Running Head: The hepatoprotective effect of antrosterol in mice 1
2
Protective effect of antrosterol from Antrodia camphorata submerged whole
3
broth against carbon tetrachloride-induced acute liver injury in mice
4 5
Guan-Jhong Huanga,Jeng-Shyan Dengb, Shyh-Shyun Huanga,Yi-Yuan Shaoc, Chin-Chu 6
Chenc, Yueh-Hsiung Kuo*, d 7
8
a
Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, 9
Taiwan 10
b
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan 11
c
Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien 12
University, Taipei, Taiwan 13
d
Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung, Taiwan 14 15 * Corresponding author 16 Dr. Kuo Yueh-Hsiung 17 Tel.: +886 4 2207-1693; Fax: +886 4 2207-1693 18
E-mail address: [email protected] 19
20
21
22
ABSTRACT 1
The hepatoprotective potential of antrosterol (ergostatrien-3-ol, ST1) from Antrodia 2
camphorata (AC) against carbon tetrachloride (CCl4)-induced liver damage was evaluated in
3
preventive models in mice. Pretreatment with ST1 markedly prevented the elevation of 4
aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver lipid peroxides in 5
CCl4-treated mice. We evaluated the activities of antioxidant enzymes [catalase (CAT),
6
superoxide dismutase (SOD) and glutathione peroxidase (GPx)] were significantly increased 7
after treatment with CCl4 in vivo. In addition, ST1 decreased the level of nitric oxide (NO)
8
production and tumor necrosis factor-alpha (TNF-α) in CCl4-treated mice. In this study,
9
these results pointed out that ST1 can inhibit lipid peroxidation, enhance the activities of 10
antioxidant enzymes, decreases the TNF-α level, nitric oxide production and inducible nitric 11
oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expressions. Therefore, we speculate 12
that ST1 protects mice from liver damage through their anti-inflammation capacity.
13
14
KEY WORDS: Chinese herb; Antrosterol; anti-inflammation; MDA; NO; TNF-α. 15
16
17
18
1. Introduction 1
The reactive oxygen species (ROS) play an important role in the degenerative or 2
pathological processes of various diseases, such as aging, cancer, coronary heart disease, 3
Alzheimer's disease, neurodegenerative disorders, atherosclerosis, cataracts, and inflammation. 4
The production of ROS can be induced by a variety of factors such as ionizing radiation, 5
exposure to drug and xenobiotics or enhanced by other factors (Bruccoleri et al., 1997). CCl4,
6
an extensively used xenobiotic, is thought to produce a highly reactive trichloromethyl radical 7
by cytochrome P450 system in liver, consequently causes lipid peroxidation that leads to
8
hepatocellular membrane damage and followed by the release of inflammatory mediators 9
from activated hepatic macrophages, which are thought to potentiate CCl4-induced hepatic
10
damage (Kuo et al., 2010). These activated macrophages released inflammatory mediators 11
including TNF-α and NO have been implicated in liver damage induced by a number of 12
different toxicants (Celik, Temur & Isik, 2007). 13
Antrodia camphorata (AC), also called A. cinnamomea, is composed of fruiting bodies, 14
mycelium and spores. The fruiting body of AC is well known in Taiwan as a traditional 15
Chinese medicine. It has been used for the treatment of food and drug intoxication, diarrhea, 16
abdominal pain, hypertension, skin itching, and cancer. The fruiting body and cultured 17
mycelia of AC contain fatty acids, lignans, phenylderivatives, sesquiterpenes, steroids, and 18
triterpenoids (Geethangili & Tzeng, 2009). The fermented culture broth had cytotoxic activity 19
against several tumour cell lines (Yeh et al., 2009), anti-inflammation (Huang et al., 2010), 1
vasorelaxation (Wang et al., 2003) and anti-hepatitis B virus activity (Lee et al., 2002). The 2
filtrate in submerged culture also had protective effects against CCl4-induced hepatic toxicity
3
and high antioxidant properties (Song & Yen, 2003). However, little information is available 4
on the protective effects against CCl4-induced hepatic toxicity effects of antrosterol (ST1)
5
(Fig. 1A). To our knowledge, this study is the first report that demonstrates the
6
anti-inflammatory effects of ST1 in liver protection and regulation of iNOS and COX-2 in 7
CCl4-induced liver injury model by AC component.
8
2. Material and methods 1
2.1. Chemicals. 2
Carbon tetrachloride was purchased from Merck (Darmstadt, Germany). Silymarin, 3
malondialdehyde, and other chemicals were purchased from Sigma Chemical Co (Steinheim, 4
Germany). Biochemical assay kits for measurement of ALT and AST contents were 5
purchased from Randox Laboratories (Crumlin, United Kingdom). TNF-α was purchased 6
from Biosource International Inc., (Camarillo, CA, USA). 7
8
2.2. Fungus material. 9
Freeze-dried powder of AC of the submerged whole broth (Batch No. MZ-247) was 10
provided by the Biotechnology Center of Grape King Inc., Chung-Li City, Taiwan, Republic 11
of China. 12
13
2.3. Isolation and Determination of the Active Compound. 14
Freeze-dried powder of AC of the submerged whole broth (1.6 kg) was extracted three 15
times with methanol (16 L) at room temperature (1 day each). The methanol extract was 16
evaporated in vacuo to give a brown residue, which was suspended in H2O (1 L), and then
17
partitioned (3 times) with 1 L of ethyl acetate. The EtOAc fraction (95 g) was 18
chromatographed on silica gel using mixtures of hexane and EtOAc of increasing polarity as 19
eluents and further purified with HPLC. ST1 (5.4 g) was eluted with 10% EtOAc in hexane, 1
and recrystallization with EtOH (Shao et al., 2008). 2
3
2.4. Animal Treatment. 4
6-8 weeks male imprinting control region (ICR) mice were obtained from the BioLASCO 5
Taiwan Co., Ltd. The animals were kept in plexiglass cages at a constant temperature of 22 6
±1°C, and relative humidity of 55 ± 5 % with 12 h dark-light cycle for at least 2 week before 7
the experiment. They were given food and water ad libitum. Animal studies were conducted 8
according to the regulations of the Instituted Animal Ethics Committee and the protocol was 9
approved by the Committee for the Purpose of Control and Supervision of Experiments on 10
Animals. Mice were randomly divided into six groups of six animals each (n = 6). Mice in the 11
normal control and negative control groups were administered with distilled water. The 12
positive control group was administered with silymarin (25 mg/kg in 1% carboxymethyl 13
cellulose about 10 mL, i.p.) once daily for 7 days. In the three experimental groups, the mice 14
were pretreated with ST1 (2.5, 5, and 10 mg/kg in 1% carboxymethyl cellulose, i.p.) once 15
daily for seven consecutive days. One hour after the last treatment, all the mice, except for 16
those in the normal control, were treated with CCl4 (1.5 mL/kg in olive oil, 20%, i.p.). 24 h
17
after the CCl4 treatment, animals were anesthetized with ethyl ether and blood samples were
collected through their carotid arteries. The mortality rate and body weight were recorded 1
daily. 2
3
2.5. Assessment of Liver Functions. 4
The blood was centrifuged at 1700×g (Beckman GS-6R, Germany) at 4°C for 30 min to 5
separate serum. ALT and AST were analyzed. Liver tissues collected from the animals were 6
fixed at 10% formalin for histopathological studies. Also, liver tissues were kept under -80°C 7
for further analysis of their enzyme levels. The biochemical parameters were analyzed by 8
using clinical test kits (Roche Cobas Mira plus, Germany). 9
10
2.6. Histopathological Examination. 11
Small pieces of liver, fixed in 10 % buffered formalin were processed for embedment in 12
paraffin. Sections of 4-5 μm were cut and stained with hematoxylin and eosin, and then 13
examined for histopathological changes under the microscope (Nikon, ECLIPSE, TS100, 14
Japan). Images were taken with a digital camera (NIS-Elements D 2.30, SP4, Build 387) at 15
original magnification of ×200. 16
2.7. Antioxidant Enzyme Activity Measurements. 1
The following biochemical parameters were analyzed to check the hepatoprotective 2
activity of ST1 by the methods given below. 3
Total superoxide dismutase (SOD) activity was determined by the inhibition of 4
cytochromec reduction (Flohe and Otting 1984). The reductionof cytochrome c was mediated 5
by superoxide anions generatedby the xanthine/xanthine oxidase system and monitored at 6
550 nm.One unit of SOD was defined as the amount of enzyme requiredto inhibit the rate of 7
cytochrome c reduction by 50%. Total catalase (CAT) activity estimation was based on that of 8
Aebi (Aebi, 1984). In brief, the reduction of 10 mM H2O2 in 20 mM of phosphate buffer (pH
9
7) was monitored by measuring the absorbance at 240 nm. The activity was calculated using a 10
molar absorption coefficient, and the enzyme activity was defined as nanomoles of dissipating 11
hydrogen peroxide per milligram protein per minute. Total GPx activity in cytosol was 12
determined as previously reported (Paglia and Valentine, 1967). The enzyme solution was 13
added to a mixture containing hydrogen peroxide and glutathione in 0.1 mM Tris buffer (pH 14
7.2) and the absorbance at 340 nm was measured. Activity was evaluated from a calibration 15
curve, and the enzyme activity was defined as nanomoles of NADPH oxidized per milligram 16
protein per minute. 17
18
2.8. Measurement of Hepatic GSH Level. 19
Hepatic GSH levels were determined by the method of Ellman with slight modification 1
(Ellman, 1959). Briefly, 720 L of the liver homogenate in 200 mM Tris-HCl buffer (pH 7.2) 2
was diluted to 1440 Lwith the same buffer. Five percent TCA (160 L) was added to it and 3
mixed thoroughly. The samples were then centrifuged at 10,000×g for 5 min at 4°C. 4
Supernatant (330 L) was taken in a tube and 660 L of Ellman's reagent (DTNB) solution 5
was added to it. Finally the absorbance was taken at 405 nm. 6
7
2.9. Lipid Peroxidation Intermediates. 8
Thiobarbituric acid reactive substances (TBARS), in particular malondialdehyde (MDA), 9
are products of the oxidative degradation of polyunsaturated fatty acids, Lipid peroxidation 10
was assayed by the measurement of MDA levels via absorbance at 535 nm on the basis of 11
MDA reacting with thiobarbituric acid, according to as previously reported (Tatum & Chow, 12
1996). Briefly, 0.4 mL of the treated cell or liver extract was mixed with 0.4 mL 13
thiobarbituric acid reagent (consisting of 0.4% thiobarbituric acid (TBA) and 0.2% butylated 14
hydroxytoluene (BHT). The reaction mixture was placed at 90°C water for 45 min, cooled, 15
added the equal volume of n-butanol, centrifuged and then the absorbance of the supernatant 16
was recorded at 535 nm. A standard curve was obtained with a known amount of 1, 1, 3, 17
3-tetraethoxypropane (TEP), using the same assay procedure. 18
1
2.10. Total Protein Assay. 2
Proteincontent in each sample was determined by a bicinchoninic acid (BCA)protein 3
assay kit (Pierce). 4
5
2.11. Measurement of Serum TNF-α Level by ELISA. 6
Serum levels of TNF- were determined using a commercially available enzyme linked
7
immunosorbent assay (ELISA) kit (Biosource International Inc., Camarillo, CA) according to 8
the manufacturer’s instruction. TNF- was determined from a standard curve. The 9
concentrations were expressed as pg/mL. 10
11
2.12. Measurement of Nitric Oxide/Nitrite Level. 12
NO production was indirectly assessed by measuring the nitrite levels in serum 13
determined by a calorimetric method based on the Griess reaction (Recknagel, Glende & 14
Britton, 1991). Serum samples were diluted four times with distilled water and deproteinized 15
by adding 1/20 volume of zinc sulfate (300 g/L) to a final concentration of 15 g/L. After 16
centrifugation at 10,000×g for 5 min at room temperature, 100 μL supernatant was applied to 1
a microtiter plate well, followed by 100 μL of Griess reagent (1% sulfanilamide and 0.1% 2
N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid). After 10 min of 3
color development at room temperature, the absorbance was measured at 540 nm with a 4
Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). By using sodium nitrite 5
to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 6
nm. 7
8
2.13. Western Blot Analysis. 9
Liver tissues were homogenized in lysis buffer (0.6% NP-40, 150 mM NaCl, 10 mM 10
HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF) at 4°C. Fifty micrograms of protein was 11
fractionated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes 12
(Millipore, Bedford, MA, USA). Membranes were incubated with primary antibodies 13
overnight at 4°C using 1:1000 dilution of goat polyclonal anti-rabbit iNOS, COX-2 and 14
-actin antibodies. The membranes were washed three times and the immunoreactive proteins 15
were detected by enhanced chemiluminescence (ECL) using hyperfilm and ECL reagent 16
(Amersham International plc., Buckinghamshire, U.K.). The results of Western blot analysis 17
were quantified by measuring the relative intensity compared to the control using Kodak 18
Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY) and 1
represented in the relative intensities. 2
3
2.14. Statistical Analysis. 4
All data were presented as mean ± standard deviation (SD) from three independent 5
experiments. Means of triplicates were calculated. Student’s t test was used for comparison 6
between two treatments. A difference was considered to be statistically significant when p < 7 0.05, p < 0.01 or p < 0.001. 8 9 3. Results 10
3.1. Effect of ST1 on hepatotoxicity in CCl4-treated mice.
11
Several hepatic enzymes in serum such as AST and ALT were used as the biochemical 12
markers for the early acute hepatic damage. The levels of AST and ALT were measured in the 13
serum to evaluate hepatic tissue damage (Fig. 1B and 1C). CCl4 administration resulted in
14
significant (p < 0.001) rise in the levels of AST and ALT when compared with the control 15
group. Intraperitoneal pre-administrations of ST1 at three different doses (2.5, 5 and 10 mg/kg) 16
significantly prevented the increased serum levels of ALT and AST. Silymarin (a 17
positive control) at a dose of 25 mg/kg also prevented the elevation of ALT and AST. In this
study, mice treated with CCl4 developed significant hepatic damage as manifested by a
1
significant increase in activities of AST and ALT that are indicators of hepatocyte damage
2
and loss of functional integrity.
3
4
3.2. Histopathology of the liver. 5
Fig. 2 showed that that CCl4 could induce histological changes including increased
6
degeneration, necrosis, hepatitis and portal triaditis. All mice except those in the control group 7
exhibited the ballooning degeneration in the centrolobular zone and the necrosis of 8
hepatocytes (Fig. 2). The CCl4-induced damage suffered more severely than other groups
9
pretreated with ST1. It seems likely that CCl4 administration cause oxidative stress in liver
10
via the generation of free radicals whereas ST1 ameliorates the liver injuries by scavenging of
11
free radicals, which is further confirmed by the reduced amount of histopathological injury.
12
13
3.3. Effect of ST1 on antioxidant enzymes activities in CCl4-treated mice liver.
14
The hepatic antioxidant enzyme activities (SOD, CAT and GPx) are shown in Fig. 3. 15
The activities of SOD, CAT and GPx were significant decreased in CCl4-treated mice,
16
comparing to the control. Mice pretreated with ST1 at 2.5, 5 and 10 mg/kg showed significant 17
increase in SOD when compared to CCl4 group. Mice pretreated with ST1 at 10 mg/kg
1
showed significant increase in CAT when compared to CCl4 group. Mice pretreated with ST1
2
at 10 mg/kg showed significant (p < 0.001) increase in GPx when compared to CCl4 group.
3
Silymarin-treated mice (The group of reference protective drug) also showed significant 4
increase in SOD, CAT, and GPx when compared to CCl4 treated mice.
5
6
3.4. Effect of ST1 on lipid peroxidation in CCl4-treated rat liver.
7
As shown in Fig. 4A, hepatic levels of TBARS were assessed as an indicator of lipid 8
peroxidation in the tissue. CCl4 treatment significantly increased the level of TBARS in the
9
liver. CCl4 alone treated mice was observed a significant increase (p < 0.001) in tissue
10
TBARS level. CCl4-induced elevation of tissue TBARS concentration was lowered
11
significantly by the i.p. pre-treatment of the mice with ST1. ST1 at 2.5, 5 and 10 mg/kg 12
significantly prevented the increase in TBARS level when compared to CCl4 group; Silymarin
13
also protected the liver from elevating TBARS levels and kept TBARS levels in normal 14
values. 15
16
3.5. Effect of ST1 on cellular GSH levels in CCl4-treated mice.
The CCl4-treatment caused significant (p < 0.001) decrease in the level of GSH in liver
1
homogenate when compared with control group (Fig. 4B). The pretreatment of ST1 at the 2
dose of 2.5, 5 and 10 mg/kg resulted in significant increase of GSH content when compared to 3
CCl4 treated mice. Silymarin (25 mg/kg) treated mice also showed significant (p < 0.001)
4
increase in GSH level in liver compared with CCl4 group.
5
6
3.6. Effect of ST1 on the serum level of TNF-α and NO in CCl4-treated mice.
7
As shown in Fig. 5A, the level of serum TNF-α was 87.45 ± 4.88 pg/mL in the control 8
group. The CCl4-treatment caused significant (p < 0.01) increase in the level of TNF-α in the
9
serum when compared with control group. The pretreatment of ST1 at the dose of 2.5, 5 and 10
10 mg/kg resulted in significant decrease of TNF-α level when compared to CCl4-treated mice.
11
Silymarin (25 mg/kg) treated mice also showed significant (p < 0.05) decrease in TNF-α level 12
in serum compared with CCl4-treated mice. As shown in Fig. 5B, the production of NO in
13
mice serum was significantly increased in CCl4-treated mice comparing to the control group.
14
However, pretreatment of ST1 reduced the NO production in CCl4-treated mice. For example,
15
NO production in the control group was 3.18 ± 0.34 μM, while it was 9.44 ± 0.79 μM with 16
CCl4 treatment. However, the NO production in the CCl4-treated mice was significantly (p <
17
0.001) decreased from 6.28 ±0.63, 4.66 ± 0.91 to 3.61± 0.66 μM with 2.5, 5, and 10 mg/kg 18
ST1 pretreatment, respectively. Silymarin (25 mg/kg) treated mice also showed significant (p 1
< 0.05) decrease of NO production in serum compared with CCl4 group.
2
3
3.7. Effect of ST1 on activities of iNOS and COX-2 in CCl4-treated mice liver.
4
We investigated the changes of the activation of iNOS and COX-2 by ST1 in 5
CCl4-treated mice (Fig. 6). The relative intensities of bands obtained from Western blot were
6
calculated with the use of the Kodak Molecular Imaging Software (Tokyo, Japan). The results 7
showed that CCl4 treatment stimulates to increase activation of iNOS and COX-2. For
8
example, in CCl4 treatment group, the relative intensity of iNOS and COX-2 band was
9
increased by 2.84- and 1.95-fold, compared to the control. However, the treatment of ST1 10
decreased the iNOS and COX-2 expression in CCl4-induced mice. Namely, the relative
11
intensities of bands about iNOS and COX-2 expressions were reduced by 1.24- and 1.16-fold 12
at 10 mg/kg of ST1, respectively, compared to CCl4 treatment alone.
13
14
4. Discussion 15
In this manuscript, we strongly speculated that ST1 can protect against diseases which
16
are caused by ROS, because it has a radical scavenging activity. The results of the present 17
study demonstrate that the pre-administration of ST1 effectively protected mice against 18
CCl4-induced acute liver damage. Administration of CCl4 to mice markedly increases serum
1
ALT and AST levels. This increase commonly reflects the severity of liver injury (Lin, Yao, 2
Lin, & Lin, 1996). The leakage of large quantities of enzymes into the blood stream was 3
associated with massive centrilobular necrosis, ballooning degeneration and cellular 4
infiltration of the liver. In the present work, substantial increases in serum ALT and AST were 5
observed after administration of CCl4, however, the increased levels of enzymes were
6
considerably reduced by pre-treatment with ST1, implying that ST1 tended to prevent 7
damage and suppressed the leakage of enzymes through cellular membranes. The dry matter 8
of fermented filtrate (DMF) from submerged cultures of AC and aqueous extracts from 9
fruiting bodies of AC have been reported to possess hepatoprotective activity against liver 10
diseases induced by CCl4 (Song & Yen, 2003). Both of them could reduce GSH-dependent
11
enzymes (GPx, GSH reductase and GSH-S-transferase), and the GSH/GSSG ratio was 12
significantly improved by the oral pretreatment of rats with DMF. Also, scientific research 13
showed that both the fruiting bodies and mycelia of AC possessed protective activity against 14
liver hepatitis and fatty liver induced by acute hepatotoxicity of alcohol (Dai et al., 2003). 15
Using acute ethanol-intoxicated rats as an experimental model, we compared the 16
hepatoprotective effects of AC, a traditional Chinese fungi drug for liver diseases, on liver 17
injury induced by ethanol (Liu et al., 2007). Treatment with AC notably prevented the 18
ethanol-induced elevation of levels of serum aspartate aminotransferase (AST), alanine 19
aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin to an extent that was 1
comparable to the standard drug silymarin. 2
Many studies have shown that the hepatoprotective effects may be associated with an 3
antioxidant capacity to scavenge reactive oxygen species (Sabis & Rocha, 2008). Antioxidant 4
enzymes (SOD, CAT and GPx) offer protection against oxidative tissue damage. Although in 5
the antioxidant paradox, CCl4 may cause oxidative stress and the consequent up-regulation of
6
antioxidant enzymes, to rendering cells more resistant to subsequent oxidative damage 7
(Halliwell & Gutteridge, 1990), we did not observed this phenomenon. Our results indicated 8
decreased SOD, CAT, and GPx levels in mice liver in response to CCl4 treatment, while ST1
9
pre-treatments kept levels of all three antioxidant enzymes (Fig. 3) to the normal ones. This 10
may be due to the inhibitory effects on cytochrome P450 and/or promotion of its
11
glucuronidation, both related to the early stage in CCl4-induced liver injury. GSH constitutes
12
the first line of defense against free radicals and is a critical determinant of the tissue 13
susceptibility to oxidative damage. It has been reported that GSH plays a key role in 14
detoxifying the reactive toxic metabolites of CCl4 and that liver necrosis begins when the
15
GSH stores are depleted (Williams, & Burk, 1990). In this study, CCl4 treatment decrease the
16
hepatic GSH levels and ST1 pre-treatments also restored the hepatic GSH levels to the normal 17
ones (Fig.4). The effect could be due either to the de novo synthesis of GSH, its regeneration, 18
or both. 19
The liver is a major inflammatory organ, and inflammatory processes contribute to a 1
number of pathological events after exposure to various hepatotoxins. Kupffer cells release 2
pro-inflammatory mediators either in response to necrosis or as a direct action by the 3
activated hepatotoxins, which are believed to aggravate CCl4-induced hepatic injury (Badger,
4
et al., 1996). TNF-α, a pleiotropic pro-inflammatory cytokine, is rapidly produced by 5
macrophages in response to tissue damage. While low levels of TNF-α may play a role in cell 6
protection, excessive amounts cause cell impairment. An increase in the TNF-α level has been 7
directly correlated with the histological evidence of hepatic necrosis and the increase in the 8
serum aminotransferase levels (Bruccoleri et al., 1997). DeCicco et al. (1998) have reported 9
the stimulation of TNF-α production in both serum and liver following CCl4 administration,
10
and it is suggested that CCl3· activates Kupffer cells to release TNF-α. TNF-α also stimulates
11
the release of cytokines from macrophages and induces the phagocyte oxidative metabolism 12
and nitric oxide production (DeCicco et al., 1998). Nitric oxide is a highly reactive oxidant 13
that is produced through the action of iNOS, and plays a role in a number of physiological 14
processes, such as, vasodilation, neurotransmission, and nonspecific host defense (Yen, Lai, & 15
Chou, 2001). Nitric oxide can also exacerbate oxidative stress by reacting with reactive 16
oxygen species, particularly with the superoxide anion and forming peroxynitrite. This study 17
confirmed a significant increase in the serum TNF-α protein expression after CCl4
18
administration. These alterations were attenuated by ST1 pre-treatment (Fig. 5), which 19
suggests that ST1 suppresses TNF-α protein secretion and/or enhances its degradation. 1
ST1 blocked the reduction of serum NO level in CCl4-treated mice. There are two
2
possible explanations for the observed decrease in NO levels after CCl4 treatment in our study.
3
First, gene expression of nitric oxide synthase (NOS) was reduced. Second, the NOS system 4
(enzyme protein, substrates, or cofactors) was damaged, thus decreasing the NO production. 5
Third, NO usage increased after CCl4 treatment. It is possible that another mechanism of
6
protective action of ST1 against CCl4- induced hepatotoxicity is due to the increased NO
7
production. Several studies have found that NO protected against CCl4-induced liver injury
8
using a NOS knockout mice or a NOS inhibitor (Wink, et al., 1996). The mechanism 9
underlying the protective effects of NO in CCl4-induced hepatotoxicity has not been
10
elucidated and may be related to its antioxidant properties. NO has also been shown to 11
interfere directly with the progression of lipid peroxidation, which may contribute to its 12
protective actions in the present work. NOis a short-lived signaling molecule capable of 13
regulating many physiological and pathological processes. Neuronal NOS (nNOS) and 14
endothelial NOS (eNOS) are constitutivelyexpressed; iNOS is triggered in many cell types by 15
cytokinessuch as TNF-α or interferon-γ. Endogenous NO, produced by an early and transient 16
activation of constitutive NOS, protects both hepatocytes and endothelial cells against 17
reperfusion injury in the liver (Liu et al., 2007). However, iNOS expression usually occurs 18
after inflammatory responses. INOS has been implicated as a mediator of cellular injuryat 19
sites of inflammation, including liver ischemia/reperfusion injury. Under this circumstance, 1
NO reacts with superoxide and generates reactive nitrogen species (ROS), thereafter 2
modifying bioorganic molecules. ROS leads to extracellular matrix (ECM)degradation and 3
the leukocyte migration across ECM proteins. 4
In conclusion, ST1 protects the liver from CCl4-induced oxidative stress and tissue
5
injuries, which might be due to the antioxidant properties and the inhibition of the 6
inflammatory response. It's possible role as a promising therapeutic in human oxidative stress 7
and inflammatory liver disease deserves consideration. The potential for using ST1 in 8
experimental designs and practical applications should be examined further. 9
10
Acknowledgements: 11
The authors want to thank the financial supports from the National Science Council 12
(NSC100-2313-B-039-004, NSC100-2815-C-039-026-B, and NSC100-2815-C-039-027-B), 13
China Medical University (CMU) (CMU100-SR-24 and CMU100-SR-25), Taiwan 14
Department of Health Clinical Trial and Research Center of Excellence 15
(DOH100-TD-B-111-004), and Department of Cancer Research Center of Excellence 16 (DOH100-TD-C-111-005) 17 18 References 19
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