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www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327
wjg@wjgnet.com © 2006 The WJG Press. All rights reserved.
BASIC RESEARCH
Wen-Chuan Lin, Wei-Lii Lin, Department of Pharmacology, by exerting a protective effect against hepatocellular ne-
China Medical University, 91 Hsueh Shih Road, Taichung 404, crosis by its free-radical scavenging ability.
Taiwan, China
C o r r e s p o n d e n c e t o : We n - C h u a n L i n , D e p a r t m e n t o f © 2006 The WJG Press. All rights reserved.
Pharmacology, China Medical University, 91 Hsueh Shih Road,
Taichung 404, Taiwan, China. wclin@mail.cmu.edu.tw
Key words: Ganoderma lucidum; Carbon tetrachloride;
Telephone: 886-4-22053366 (ext. 8306) Fax: 886-4-2205-3764
Received: 2005-07-07 Accepted: 2005-07-20 Liver fibrosis
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266 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 14, 2006 Volume 12 Number 2
of G. lucidum on chronic CCl4-induced liver fibrosis. in 100 mL/L neutral formalin for the preparation of
pathological sections; (2) frozen directly in liquid nitrogen
for transcript analysis; (3) after weighing, the liver was
MATERIALS AND METHODS
completely dried at 100 ºC for the determination of
Preparation of test substance collagen content; and (4) remaining samples were stored at
Crude G. lucidum extract (GLE), which also contains -80 °C as reserves.
cracked spores of G. lucidum, was obtained from the
Taiwan branch of the American company NuSkin Assessment of liver functions
Phar manex. GLE was suspended in distilled water Whole blood was centrifuged at 4 700 r/min at 4 ºC for
and administered orally to each rat at a volume of 1 10 min to separate the plasma. Aspartate aminotransferase
mL/100 g body weight. To guarantee reproducibility (AST) and alanine aminotransferase (ALT), albumin and
of pharmacological experiments, we assayed the total total protein were determined spectrophometrically with
triterpene content of GLE. an automatic analyzer (Cobas Mira; Roche, Rotkreuz,
Switzerland) using commercially available kits (Roche
Determination of total triterpenes in GLE by HPLC Diagnostics).
GLE (100 mg) was extracted with ethyl acetate and then
evaporated to dryness under vacuum. The residue was Assays for hepatic protein, lipid peroxidation and
dissolved in methanol and diluted to 2 mL. The sample hydroxyproline
solutions were filtered through a 0.45-µm filter before Livers were homogenized in 9 vols ice-cold 0.15 mol/L
HPLC analysis as follows. HPLC instrument: Waters KCl and 1.9 mmol/L ethylenediaminetetraacetic acid. Liver
2690 separation unit plus Waters 996 PDA; column: protein concentration was measured according to Lowry
Phenomenex Lunca C18(2); flow rate: 1.0 mL/min; et al [16] using bovine serum albumin as standard. Lipid
detection: absorption at 252 nm; gradient solvent system: peroxidation was measured by the methods of Ohkawa
CH3CN+0.1% trifluoroacetic acid. The total peak area for et al[17] using 2-thiobarbituric acid. Lipid peroxidation was
a retention time of 8.0-38.0 min was used to calculate total expressed as the amount of malondialdehyde/mg protein.
triterpenes. The peak area of ganoderic acid A (Shanghai Hydroxyproline determination followed a method
R&D, Pharmanex) was used as standard. This method designed by Neuman and Logan[18]. After hydrolysis, dried
showed that the total triterpene content of GLE was over liver tissue was oxidized by H2O2 and colored by p-dimeth
6%. ylaminobenzoaldehyde; and absorbance was determined at
540 nm. The amount of hydroxyproline was expressed as
Animals µg/g tissue.
Male Wistar rats were obtained from the National
Laborator y Animal Breeding and Research Center, RNA extraction and RT-PCR analysis
National Science Council, and fed with a standard Total RNA was isolated from rat livers using the acid
laboratory diet and tap water ad libitum. Experimental guanidum thiocyanate-phenol-chloroform extraction
animals were housed in an air-conditioned room at 22-25 ºC methods, as described by Chomczynski and Sacchi[19]. A
and a 12 h light/dark cycle. Rats were allowed free access total of 5 µg RNA from each liver sample was subjected
to powdered feed and mains water that was supplied to reverse transcription (RT) by using MMuLV reverse
through an automatic watering system. When they reached transcriptase in a 50 µL reaction volume. Aliquots of the
250-300 g, forty rats were divided randomly into 4 groups, reverse transcription mixture were used for amplification
such as control, model and two GLE treatment groups, by polymerase chain reaction (PCR) of fragments specific
according to body weight 1 d before administration of to transforming growth factor-β1 (TGF-β1), methionine
the test substance. All animals received humane care and adenosyltransferase 1A (MAT1A) and MAT2A using the
the study protocols were in compliance with Institutional primer pairs listed in (Table 1). The levels of expression of
Guidelines for the use of laboratory animals. all transcripts were normalized to that of glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) mRNA in the same
CCl4-induced liver fibrosis tissue sample. The primer pairs for TGF-β1 and GAPDH
Liver fibrosis was induced by oral administration of 0.2 were designed by Wolf et al[20]. In addition, the primer
mL/100 g body weight of CCl4 (200 mL/L; diluted in pairs for MAT1A and MATA2A were designed using the
olive oil) twice a week for 8 wk. Animals received CCl4 Primer select program[21]. The identities of the resultant
only (model group), CCl4 with GLE (600 or 1 600 mg/kg PCR products were confirmed by sequence analysis. The
per day) throughout whole experimental period. During cycling parameters were 30 min at 55 ºC for cDNA first
CCl4 administration, the time interval between CCl4 and strand synthesis, and 5 min at 95 ºC, 1 min at 55 ºC and 1
GLE administration was at least 5 h to avoid disturbance min at 72 ºC for 32 cycles in a Perkin Elmer 9700 Gene
in absorption of each substance. At the end of the Amp PCR system. The PCR product was electrophoresed
experimental period, rats were sacrificed under ether on a 20 g/L agarose gel recorded by Polaroid film, and the
anesthesia and blood was withdrawn from the abdominal bands were quantitated by using densitometry.
artery. Liver and spleen were quickly removed, weighed
after washing with cold normal saline and removing excess Pathological examinations
moisture. The largest lobe of liver was divided into four After for malin fixation, tissue samples were sliced,
parts, which were then used as follows: (1) submerged embedded in a standard manner and stained with Sirius
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Lin WC et al. G. lucidum on CCl4-induced liver fibrosis 267
Table 1 Primer sequences for PCR amplification Table 2 Effect of GLE on weight of liver and spleen in
CCl4-treated rats
mRNA Primer sequences Length (bp)
Group Dose (mg/kg) Liver (g) Spleen (g)
TGF-β1 Sense 5' TAT AGC AAC AAT TCC TGG CG 3' 162
Antisense 5' TGC TGT CAC AGG AGC AGTG 3' Control – 15.1 ± 1.8 0.90 ± 0.11
MAT1A Sense 5' AAA TGA AGA GGA TGT TGG TG 3' 264 CCl4+H2O – 16.6 ± 3.3 2.58 ± 0.32b
Antisense 5' ATT GTG TTG GCA CAG AGA GAT GA 3' CCl4+GLE 600 16.7 ± 2.2 2.21 ± 0.36
MAT2A Sense 5' ATG CTG TCC TTG ATG CAC 3` 400 1600 18.2 ± 2.0 2.09 ± 0.56a
Antisense 5' GCG TA A CCA AGG CAA TG 3'
GAPDH Sense 5' CTT CAT TGA CCT CAA CTA CAT GGT CTA 3'
a
P<0.05 vs CCl4 + H2O group; bP<0.001 vs control group.
Antisense 5' GATG ACA AGC TTC CCA TTC TCA G 3' 99
a
P < 0.05 vs CCl4 + H2O group; bP < 0.001 vs control group.
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268 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 14, 2006 Volume 12 Number 2
a
P < 0.05, bP < 0.01 vs CCl4 + H2O group; dP < 0.001 vs control group.
Carbon tetrachloride,CCl4
Control H 2o GLE 1600 mg/kg GLE 600 mg/kg
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
bp
Table 6 Effect of GLE on the mRNA expression of he-
patic TGF-β1, MAT1A and MAT2A 300 TGF-β1
100 GAPDH
Group Dose TGF-β1/GAP- MAT1A/GAP- MAT2A/GAP
(mg/kg) DH ratio DH ratio DH ratio
300
Control – 0.18 ± 0.03 4.34 ± 0.0.81 0.29 ± 0.02 MAT1A
CCl4 + H2O – 22.17 ± 7.20f 1.75 ± 0.46f 4.95 ± 0.21f 100 GAPDH
CCl4 + GLE 600 16.99 ± 3.26a 3.07 ± 1.15a 1.68 ± 0.14d
1600 4.83 ± 0.48d 3.83 ± 1.35b 0.56 ± 0.03d MAT2A
300
a
P<0.05, bP<0.01, dP<0.001 vs CCl4 + H2O group; fP<0.001 vs control 100 GAPDH
group.
Figure 1 Effect of GLE on mRNA expression of TGF-β1, MAT1A and MAT2A in
hepatic tissue. M: DNA marker
MAT2A mRNA. In contrast, the level of MAT1A mRNA
was significantly decreased by CCl4 treatment. However,
treatment with GLE significantly increased the level of decays at birth to negligible levels and, in the adult liver,
MAT1A mRNA. increases during regeneration after partial hepatectomy[26
–28]
. Thus, in response to liver injury, MAT1A expression
Pathological changes is switched off and MAT2A expression is switched on.
As shown in Figure 2, CCl4 induced liver lesions in rats. Consistent with this, the expression of MAT1A was found
Sirius red stain showed clear nodular fibrosis (Figure 2B). to be reduced in the livers of rats with chronic CCl4 injury,
Treatment with GLE (1 600 mg/kg) showed a marked whereas the expression of MAT2A increased. In this study,
improvement in the pathological changes to these tissues we also found that the changes in MAT expression in
(Figure 2C and Table 7). chronic CCl4-injured rats were reduced by GLE treatment.
These results further support the fact that GLE possesses
a hepatoprotective effect.
The liver synthesizes not only the protein it needs, but
DISCUSSION
also produces numerous export proteins. Among the latter,
The present study revealed the beneficial effect of GLE plasma albumin is the most important[29]. Export proteins
in prevention of liver fibrosis induced by CCl4 treatment. are synthesized on polyribosomes bound to the rough
An improvement brought about by GLE was also seen in endoplasmic reticulum of the hepatocytes. In contrast,
plasma biochemical parameters. protein destined for intracellular use is synthesized on
CCl4 treatment caused hepatocellular damage in rats, free polyribosomes rather than bound polyribosomes[29].
as indicated by a drastic increase in both plasma ALT In this experiment, CCl4 induced liver fibrosis in rats and
and AST levels after CCl 4 administration. Rats treated it appeared to cause a decrease in both hepatic protein
with GLE showed a protection against CCl 4-induced and plasma albumin contents. GLE clearly reduced the
hepatotoxicity, with the levels of both plasma AST and decrease in protein content in the liver and albumin
ALT being reduced. content in the plasma; thus it was shown to ameliorate the
It is well known that adenosylmethionine-dependent decline in liver synthesis function caused by CCl4-induced
methylation is central to many biological processes [23]. fibrosis.
Methionine adenosyltransferase (MAT) is a key enzyme for Immunoglobulin is synthesized by immunocytes and
liver methionine metabolism, which catalyzes the synthesis hyperglobulinemia is found in hepatocellular disorders,
of S-adenosylmethionine[24]. In mammalian tissue, three appearing as an inflammatory reaction of liver[30]. In the
different forms of MAT (MAT I/III and MAT II) have present experiments, we observed CCl4-induced chronic
been identified, which are the product of two different liver lesions in rats and also a decrease in A/G ratio. GLE
genes (MAT1A and MAT2A). MAT1A is primarily could clearly lessen the decrease in the A/G ratio caused
restricted to adult liver[25]. MAT2A is high in fetal liver, by CCl4, thereby exhibiting suppressive actions on liver
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Lin WC et al. G. lucidum on CCl4-induced liver fibrosis 269
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