Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by
carbon tetrachloride in rats.
Hitoshi Ikeda1, 2, Yukio Kume1, Kazuaki Tejima1, 2, Tomoaki Tomiya2, Takako
Nishikawa2, Naoko Watanabe2, Natsuko Ohtomo2, Masahiro Arai3, Chihiro Arai1,
Masao Omata2, Kenji Fujiwara4, Yutaka Yatomi1
1Department of Laboratory Medicine and
2Department of Gastroenterology, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
3Toshiba Hospital, 6-3-22 Higashiooi, Shinagawa-ku, Tokyo 140-8522, Japan
4Yokohama Rohsai Hospital, 3211 Kodukue-chou, Koh-hoku-ku, Yokohama-shi,
Kanagawa 222-0036, Japan
Running head: Rho-kinase inhibitor and CCl4
Corresponding author: Hitoshi Ikeda, M.D., Ph.D.
Department of Laboratory Medicine, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, JAPAN
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Articles in PresS. Am J Physiol Gastrointest Liver Physiol (August 30, 2007). doi:10.1152/ajpgi.00210.2007
Copyright © 2007 by the American Physiological Society.
Phone 81-3-5800-8730, Fax 81-3-5689-0495
E mail: email@example.com
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A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention.
Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride
(CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion
injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis, but also
reduced serum alanine aminotransferase level in CCl4-induced liver fibrosis, we
wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect.
We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor,
HA-1077, reduced serum alanine aminotransferase level in rats with acute liver injury
induced by CCl4 with the improvement of histological damage and the reduction of the
number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077
reduced apoptosis evaluated by quantitativedetermination of cytoplasmic
histone-associated DNA oligonucleosome fragments with the reduction of caspase 3
activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation
of Akt, and wortmannin, an inhibitor of PI3-kinase/Akt pathway, abrogated the
reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin
abrogated the reduction of serum alanine aminotransferase level by HA-1077 in rats
with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt
Page 3 of 40
pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in
vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver
injury induced by CCl4 in rats, and merits consideration as a hepatocyte-protective agent
in liver injury, considering its direct anti-apoptotic effect on hepatocytes in vitro.
Key words: HA-1077, fasudil, Y-27632, Rho
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Rho-kinase plays an important role in many downstream effects of the small
GTP-binding protein, Rho. Rho-kinase was originally identified as a regulator of actin
cytoskelton organization and thereby mediates cell adhesion, motility and contractility
(12, 16, 17). Since its discovery, there has been growing evidence that Rho-kinase may
also be involved in regulating various gene expressions and signaling pathways, which
suggests that Rho-kinase mediates other cellular functions such as proliferation or
Rho-kinase inhibition is now known to be useful for the treatment of ischemic
disease including cerebral vasospasm after subarachnoid hemorrhage, angina pectoris or
pulmonary hypertension based on the inhibitory action on cell contractility (1, 33, 34).
Furthermore, recent accumulating evidence reveals that Rho-kinase inhibition improves
various kinds of diseases besides the ischemic disease; Rho-kinase inhibition attenuates
angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by
inhibiting apoptosis and proteolysis (43), aldosterone-induced renal injury in rats (38),
or development of diabetes and nephropathy in insulin-resistant diabetic rats (15).
Regarding liver damage, the administration of Rho-kinase inhibitor leads to
improvement of liver fibrosis in rats induced by carbon tetrachloride (CCl4) (21, 22) or
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dimethylnitrosamine (39). This improvement of liver fibrosis by Rho-kinase inhibitor
has been explained by its effects on hepatic stellate cells; inhibition of cell contraction
(13, 45) and migration (41, 45), and stimulation of apoptosis (10). Of interest, however,
in the experiment to determine the effect of Rho-kinase inhibitor on CCl4-induced liver
fibrosis, not only the improvement of liver fibrosis, but also the reduction of serum ALT
level were reported, where the authors speculated a direct action of Rho-kinase inhibitor
to prevent the hepatocyte damage caused by CCl4(21). To examine this possibility, we
investigated whether Rho-kinase inhibitor could prevent the hepatocyte damage in
acutely CCl4-intoxicated rats.
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Materials and Methods
Animals. Male Sprague-Dawley rats (Shizuoka Laboratory Animal Center,
Shizuoka, Japan) were given a standard pellet diet and water ad libitum, and used in all
experiments. All animals received humane care and the experimental protocol was
approved by Animal Research Committee of the University of Tokyo.
Materials. HA-1077 was purchased from BIOMOL Research Laboratories.
Anti-Bcl-2, anti-Bcl-x, anti-Akt and anti-phospho-Akt (Ser472/473/474) were obtained
from BD Biosciences, anti-Bax (N-20), and anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (V-18) from Santa Cruz Biotechnology.
Acute liver injury in rats. Rats, weighing 140-180 g, were randomized into the
following treatment groups: (1) vehicle; (2) HA-1077 (10 mg/kg body weight) (44); (3)
CCl4 (3.0 mL/kg body weight as a 20% solution in olive oil) (7); (4) CCl4 + HA-1077;
(5) CCl4 + wortmannin (15 µg/kg body weight) (44); (6) CCl4 + HA-1077 +
wortmannin. CCl4 was administered subcutaneously, and HA-1077 and wortmannin
were administered intraperitoneally (44) at the same time of CCl4 injection.
Determination of liver function. Blood samples were collected from rats
through the inferior vena cava at 24 hours after administrations of CCl4, HA-1077 or
wortmannin. The serum level of alanine aminotransferase (ALT) was determined using
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an automated analyzer (Hitachi 7170; Hitachi Instruments Service Co., Ltd., Tokyo,
Histological examination. Liver histology was studied in formalin-fixed and
paraffin embedded specimens of the liver stained with haematoxylin and eosin by a
light microscope (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan) with
magnifications 100?, 200? and 400?, and a digital camera (Nikon DXM1200F).
Cells. Hepatocytes were isolated from rats weighing 140-180 g by in situ
perfusion of the liver with type I collagenase (29). The isolated cells were seeded at a
density of 5 × 104 cells/cm2 and cultured in Williams' medium E (ICN Biomedicals Inc.,
Costa Mesa, CA) with 10% fetal calf serum (GIBCO, Grand Island, NY), 0.9 µM
dexamethasone (Takeda Pharmaceutical Industries, Osaka, Japan) and 10 µM Actrapid
MC insulin (Novo Industri A/S, Copenhagen, Denmark). At 2 hours after seeding, the
medium was replaced with serum- and hormone-free Williams' medium E containing
0.2 µg/mL aprotinin (Sigma Chemical Co)(24).
TdT-mediated dUTP nick-end labeling (TUNEL) staining. For the liver
specimen, sections 3 µm thick were collected on glass slides coated with poly-L-lysine,
and apoptotic cells were detected using ApopTag Peroxidase In Situ Apoptosis
Detection Kit (Chemicon). The number of TUNEL-positive cells was determined as the
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mean in five different areas at 400-fold magnification in each section. For the cultured
hepatocytes, the cells at day 1 on Lab Tek slides (4 wells) were incubated with HA-1077
for 24 hours. Apoptotic cells were detected by an in situ cell death detection kit (Roche
Applied Science) using the manufacturer’s instructions.
Cytoplasmic histone-associated DNA fragments assay. Cultured hepatocytes at
day 1 were incubated with various concentrations of HA-1077 for 24 hours. Cell Death
Detection ELISA (Roche Applied Science) was used to quantitatively determine
cytoplasmic histone-associated DNA fragmentsassociated with apoptotic cell death.
Caspase 3 activity assay. Caspase 3 activity was measured using a colorimetric
CaspACE kit (Promega, Madison, WI) according to the manufacturer’s instructions.
Briefly, after homogenization of liver tissue in cell lysis buffer (100 mg/mL),
homogenates were centrifuged for 20 min at 10,000 g, and the supernatant was used for
the measurement of caspase 3 activity. Caspase 3 activity was also measured in the
cultured hepatocytes at day 1, which were incubated with HA-1077 for 24 hours.
Immunoblot analysis. After experimental treatment, the medium was discarded,
and hepatocytes were incubated in 50 mM Tris pH 7.5, 250 mM NaCl, 2 mM EDTA,
0.5% Nonidet P-40, 50 mM NaF, 0.1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 2 µg/mL
leupeptin, 2 µg/mL pepstatin and 2 µg/mL aprotinin. Samples containing the same
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amount of protein were separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis under reducing conditions, which were transferred to a sheet of
polyvinylidene difluoride membrane (Amersham). To block nonspecific binding, the
membrane was soaked in blocking agent derived from skim milk (Blockace; Snow
Brand Milk Product Co., Ltd., Sapporo, Japan) for 1 hour at room temperature. Then, it
was incubated with primary antibody (dilution 1:1000, antibodies used: Bcl-2, Bax, Akt,
phospho-Akt, and dilution 1:2000, antibodies used: GAPDH) overnight at 4°C. The
membrane was incubated with horseradish peroxidase-conjugated secondary antibody
(dilution 1:1000) for 1 hour at room temperature. Immunoreactive proteins were
visualized using a chemiluminescence kit (Amersham), and recorded in a
chemiluminescence recording system (LAS 1000; Fuji-film, Tokyo, Japan).
Statistical analysis. When indicated, statistical analysis was performed by
Student's t-test, and P < 0.05 was considered significant.
Page 10 of 40
Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced
by CCl4. To determine whether Rho-kinase inhibition could prevent hepatocyte damage
in CCl4-intoxicated rats, we employed the model of acute liver injury induced by CCl4;
3.0 mL/kg CCl4 was administered in rats with or without Rho-kinase inhibitor,
HA-1077 at 10 mg/kg body weight, and serum ALT level and liver histology were
examined at 24 hours after the administration. Because HA-1077 at 10 mg/kg body
weight was used to show that inhibition of Rho-kinase leads to cardiovascular
protection in rats in vivo (44), the equivalent dose of HA-1077 was examined in rats in
this study. As demonstrated in Figure 1, serum ALT level was increased to 593 ± 174
IU/L (mean ± SEM, n=8) by CCl4 administration, whereas it was significantly reduced
to 82 ± 14 IU/L by cotreatment with HA-1077 (P < 0.05, mean ± SEM, n=8). On the
other hand, serum ALT level of untreated rats was 46 ± 5 IU/L (mean ± SEM, n=3), and
the administration of HA-1077 alone did not alter serum ALT level (38 ± 2 IU/L, mean
± SEM, n=3). The ballooning of hepatocytes is one of the earliest, most frequent and
most conspicuous changes seen in the liver injured by CCl4(31). Histological analysis
showed that the cotreatment with HA-1077 reduced this change of ballooning of
hepatocytes (Figure 2A and 2B).
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Because hepatocyte damage has been attributed to apoptosis in addition to
necrosis in acute liver injury induced by CCl4(31), we examined apoptotic cells in the
liver of CCl4-intoxicated rats with or without treatment of HA-1077. Figure 2C, 2D and
2E depict TUNEL-positive cells in the livers of CCl4-intoxicated rats with or without
treatment of HA-1077. TUNEL-positive cells appeared less apparent in the livers of
CCl4-intoxicated rats without HA-1077 treatment (Figure 2D) compared to those with
HA-1077 treatment (Figure 2C); TUNEL staining was noted in the ballooned
hepatocytes in the CCl4-intoxicated livers without HA-1077 treatment (Figure E). As
shown in Figure F, HA-1077 significantly reduced the number of TUNEL-positive cells
in the livers of CCl4-treated rats (P < 0.01, n = 3). Because caspase 3 plays a key role in
various forms of apoptosis, we investigated whether this enzyme activity could be
altered in the CCl4-intoxicated livers with HA-1077 treatment. As demonstrated in
Figure 2G, HA-1077 treatment significantly reduced caspase 3 activity in the
CCl4-intoxicated livers (P < 0.01, n = 3).
Rho-kinase inhibitior reduces hepatocyte apoptosis in vitro with activation of
PI3-kinase/Akt pathway. Next, we examined if Rho-kinase inhibitor could directly
prevent hepatocyte damage using rat hepatocytes in vitro. Rat hepatocytes in primary
culture do not survive for long periods of time in the absence of serum, unless
Page 12 of 40
extracellular matrices, growth factors and/or coculture are employed (8, 30, 36). It has
been shown that aprotinin, a protease inhibitor, is capable of maintaining viability of rat
primary hepatocytes in culture for over two days (2, 8). We used this relatively simple
serum-free culture system with aprotinin alone to check a possible direct action of
Rho-kinase inhibitor on hepatocytes. First, we checked the status of the cells cultured
with serum-free medium with 0.2 µg/mL aprotinin. Although aprotinin did maintain
hepatocytes for over 2 days, the significant amount of the cells cultured in the medium
with aprotinin alone underwent apoptosis compared with those cultured in the medium
with fetal calf serum; histone-associated DNA fragmentation was detected 1.6-fold
more in the cells cultured with aprotinin alone than those with fetal calf serum (data not
shown). Then, we examined the effect of HA-1077 on hepatocyte apoptosis using this
serum-starved culture system with aprotinin; rat hepatocytes in culture (day 1) were
treated with HA-1077 for 24 hours and the fragmentation of DNA into
oligonucleosomal lengths was examined by TUNEL technique. The treatment with 30
µM HA-1077 appeared to decrease the number of cells containing fragmented DNA
(Figure 3B) compared with the untreated cells (Figure 3A), and we quantitatively
determined cytoplasmic histone-associated DNA oligonucleosome fragmentsassociated
with apoptotic cell death. The treatment with HA-1077 reduced histone-associated DNA
Page 13 of 40
fragmentation in a concentration-dependent manner, as shown in Figure 3C. At a
concentration of 10 µM, HA-1077 significantly reduced histone-associated DNA
fragmentation by 25%, and 30 µM by 35%. HA-1077 at 30 µM was used to examine the
effect of Rho-kinase inhibition on endothelial cells (44) and 10 µM HA-1077 was
specific to inhibit Rho-kinase in smooth muscle cells (23). We then investigated whether
caspase 3 activity could be altered in the reduction of DNA fragmentation by HA-1077.
When the cells were treated with 30 µM HA-1077 for 24 hours, caspase 3 activity was
significantly reduced as demonstrated in Figure 4A. These results suggest that
Rho-kinase inhibition may increase hepatocyte survival cultured.
Next, possible mechanism by which Rho-kinase inhibition reduces hepatocyte
apoptosis was examined by quantifying any associated changes in cellular content of
proteins that enhance or inhibit apoptosis of Bcl-2 family. As shown in Figure 4B,
immunoblot analysis of Bcl-2, Bax or Bcl-x extracted from hepatocytes showed that in
the presence of 30 µM HA-1077 Bcl-2 expression was increased, whereas Bax or Bcl-x
expression was not altered. Thus, it is likely that Bcl-2 plays some role in the reduction
of hepatocyte apoptosis by Rho-kinase inhibition.
Because PI3-kinase/Akt is considered a key factor for cell apoptosis (6), we
examined a possible involvement of activation of PI3-kinase/Akt pathway in the
Page 14 of 40
HA-1077-induced survival in cultured hepatocytes. As shown in Figure 5, a sharp
increase in the level of phosphorylation of Akt was determined at 15 minutes after the
addition of 30 µM HA-1077. Then, we examined an effect of wortmannin, the
PI3-kinase/Akt inhibitor, on the reduction of apoptosis by Rho-kinase inhibition. As
depicted in Figure 6 in the presence of 30 nM wortmannin, the reduction of apoptosis
by 30 µM HA-1077 was abrogated. These results suggest that the activation of
PI3-kinase/Akt pathway is involved in the mechanism of the reduction of apoptosis by
PI3-kinase/Akt inhibitor abrogates the preventive effect of Rho-kinase inhibitor
on hepatocyte damage in acute liver injury induced by CCl4. Because the activation of
PI3-kinase/Akt pathway was found to be involved in the anti-apoptotic effect of
Rho-kinase inhibitior on hepatocytes in vitro, we next examined the significance of
activation of PI3-kinase/Akt pathway in the hepatocyte protection by Rho-kinase
inhibitor in CCl4-induced acute liver injury in vivo; an effect of wortmannin, 15 µg/kg
body weight, on this acute liver injury model by CCl4 was determined in the presence or
absence of HA-1077. Wortmannin at 15 µg/kg body weight was previously used to
inhibit PI3-kinase/Akt pathway in rats in vivo (44). As shown in Figure 1, wortmannin
abrogated the reduction of serum ALT level caused by cotreatment with HA-1077 (322
Page 15 of 40
± 114 IU/L, mean ± SEM, n = 8). It is also notable that wortmannin alone did not alter
serum ALT level in acute liver injury model by CCl4 (545 ± 333 IU/L, mean ± SEM, n =
8). This result indicates that the activation of PI3-kinase/Akt pathway is necessary for
the hepatocyte protection by Rho-kinase inhibitor in CCl4-induced acute liver inlury.
Page 16 of 40
The current study indicates that Rho-kinase inhibitor prevents hepatocyte
damage in acute liver injury induced by CCl4 administration in rats, where histological
analysis revealed the reduction of the number of apoptotic cells with reduced caspase 3
activity in the liver. Thus, as speculated previously (21), the reduction of serum ALT in
CCl4-induced liver fibrosis in rats by Rho-kinase inhibitor may be mediated at least in
part by its direct protective effect on hepatocytes. In the course of clarifying the
mechanism underlying this preventive effect of Rho-kinase inhibitor on hepatocyte
damage, we have found that Rho-kinase inhibitor inhibits directly apoptosis of
hepatocyts in rats cultured in serum-free condition.
The reports showing the significance of Rho/Rho-kinase pathway in
hepatocytes have been scarce. Unlike fibroblasts or smooth muscle cells, no apparent
changes in cell shape or cell adhesion were detected in cultured rat hepatocytes with the
addition of the stimulator or inhibitor of Rho or Rho-kinase (not shown), which might
suggest that the role of Rho/Rho-kinase would be minor in hepatocytes. However,
GTP-bound RhoA, the active form of Rho, is expressed in cultured rat hepatocytes,
though it is relatively small amounts, and furthermore, Rho activation is involved in the
regulation of growth factor-induced DNA synthesis of those cells (11), suggesting that
Page 17 of 40
Rho/Rho-kinase pathway might play some role in hepatocytes. In line with this
speculation, we have found that Rho-kinase inhibition leads to reduction of apoptosis in
cultured rat hepatocytes in serum-free condition. This was found to quantitatively
determine cytoplasmic histone-associated DNA fragmentsassociated with apoptotic cell
death and was confirmed by reduction of caspase 3 activity and enhanced expression of
Bcl-2. Activation of Akt by Rho-kinase inhibition seems important to this finding,
because a specific inhibitor of PI3-kinase/Akt pathway, wortmannin, abrogated the
protective effect of Rho-kinase inhibition on apoptosis of cultured hepatocytes.
As to the contribution of Rho/Rho-kinase pathway to apoptosis, it has been first
discovered that Rho/Rho-kinase pathway is activated during the execution phase of
apoptosis to stimulate apoptotic membrane blebbing (4, 19, 28). Then, the reports of
participation of Rho/Rho-kinase pathway on not only the execution phase but also
initial phase of apoptosis have been accumulating (18, 25, 26, 32), in which there has
been a distinct effect. Rho-kinase inhibition enhanced apoptosis in rat smooth muscle
cells (32) or human endothelial cells (18), and in contrast Rho-kinase inhibition reduced
apoptosis in mouse myocytes (3) or mouse vascular wall (43). We demonstrated that
Rho-kinase inhibition enhanced apoptosis in rat hepatic stellate cells in the previous
study (10), and in the current study we have found that Rho-kinase inhibition reduced
Page 18 of 40
apoptosis in rat cultured hepatocytes induced by serum-free condition. The positive or
negative regulation of cell survival by Rho-kinase inhibition seems to depend on cell
types. The mechanism to explain this distinct effect should be further clarified.
Besides the improvement of liver fibrosis (21, 22, 39), it is reported that the
administration of Rho-kinase inhibitor leads to reduction of hepatic
ischemia-reperfusion injury in rats (7, 9, 14, 20, 35, 40) and improvement of septic liver
injury in mice (42). These effects are explained by the facts that Rho-kinase inhibitor
suppresses infiltration of polymorphonuclear leukocytes, production of inflammatory
cytokines (14, 35, 40, 42) or generation of reactive oxygen species (35), and improves
microcirculatory disruption through inhibition of hepatic stellate cell contraction (20).
Because the mechanism of CCl4-induced liver injury involves inflammatory cytokines
(5) or reactive oxygen species (37), the prevention of CCl4-induced acute liver injury by
Rho-kinase inhibitor in the current study may be explained by the same mechanism i.e.
the suppression of inflammatory cytokines or reactive oxygen species. However, the
finding that wortmannin cancelled the hepatocyte-protective effect of Rho-kinase
inhibitor in CCl4-induced acute liver injury (Figure 1) suggests that an anti-apototic
effect of Rho-kinase inhibitor on hepatocytes might operate at least in part, considering
the in vitro finding that Akt activation is a key to Rho-kinase inhibitor-induced
Page 19 of 40
anti-apoptosis in hepatocyte. Partial cancellation of Rho-kinase inhibitor-induced
hepatocyte protection by wortmannin may be in line with this speculation.
In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver
injury induced by CCl4 in rats, and merits consideration as a hepatocyte-protective agent
in liver injury due to not just CCl4 but other insults, considering its direct anti-apoptotic
effect on hepatocytes in vitro.
Page 20 of 40
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Figure 1. Effect of HA-1077 on serum ALT level in rats treated with CCl4 in the
presence or absence of wortmannin. Rats were randomized into the following treatment
groups: (1) vehicle; (2) HA-1077 (10 mg/kg body weight); (3) CCl4 (3.0 mL/kg body
weight); (4) CCl4 + HA-1077; (5) CCl4 + wortmannin (15 µg/kg body weight); (6)
CCl4 + HA-1077 + wortmannin. HA-1077 and wortmannin were administered
intraperitoneally at the same time of CCl4 subcutaneous injection. Twenty-four hours
after the injections rats were sacrificed and serum ALT level was determined. Columns
and bars represent the means ± SEM of 8 samples (3 samples for untreated control and
HA-1077 alone). An asterisk indicates a significant difference from the rats treated with
CCl4 alone (P < 0.05). N.S., not significant.
Figure 2. Effect of HA-1077 on liver histology and TUNEL staining on liver sections in
CCl4-treated rats. Rats received CCl4 (3.0 mL/kg body weight) injection with or without
HA-1077 treatment (10 mg/kg body weight), and were sacrificed 24 hours later.
Representative photomicrographs of liver histology stained with haematoxylin and
eosin and TUNEL staining on liver sections in rats treated with CCl4 alone (A, C, E)
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and combination of CCl4 and HA-1077 (B, D) are shown. Bars; A, B, C and D, 100 µm;
E, 50 µm. The number of TUNEL-positive cells was determined as the mean in five
different areas at 400-fold magnification (high power field; HPF) in each section (F).
Caspase 3 activity in the CCl4-intoxicated livers with or without HA-1077 treatment
was measured using a colorimetric CaspACE kit (G). Columns and bars represent the
means ± SD of three rats. An asterisk indicates a significant difference from the rats
treated with CCl4 alone (P < 0.01).
Figure 3. Effect of HA-1077 on DNA fragmentation in cultured rat hepatocytes.
Hepatocytes (day 1) were incubated with various concentrations of HA-1077 up to 30
µM for 24 hours. The cells cultured with (B) or without (A) 30 µM HA-1077 were
examined for containing fragmented DNA by TUNEL technique using an in situ cell
death detection kit. Representataive photographs of TUNEL staining are shown. Bars; A,
B, 200 µm. (C) Histone-associated DNA fragmentation was determined using a cell
death detection enzyme-linked immunosorbent assay kit. The results are expressed as
fold increase compared to the untreated control. Each value represents the mean ± SD of
three experiments. At a concentration of 10 µM and 30 µM, HA-1077 significantly
reduced histone-associated DNA fragmentation (P < 0.05).
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Figure 4. Effect of HA-1077 on caspase 3 activity and Bcl-2, Bax or Bcl-x expression in
cultured rat hepatocyts. Hepatocytes were cultured with 30 µM HA-1077 for 24 hours.
Caspase 3 activity was determined using a colorimetric CaspACE kit. Columns and bars
represent the means ± SD of three experiments, and an asterisk indicates a significant
difference from the untreated control (P < 0.01) (A). Bcl-2, Bax or Bcl-x expression in
the cells was determined by immunoblotting. GAPDH expression was also analyzed as
a control for protein loading. Representataive immunoblot of three experiments is
Figure 5. Effect of HA-1077 on Akt expression in cultured rat hepatocytes. Hepatocytes
were incubated with 30 µM HA-1077 for various periods of time up to 2 hours. Then,
Akt, or phosphorylated Akt expression in the cells was determined by immunoblotting.
GAPDH expression was also analyzed as a control for protein loading. Representataive
immunoblot of three experiments is shown.
Figure 6. Effect of wortmannin on reduction of histone-associated DNA fragmentation
by HA-1077 in cultured rat hepatocytes. Hepatocytes were incubated with 30 µM
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HA-1077 in the presence or absence of 30 nM wortmannin for 24 hours.
Histone-associated DNA fragmentation was determined using a cell death detection
enzyme-linked immunosorbent assay kit. The results are expressed as fold increase
compared to the untreated control. Columns and bars represent the means ± SD of three
experiments. An asterisk indicates a significant difference from the untreated control
cells (P < 0.01). N.S., not significant.
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