3.15 M perchloric acid
and 1 ml of 10% p-dimethylamino-benzaldehyde and incubated at 60oC for 20
min. Finally, the reaction mixture was cooled in an ice bath for 5 min. and the
obtained color was measured spectrophotometrically at 550 nm against reagent
blank. Standard hydroxyproline solutions of concentrations 5, 10, 15, 20, 25 and 30
g/ml, were used for construction of the standard curve.
D) Assessment of oxidative stress:
Lipid peroxidation, a major indicator of oxidative stress, was assayed
by measuring malondialdehyde (MDA) in serum (37). Serum level of catalase was
determined according to the method of Goth (38).
E) Determination of PAF in liver tissues:
PAF was extracted and purified form liver as described previously (39, 40).
PAF was determined using high performance thin layer chromatography
(HPTLC) (41) as described below.
For lipid extraction, 100 mg of liver were homogenized in 9.5 ml of
methanol-chloroform-water mixture (2:1:0.8 v/v). The homogenates were kept at
-4-
room temperature for one hour, mixed with 5 ml chloroform-water (1:1 v/v), and
after one hour the mixture was centrifuged (3000 rpm for 15 min). The lower
chloroformic layer containing hepatic lipids, including PAF, was aspirated into
glass tubes and stored at -20oC.
For purification, the chloroformic layer was dried under nitrogen and
the obtained residue was dissolved in 200 l of chloroform and finally purified by
Sep-Pak columns (Strata, phenomenex, USA) using 3 ml of acetone-chloroform
(2:3 v/v). The eluate was dried under reduced pressure at room temperature and
then quantitatively transferred with chloroform into a small vial and evaporated
until dryness under nitrogen.
For PAF determination, the residue was redissolved in 100 l
chloroform and spotted on precoated HPTLC (Merck, Germany) using
chloroform-methanol-water (65:53:6 v/v/v) as the developing solvent (41). Spots
on a HPTLC were quantitized spectroscopically by transmission at 254 nm. The
layer was passed through a beam of light, and the transmitted energy was
measured by densitometer (Wilmington, FNC, USA). Authentic PAF (C16); 1-Opalmityl-2-acetyl-sn-glycero-3- phosphocholine (Sigma, GmbH, Germany) was
dissolved in chloroform to produce a concentration range of 2.5 to 50 fmoles and
assayed by the same method.
PAF concentrations on HPTLC fractions of unknown samples were
calculated directly from PAF C16 standard curve and expressed pmole equivalent
of PAF C16/ mg liver tissue. Total PAF for a given sample represented the sum of
PAF activity detected in all HPTLC fractions for that sample.
Statistical analysis:
Data were expressed as mean  standard error of mean (SEM).
Statistical analysis was performed using Instat-3 computer program (Graph pad
software Inc, San Diego, CA, USA). The groups were compared using one way
analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison
test.
Results
Values of the analyzed parameters and the statistical differences in the
groups are shown in Table (1) and Figure (1). During the experiment, one rat died
from each of the following groups; CCl4, Mirazid® and Pegasys®, while three rats
died from Thioproline group.
a) Liver fibrosis markers:
Three weeks after CCl4 administration; levels of serum transaminases
(ALT, AST) and hepatic hydroxyproline were significantly higher (p<0.001) in
group 1 (rats received CCl4 only) than those of control. Groups 2 to 5 (OMNI®,
Mirazid®, Thioproline and Pegasys®) showed significant reductions (p<0.001) in
serum ALT and hepatic hydroxyproline levels in comparison with CCl 4 group and
insignificant differences were observed as compared to control group. On the
other hand, serum AST decreased significantly (p<0.01) only in OMNI®-treated
-5-
group as compared to CCl4 group, whereas, Mirazid®, Thioproline and Pegasys®
did not significantly affect AST as compared to CCl4 group. No significant
differences were found among drug-treated groups in ALT and hydroxyproline
levels.
b) Oxidative stress markers:
Serum MDA levels were significantly higher (p<0.001) in CCl4 group
than in control. OMNI®, Mirazid®, Thioproline and Pegasys® significantly
reduced (p<0.001) serum MDA levels as compared to CCl4 with no significant
differences in comparison with control. On the other hand, serum catalase levels
decreased significantly (p<0.05) in CCl4 group as compared to control. All tested
drugs significantly increased catalase levels as compared to CCl4 group (p<0.01).
No significant differences were observed in levels of MDA and catalase among
tested drug groups.
C) Hepatic PAF levels:
A highly significant increase in hepatic PAF levels was observed in CCl4
group as compared to control (24.242.01 Vs 8.811.09 pmol equiv./mg, p<0.001).
A highly significant reductions (11.84±0.22, 14.5±1.00, 13.17±0.54 and 14.26±1.09
pmol equiv./mg, p<0.001) were detected by OMNI®, Mirazid®, Thioproline and
Pegasys®, respectively, as compared to CCl4 group. OMNI® and Thioproline
groups showed no significant differences in hepatic PAF as compared to control,
while PAF levels in Mirazid® and Pegasys® groups were still significantly higher
(p<0.01) than those of control. No significant differences were shown among tested
drug groups.
As shown in Table (2), positive correlation was found between levels of
PAF and MDA in CCl4 and tested drug groups. The correlation was significant in
CCl4, OMNI® and Thioproline groups, while insignificant correlation was seen in
Mirazid® and Pegasys® groups. On the other hand, negative correlation was
observed between levels of PAF and catalase, significantly in CCl4 and OMNI®
groups and insignificantly in Mirazid®, Thioproline and Pegasys® groups.
Discussion
Metabolism of CCl4 by hepatocytes and/or endogenous macrophage
like Kupffer cells (42), resulted in severe hepatic necrosis and fibrosis as indicated
by elevated liver enzymes (ALT and AST) and hepatic concentration of
hydroxyproline. In this established model, liver injury is derived from a mixture of
free radicals and reactive oxygen species (43, 44), lipid peroxidation (45, 46) and
activated Kupffer cells (47), each of which has an underlying role in resulting liver
damage. The administration of OMNI®, Mirazid®, Thioproline and Pegasys® to
CCl4-intoxicated rats for 3 weeks limited the metabolism of CCl4 and improved the
biochemical parameters. These results were obvious from their effect on lowering
-6-
liver enzyme (ALT), hepatic hydroxyproline and serum MDA in addition to
increasing serum catalase (Table 1).
The antioxidant activity of OMNI® is derived mainly from curcumin
which is a very potent antioxidant (18, 48-50) and glycyrrhizin which antioxidant
activity (51-53) and its role in reducing liver fibrosis (54) has been established.
Thioproline (thiazolidine-4-carboxylic acid) is a well known intracellular
sulfhydryl antioxidant and free radical scavenger (27, 55). Diet supplementation
with this antioxidant slowed the aging process, prolonged life span and stimulated
the immune system in old mice (55) and reversed age-related behavioral
dysfunction in prematurely-aging mice (56).
Many studies suggest an antioxidant and anti-fibrotic activity of
interferon alpha (IFN-) (29,32,57-59). Lu et al.(58) suggested that, IFN- may
enhance biological defense activities against oxidative stress and function as a
potent fibro-suppressant by protecting hepatocytes and hepatic stellate cells from
lipid peroxidation. In the study of Serejo et al. (29), IFN- promoted a long term
inhibition of oxidative stress with concomitant improvement of activity of fibrosis.
Madro et al. (32) found positive effects of high doses of IFN- in improving liver
function and reducing liver fibrosis induced by CCl4 in rats.
Platelet activating factor (PAF) is a potent pro-inflammatory and
hypotensive phospholipid (1-6). Inappropriate or excessive PAF has the potential
to modify liver function, and PAF is an established hepatotoxic agent (9, 11). Our
study aimed to estimate hepatic levels of PAF in CCl4-induced liver fibrosis in rats.
In addition, the effect of different hepatoprotective drugs (OMNI®, Mirazid®,
Thioproline and Pegasys®) on these levels was also studied.
The results showed a highly significant increase in hepatic PAF levels in
CCl4 group as compared to control (Fig. 1). This is in agreement with the results
obtained by Yang et al. (60). It has been found that, Kupffer cells isolated from
cirrhotic rats release significantly more PAF than those obtained from control rats
(61). Hepatic endothelial cells and systemic macrophage recruited to injured liver
may also contribute to elevated hepatic PAF levels. Marathe et al. (15) found that,
the livers of rats metabolizing CCl4 contain inflammatory phospholipids, mainly
PAF. They also found that, oxidants, which stimulate cellular PAF generation in
vitro, such as H2O2, (62) lead to the accumulation of significant amounts of hepatic
PAF. In addition, CCl4 metabolism by cytochrome P-450 generates the reactive
trichloromethyl (CCl3) radical and this event generates superoxide and H2O2 (45).
H2O2 stimulates the synthesis of PAF (62,63) and PAF is synthesized by stimulated
macrophages and Kupffer cells (61). All these findings coincide with our results in
CCl4 group that revealed a strong positive correlation between PAF and MDA as
well as a significant negative correlation between PAF and catalase (Table 2).
It is also postulated that, the elevated hepatic PAF in this model is
related to reduced PAF metabolism by the liver due to low secretion of PAF-
-7-
actylhydrolase, an enzyme predominantly responsible for hydrolysis of PAF to
lysoPAF (64).
All tested drugs (OMNI®, Mirazid®, Thioproline and Pegasys®)
exerted a significant reduction of hepatic PAF after 3 weeks of treatment (Fig. 1).
These results may add further explanation to the anti-fibrotic activity of these
drugs. However, the levels of hepatic PAF were still higher than those of control
(significantly in Mirazid® and Pegasys® groups and insignificantly in OMNI® and
Thioproline groups). This may suggest the need for either higher doses or
prolonged time of treatment.
The effect of the tested drugs on hepatic PAF could be attributed to
their inhibitory effect on PAF production, through their antioxidant activity
revealed by lowering MDA levels and increasing catalase (Table 1). This
explanation could be confirmed by the positive correlation observed in the tested
drug groups between levels of PAF and MDA as well as the negative correlation
found between PAF and catalase (Table 2). Higher catalase levels achieved by
these drugs leads to H2O2 scavenging limiting its role in PAF production (62,63).
Most likely, the drugs in this study may affect the activity of
cholinephosphotransferase (CPT) or PAF-acetylhydrolase; the enzymes which are
responsible for synthesis and degradation of PAF, respectively. It has been found
that, CPT was reduced significantly in renal cell carcinoma patients who had
received IFN- compared with those who had not (65). These findings suggest that
IFN- may modulate the production of PAF. Further studies are recommended to
study the modulatory effect of these drugs on the enzymes that regulate PAF
levels.
Conclusion
Hepatic PAF level showed an increase in CCl4-induced liver fibrosis in
rats. The treatment of intoxicated rats with OMNI®, Mirazid®, Thioproline and
Pegasys® daily for 3 weeks modulated the toxic effect of CCl4 via either
antioxidant activity and/or lowering PAF contents of liver tissues. This study may
add further explanation to the anti-fibrotic activity of the tested compounds via
reducing hepatic PAF levels.
Acknowledgment
We thank Mr. Ahmad Abbas; the technician of Pharmacognosy
Department, Faculty of Pharmacy, Mansoura University for technical assistance,
care of animal house and his effort throughout the whole work.
-8-
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Table (1):
Group
Marker
CCl4
(n=8)
OMNI®+CCl4
ALT
AST
H. proline
MDA
CAT
(IU/ml)
(IU/ml)
(g/ml)
(nmol/ml)
(kU/L)
a
151.214.3
a
b
76.68.2
b
b
70.04.2
a
219.69.9
a
22.051.07
a
17.70.84
164.69.6
b
12.90.42
b
205.810.6
b
b
10.630.68
a
219  4.27
b
289.7 
15.2
b
295.5 
(n=9)
Mirazid®+
14.50.7
11.060.7
CCl4 (n=8)
Thio+CCl4
11.7
b
b
(n=6)
Pegasys®+CCl4
(n=9)
252.618
b
13.90.52
b
13.350.9
292.6.4 
13.5
b
b
(n=8)
Control
71.258.0
b
73.125.6
194.521.7
b
13.40.44
b
11.260.38
302.6 
11.7
b
b
58.574.7
b
159.36.4
b
12.50.73
b
10.70.26
291.5 
10.95
n=number of rats
a=significance versus control.
b=significance versus CCl4 group.
Thio=Thioproline, ALT=alanine aminotransferase, AST=aspartate aminotransferase,
MDA=malondialdehyde, CAT= catalase, H. proline=hydroxyproline.
- 14 -
Table (2):
Correlation between
Correlation between PAF
PAF and MDA
and Catalase
Significant, p<0.01
Significant, p<0.05
r =0.87
r = -0.81
Significant, p<0.05
Significant, p<0.01
r =0.79
r = -0.76
Not significant
Not significant
r =0.67
r = -0.50
Thioproline+CCl4
Significant, p<0.05
Not significant
(n=6)
r =0.82
r = -0.55
Not significant
Not significant
r =0.52
r = -0.57
CCl4 group (n=8)
OMNI®+CCl4 (n=9)
Mirazid®+CCl4 (n=8)
®
Pegasys +CCl4 (n=8)
- 15 -
Table (1): Markers of liver fibrosis and oxidative stress in OMNI ®,
Mirazid®, Thioproline and Pegasys® groups as compared to
both control and CCl4 groups 3 weeks after treatment. Values
were expressed as meanSEM.
Table (2): Correlation between levels of PAF and levels of both MDA and
catalase in CCl4, OMNI®, Mirazid®, Thioproline and Pegasys®
groups.
- 16 -
Figure (1):
30
PAF (pmol equiv./mg)
a
20
ab
b
ab
b
b
10
0
CCl4
OMNI® Mirazid® Thio Pegasys®Control
+ CCl4 + CCl4
+ CCl4
+ CCl4
- 17 -
Legends to figures:
Figure (1): Hepatic platelet activating factor (PAF) levels in OMNI ®,
Mirazid®, Thioproline and Pegasys® groups as compared to
both control and CCl4 groups 3 weeks after treatment. Values
were expressed as meanSEM.
a = Significance versus control group.
b = Significance versus CCl4 group.
- 18 -