Extract
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Introduction
Bacterial lipopolysaccharide (LPS) is a toxic component
of cell walls of Gram-negative bacteria and is widely present
in the digestive tracts of humans and
animals[1]. Humans are constantly exposed to low levels of LPS through infection.
Gastrointestinal distress and alcohol consumption often
increase the permeability of LPS from the gastrointestinal tract
into the blood[2]. In humans, nanograms of LPS injected into
the blood stream can result in the physiological
manifestations of septic shock[3]. The liver is a highly responsive organ
in systemic inflammation caused by LPS. Hepatic dysfunction
after sepsis is a frequent event that is characterized by the loss
of synthetic function and hepatocellular
necrosis[4].
D-Galactosamine (GalN) is an amino sugar selectively
metabolized by the hepatocyte, which induces a depletion
of the uridine triphosphate (UTP) pool and thereby an
inhibition of RNA synthesis[5]. When given together with a low
sublethal dose of LPS, GalN highly sensitizes animals to
develop lethal liver injury mimicking fulminant
hepatitis[6]. GalN/LPS-induced liver injury is characterized by apoptosis
of hepatocytes, widespread destruction of liver architecture,
and erythrocyte agglutination. In the GalN/LPS model,
TNF-α is the major mediator leading to apoptotic liver
injury[7]. Nitric oxide may also play a role in GalN/LPS-induced
apo-ptotic liver injury[8,9]. Recent studies showed that hydrogen
peroxide and reduced glutathione depletion sensitize primary
mouse hepatocytes to TNF-α-induced
apoptosis[10_12]. Furthermore, rosmarinic acid, an exogenous antioxidant,
protects against D-GalN/LPS-induced hepatic
apoptosis[13], suggesting that reactive oxygen species (ROS) may be involved
in TNF-α-mediated apoptotic liver injury.
N-acetylcysteine (NAC) is a glutathione (GSH)
precursor and direct antioxidant. As a potent antioxidant, NAC
directly scavenges hydrogen peroxide, hydroxyl free radicals,
and hypochloric acid in
vitro[14]. NAC also decreases free
radical levels by increasing GSH
synthesis[15,16]. Several
studies have indicated that NAC inhibits LPS-induced
inducible nitric oxide synthase, TNF-α expression, and
NF-κB activity[17,18]. Our earlier studies showed that pretreatment
with NAC prevents the LPS-induced downregulation of the
pregnane X receptor and P450 3A11 expression in mouse
liver, placenta, and fetal liver[19_21]. Furthermore,
pretreatment with NAC protects mice against LPS-induced
intrauterine fetal death and intrauterine growth
retardation[22]. Recently, we found that pretreatment with NAC attenuates
acute ethanol-induced liver damage in
mice[23]. Clinically, NAC has been successfully used in adult respiratory
distress syndrome[24]. In the present study, we investigated
the effect of NAC on LPS-induced apoptotic liver damage in
GalN-sensitized mice. Our results showed that NAC
protects mice against GalN/LPS-induced apoptotic liver injury
via its antioxidant and anti-apoptotic effects.
Materials and methods
Chemicals LPS (Escheria coli LPS, serotype 0127:B8),
GalN, DL-buthionine-(SR)-sulfoximine (BSO), NAC, and other
reagents were purchased from Sigma Chemical Co (St Louis,
MO, USA) unless otherwise stated.
Animals and treatments Female CD-1 mice (6_8
week-old, 24_26 g) were purchased from Beijing Vital
River Laboratory Animal Co Ltd (Beijing, China) whose foundation
colonies were all introduced from Charles River Laboratories
(Wilmington, USA, MA) The animals were allowed free
access to food and water at all times and were maintained on
a 12 h light/dark cycle in a controlled temperature (20_25 °C)
and humidified (50%±5%) environment for a period of 1 week
before the experiments. All animal procedures followed the
guidelines for humane treatment set by the Association of
Laboratory Animal Sciences and the Center for Laboratory
Animal Sciences at Anhui Medical University (Hefei, China).
To investigate the protective effects of NAC, the mice
were administered with NAC (150 mg/kg, ip) 30 min before
GalN/LPS (700 mg/10 µg/kg, ip). The doses of NAC used in
the present study referred to those used in previous
studies[25]. The control mice received saline. Some mice were killed 1.5 h
after GalN/LPS administration. Serum was collected for the
measurement of TNF-α. The remaining mice were killed 8 h
after GalN/LPS. Serum was collected for the measurement of
alanine aminotransferase (ALT) and nitrate plus nitrite. The
livers were dissected for the measurement of GSH content,
caspase-3 activity, DNA extraction, and histological
examination.
Evaluation of hepatotoxicity Serum ALT activity was
assayed as a marker of hepatotoxicity using a commercially
available kit (Nanjing Jiancheng Institute of Biological
Engineering and Technology, Nanjing, China) according to the
manufacturer's instructions. A portion of liver was fixed in
10% formalin, processed by standard histological
techni-ques, attained with hematoxylin-eosin (HE), and examined
for morphological evaluation of liver injury.
DNA fragmentation analysis The liver tissues were
homogenized and incubated in 100 mmol/L Tris-HCl (pH 8.0),
25 mmol/L EDTA, 0.5% SDS, and 0.1 µg/mL proteinase K at
60 °C for 3 h. DNA was extracted with phenol/chloroform
(1:1) and chloroform/isoamyl alcohol (1:24).
The extracted DNA was precipitated and digested in 10 mmol/L
Tris-HCl (pH 5.0) containing 1 mmol/L EDTA and 10 µg RNase for 1 h at 37 °C.
5 µg DNA per sample was electrophoretically separated on
1.5% agarose gel containing 0.5 µg/mL ethidium bromide.
The DNA pattern was examined by UV transillumination.
Determination of caspase-3 activity The hepatic
caspase-3 activity was determined based on a colorimetric
method[26]. Briefly, the liver homogenates were prepared in lysis buffer
containing 100 mmol/L HEPES
(N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid), pH7.5, 20%
(v/v) glycerol, 0.5 mmol/L EDTA, and 5 mmol/L dithiothreitol. The protease assay
mixture included 240 µL reaction buffer (100 mmol/L HEPES, pH
7.5, 20% glycerol, 0.5 mmol/L EDTA, and 5 mmol/L
dithio-threitol), 30 µL of 1 mmol/L
acetyl-Asp-Glu-Val-Asp-p-nitroanilide in DMSO, and 30 µL liver homogenates. The
samples were incubated at 37 °C for 1 h, and the
enzyme-catalyzed release of p-nitroanilide was measured at 405 nm in
an ultra-microplate reader (Bio-Tek instruments, Winnoski,
Vermont, USA). Caspase-3 activity was expressed as
nmol/mg protein. The protein content was measured according to
the method of Lowry et al[27].
Determination of GSH content GSH was determined
based on the method of
Griffith[28]. Proteins of 0.4 mL liver
homogenates were precipitated by the addition of 0.4 mL of
a metaphosphoric acid solution. After 40 min, the protein
precipitate was separated from the remaining solution by
centrifugation at 4200×g at 4 °C for 5 min. 400 µL of the
supernatant was combined with 0.4 mL of 300 mmol/L
Na2HPO4, and the absorbance at 412 nm was read against a
blank consisting of 0.4 mL supernatant plus 0.4 mL
H2O. Then, 100 µL DTNB (5,5-dithio-bis-2-nitrobenzoic acid)
(0.02% [w/v], 20 mg DTNB in 100 mL of 1% sodium citrate)
was added to the blank and sample. The absorbance of the
sample was read against the blank at 412 nm. The GSH
content was determined using a calibration curve prepared with
an authentic sample. The GSH values were expressed as
nmol/mg protein. The protein content was measured
according to the method of Lowry et
al[27].
Analysis of serum nitrite plus nitrate
concentration The stable end products of L-arginine-dependent nitric oxide
synthesis and nitrate plus nitrite, were measured in the
serum using a colorimetric method based on the Griess
reaction[29]. Briefly, aliquots of serum were added to 35%
sulfosalicylic acid and vortexed every 5 min for 30 min to
depro-teinize the samples. The samples were then centrifuged at 10
000×g at 4 °C for 15 min.
An aliquot of the supernatant was taken for the nitrite plus
nitrate analysis. Twenty microliters of the serum sample was mixed
with 20 µL of 0.31 mol/L phosphate buffer, pH7.5, 10 µL of 0.1 mmol/L FAD (flavin adenine
dinucleotide), 10 µL of 1 mmol/L NADPH, 10 mL of nitrate
reductase (10 U/mL), and 30 µL of
ddH2O in a 96-well plate. The reaction was
allowed to proceed for 1 h in dark. To each
sample, 1 µL of lactate dehydrogenase
(1500 U/mL) and 10 µL of 100 mmol/L pyruvic acid were added
and incubated for 15 min at 37 °C. The samples were then mixed
with an equivalent volume of Griess reagent and
incubated for an additional 10 min at room temperature. Nitrite levels
were determined colorimetrically at 550 nm with a Universal microplate reader
(Bio-Tek Instruments, Winnoski, Vermont, USA) and a
sodium nitrite standard curve.
Measurement for TNF-α Serum TNF-α levels were
measured by ELISA (R&D, Minneapolis, MN, USA), following
the manufacturer's instructions.
Statistical analysis Quantified data were expressed as
mean±SEM. ANOVA and the Student-Newman-Keuls
post-hoc test were used to determine differences between the
treated animals and the control and statistical significance.
Results
Effects of NAC on GalN/LPS-induced liver
injury To investigate the effects of NAC pretreatment on
GalN/LPS-induced apoptotic liver injury, the mice were pretreated with
NAC before GalN/LPS. The results showed that neither GalN
nor LPS alone is lethal at low concentrations. Co-injection
of GalN and LPS into mice resulted in a lethal liver injury
mimicking fulminant hepatitis, in which 6 of the 10 mice died
within 12 h. Serum aspartate ALT activity was markedly
increased at 8 h after GalN/LPS (Table 1). A histological
examination showed massive necrosis of parenchymal
hepatocytes with marked hemorrhage in the liver of
GalN/LPS-treated mice (Figure 1E). Pretreatment with NAC significantly
attenuated GalN/LPS-induced elevation of serum ALT
activities. In addition, GalN/LPS-induced histological injury
was obviously improved in NAC-pretreated mice (Figure 1F).
To investigate the role of GSH on NAC-mediated protection
against GalN/LPS-induced liver injury, the mice were
pretreated with BSO, an inhibitor of GSH synthesis, to inhibit
hepatic GSH synthesis. The results showed that the
protective effects of NAC pretreatment on GalN/LPS-induced liver
injury were not counteracted by BSO (Table 1; Figure 1H).
Effects of NAC on GalN/LPS-induced hepatic apoptosis
Hepatic apoptosis was clearly confirmed in the
GalN/LPS-treated mice, as indicated by DNA laddering on agarose gel
electrophoresis (Figure 2, lanes 5 and 6). NAC pretreatment
significantly attenuated GalN/LPS-induced hepatic DNA
fragmentation (Figure 2, lanes 3 and 4). The effects of NAC
pretreatment on hepatic caspase-3 activities in the
GalN/LPS-treated mice were further analyzed. Hepatic caspase-3
activity was markedly increased in the GalN/LPS-treated mice
(Table 1). Consistent with the attenuation of DNA laddering,
NAC pretreatment significantly inhibited hepatic caspase-3
activities in the GalN/LPS-treated mice. To investigate the
role of GSH on NAC-mediated protection against
GalN/LPS-induced hepatic apoptosis, the mice were pretreated with
BSO, an inhibitor of GSH synthesis, to inhibit hepatic GSH
synthesis. The results showed that the inhibitive effects of
NAC pretreatment on hepatic caspase-3 activity were not
counteracted by BSO. In addition, BSO did not influence
the protective effects of NAC pretreatment on
GalN/LPS-induced hepatic DNA fragmentation (Figure 2, lanes 1
and 2).
Effects of NAC on LPS-induced GSH depletion
The
effects of NAC on LPS-induced hepatic GSH depletion are
presented in Table 1. The results showed that GalN/LPS
dramatically decreased hepatic GSH content. Pretreatment
with NAC significantly attenuated hepatic GSH depletion.
By contrast, BSO aggravated GalN/LPS-induced hepatic GSH
depletion.
Effects of NAC on serum TNF-α in GalN/LPS-treated
mice In the GalN/LPS model, TNF-α is the major mediator
leading to liver injury[7]. To investigate the effects of NAC
pretreatment on TNF-α production, the mice were pretreated
with NAC 0.5 h before GalN/LPS. A preliminary study showed
that TNF-α was at peak value at 1.5 h and returned to basal
level 6 h after GalN/LPS treatment (data not shown).
Therefore, serum TNF-α concentration was measured 1.5 h
after GalN/LPS administration. As expected, there was a
significant increase in serum TNF-α level in the GalN/LPS-treated
mice (Table 2). However, pretreatment with NAC did not
decrease the elevation of serum TNF-α level in the
GalN/LPS-treated mice.
Effects of NAC on LPS-induced NO production
To
investigate the effect of NAC on LPS-induced nitric oxide
production in the GalN-sensitized mice, the stable end
products of L-arginine-dependent nitric oxide synthesis and
nitrate plus nitrite, were measured at 8 h after GalN/LPS. There
was a significant increase for serum nitrate plus nitrite levels
in the GalN/LPS-treated mice (Table 2). However,
pretreatment with NAC had no effect on LPS-evoked elevation of
nitric oxide levels.
Discussion
GalN depletes UTP primarily in the liver, resulting in
decreased RNA synthesis in
hepatocytes[5]. When given together with a low sublethal dose of LPS, GalN highly
sensitizes animals to develop lethal liver injury mimicking
fulminant hepatitis[6]. In the present study, we showed that
co-injection of GalN and LPS into mice produced fulminant
hepatitis with severe hepatic congestion, resulting in rapid
death. Serum ALT levels were markedly increased 8 h after
GalN/LPS, and massive necrosis of parenchymal hepatocytes
with marked hemorrhage was observed in the histological
sections of the liver from the GalN/LPS-treated mice. NAC is
an antioxidant and a GSH precursor. A previous study
showed that pretreatment with NAC attenuated organ
dysfunction during endotoxemia and protected against
LPS-induced liver injury[30]. In the present study, we investigated
the effects of pretreatment with NAC on LPS-induced liver
injury in GalN-sensitized mice. We found that pretreatment
with NAC significantly reduced serum ALT levels in
GalN/LPS-treated mice. In parallel, NAC pretreatment significantly
attenuated GalN/LPS-induced hepatic necrosis and
conges-tion.
GalN/LPS-induced liver injury is characterized by
apoptosis of hepatocytes. A previous study showed that
treatment with YVAD-CMK (Acetyl-Tyr-Val-Ala-Asp-chloromethy- lketone), a potent tetrapeptide inhibitor of the
interleukin (IL)-1β conterting enzyme family, protects from
LPS-induced apoptotic liver injury in GalN-sensitized
mice[31]. In the present study, hepatic apoptosis was clearly
confirmed in the GalN/LPS-treated mice, as indicated by strong
DNA laddering on agarose gel electrophoresis. Moreover,
co-injection of GalN and LPS significantly increased
caspase-3 activity in the mouse liver. NAC is an anti-apoptotic
mediator[32,33]. The present study showed that NAC pretreatment
significantly inhibited hepatic caspase-3 activity in
GalN/LPS-treated mice. NAC pretreatment significantly
attenuated GalN/LPS-induced hepatic DNA fragmentation. These
results indicate that the protective effect of NAC
pretreatment against GalN/LPS-induced apoptotic liver injury might
be, at least in part, mediated by its anti-apoptotic effects.
In the GalN/LPS model, TNF-α is the major mediator of
liver injury[7]. Several studies have demonstrated that NAC
significantly inhibits the LPS-induced release of
TNF-α in Kupffer cells[15,34_36]. In the present study, we investigated
the effect of NAC pretreatment on GalN/LPS-induced
TNF-α production. As expected, LPS significantly increased serum
TNF-α level in GalN-sensitized mice. However, the present
study showed that the LPS-induced elevation of the
TNF-α level was not significantly reduced by NAC pretreatment.
Therefore, the present study does not determine whether
NAC-mediated protection against GalN/LPS-induced liver
injury is due to the inhibition of TNF-α production.
A recent study indicated that ROS are involved in
GalN-induced sensitization against TNF-α-induced hepatocyte
apoptosis[37]. Concomitant TNF-α exposure and ROS, either
extrinsically generated by non-parenchymal or inflammatory
cells or intrinsically generated in hepatocytes, may act in
concert to promote apoptosis and liver
injury[10]. Moreover, rosmarinic acid, an exogenous antioxidant, protects against
D-GalN/LPS-induced hepatic
apoptosis[13]. In addition,
reduced GSH depletion has also been demonstrated to
sensitize primary mouse hepatocytes to TNF-α-induced
apoptosis[11,12]. NAC is not only a direct antioxidant, but
also a GSH precursor. NAC acts as a free radical scavenger,
directly scavenges hydrogen peroxide, hypochloric acid, and
hydroxyl radical. On the other hand, NAC acts as a
precursor of GSH to facilitate intracellular GSH
synthesis[14]. In the present study, we analyzed the effects of NAC on hepatic
GSH content in GalN/LPS-treated mice. As expected,
pretreatment with NAC significantly attenuated
GalN/LPS-induced hepatic GSH depletion. However, BSO, an inhibitor
of GSH synthesis, did not influence NAC-mediated
protection against GalN/LPS-induced apoptotic liver injury,
although GalN/LPS-induced hepatic GSH depletion was
aggravated by BSO in NAC-pretreated mice. These results
suggest that the NAC-mediated protection against
GalN/LPS-induced apoptotic liver injury is not attributed to
increased GSH synthesis, but most likely due to its strong
ROS-scavenging effect.
Nitric oxide plays an important role in GalN/LPS-induced
apoptotic liver injury[8,9]. Several studies demonstrated that
NAC inhibits LPS-evoked inducible nitric oxide synthase
expression and nitric oxide release in
macrophages[17,38]. In the present study, we investigated the effects of NAC
pretreatment on nitric oxide production. We found that serum
nitrate plus nitrite, the stable end products of
L-arginine-dependent nitric oxide synthesis, significantly increased at
8 h after GalN/LPS administration. However, NAC
pretreatment had no effect on GalN/LPS-evoked nitric oxide
production. These results suggest that the protective effect
of NAC pretreatment against GalN/LPS-induced apoptotic
liver injury is not be mediated by the inhibition of nitric oxide
release.
In summary, the present study indicated that
pretreatment with NAC has a beneficial effect on GalN/LPS-induced
apoptotic liver injury. The protective effect of NAC
pretreatment may be mediated by its strong ROS-scavenging
and anti-apoptotic effects.
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