Elsevier

Journal of Hepatology

Volume 34, Issue 4, April 2001, Pages 529-536
Journal of Hepatology

A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats

https://doi.org/10.1016/S0168-8278(00)00059-3Get rights and content

Abstract

Background: p160ROCK is a direct Rho target which mediates Rho-induced assembly of focal adhesions and stress fibers. We previously reported that Rho signaling pathways are involved in the activation of hepatic stellate cells (HSC) in vitro. The aim of the present study was to test the hypothesis that an inhibitor specific for p160ROCK (Y27632) could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats.

Methods: Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of α-smooth muscle actin (α-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver.

Results: Y27632 treatment significantly decreased the occurrence of DMN-induced hepatic fibrosis and reduced the collagen and hydroxyproline content and α-SMA expression in the liver. The expression of α-SMA in HSC was also suppressed in vitro.

Conclusions: These findings indicate that inhibitors of the Rho-ROCK pathway might be useful therapeutically in hepatic fibrosis.

Introduction

A small GTPase, Rho, is known to regulate the organization of the actin cytoskeleton by promoting the assembly of focal adhesions [1] and actin stress fibers [2]. In addition to its effects on the cytoskeleton, Rho is involved in regulating c-fos expression through the serum response element located in the promotor region [3], [4]. It is activated by a variety of growth factors in several systems, including the G protein-coupled receptor agonists lysophoaphatidic acid (LPA), bombesin, norepinephrine and endothelin-1 (ET-1) [5], [6], [7], [8].

Recently, several putative Rho target molecules have been isolated [9]. Among them, a family of Rho-associated serine/threonine kinase isozymes named p160ROCK [10] and ROKα/Rho-kinase/ROCK-II [11], [12], [13] has been identified as a new class of Rho effectors. They were able to induce focal adhesions and stress fibers in cultured fibroblasts and epithelial cells [5]. Kimura et al. [14] showed that myosin phosphatase was phosphorylated and inactivated in vitro by Rho-kinase. This led them to suggest that it thereby regulated myosin light chain (MLC) phosphorylation. Uehata et al. [15] recently identified a new pyridine derivative which acted as a specific inhibitor of the ROCK/ROK family of protein kinases and has been designated Y27632. This compound inhibited smooth muscle contraction both in vitro and in vivo, as well as the formation of stress fibers and focal adhesions induced by p160ROCK in cultured cells. These findings suggested that ROCK mediates the effect of Rho on the formation of focal adhesions and stress fibers at least partly through the regulation of the actomyosin system. We previously reported that RhoA is present in activated hepatic stellate cells (HSC) and that Rho signaling pathways play an important role in the activation of HSC [16], [17]. Our previous studies also indicated that Y27632 reduces cell growth and type I collagen production in rat HSC [18]. Therefore, we assayed the therapeutic effects of Y27632 on rat hepatic fibrosis induced by dimethylnitrosamine (DMN) [19].

Section snippets

Animals and reagents

Male Wistar Kind A rats (200–250 g) (Laboratory of Animal Experiments, Kyushu University) were used in this experiment. Y27632 was donated by Yoshitomi Pharmaceutical Industries, Saitama, Japan. l-Alanine aminotransferase (ALT) activity in the serum was assayed by a GPT-OA test (Wako, Tokyo, Japan). All other chemicals and reagents were of analytic grade and were obtained from standard commercial sources.

Treatment of animals

DMN (1 g/ml) (Sigma, St. Louis, MO) was diluted ten times with saline (final concentration

Y27632 prevents body and liver weight loss induced by DMN

The effect of oral administration of Y27632 on body and liver weights of rats with and without i.p. injection of DMN is shown in Fig. 1 and Table 1. Treatment with DMN caused a significant decrease in rat body and liver weight (DMN-S group) compared with control animals (S-S group). Oral Y27632 essentially prevented this DMN-induced rat body and liver weight loss (DMN-Y group). There was no significant difference between the S-S/S-Y and DMN-Y groups. There was no significant difference in serum

Discussion

In the present study we have demonstrated a beneficial effect of Y27632 on hepatic fibrosis in the DMN induction model in rats. The mechanism by which this new compound provides significant protection against hepatic fibrosis is not completely clear. However, Y27632 seems to attenuate HSC activation directly. This is important because the HSC are the major cellular source of ECM in hepatic fibrosis and are transformed into myofibroblast-like cells in disease, which then specifically express

Acknowledgments

This work was supported by a Grant-in Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan (10670485).

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