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.
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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
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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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