Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes

https://doi.org/10.1016/j.ejphar.2008.04.066Get rights and content

Abstract

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide isolated from Laennec, and Laennec is a hydrolyzate of human placenta. Evidence has indicated that JBP485 exhibits potent anti-hepatitis activity. In this study, we investigated the protective effect and possible mechanisms of action of JBP485 in Concanavalin A (Con A)-induced hepatotoxicity in vitro. Two in vitro models were established. Model I: primary cultured female rat hepatocytes were only incubated with Con A (50 μg/ml); model II: co-culture system of hepatocytes and autologous splenic lymphocytes, both were stimulated with Con A (20 μg/ml). JBP485 (25 μM) was pre-incubated with the two models. Our results showed that JBP485 reduced cellular aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) leakage following the application of Con A in both of the models. Potential protective mechanisms were elucidated by measuring DNA fragmentations, immunocytochemistry and RT-PCR. We showed that DNA fragmentations in hepatocytes were attenuated in the JBP485 pre-incubated groups, and at the same time, immunocytochemistry and RT-PCR indicated that expression levels of caspase-3 protein and mRNA in the JBP485 treated groups were decreased compared with those in the untreated groups. Moreover, intercellular adhesion molecule-1 (ICAM-1) was also down-regulated by this dipeptide. The results indicate that JBP485 exhibits hepatoprotective effect through inhibition of hepatocyte apoptosis and ICAM-1 expression.

Introduction

Hepatitis is an inflammatory liver disease induced by various causes, such as virus infection, alcohol, autoimmune response, drugs or xenobiotics injury, etc. Although many unsolved problems still remain in the related mechanisms, it is generally accepted that activated T cells are involved in both acute and chronic hepatitis (Napoli et al., 1996, Chisari, 1997, Lapierre et al., 2007). Cytokines secreted by lymphocytes dominantly modulate the immune reaction in the liver, which ultimately results in liver dysfunction, hepatic cirrhosis, and even liver failure. Accordingly, several types of drugs have been developed to treat hepatitis, e.g. interferon and purine analog for viral hepatitis, prednisone and azathioprine for the autoimmune chronic hepatitis. In addition, silymarin and glycyrrhizin have frequently been used as adjunct liver-protective drugs (Ye and Lu, 2004). However, a new compound, cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485, Fig. 1) has been developed. Liu et al. (2000) were the first to report that JBP485 exhibited potent anti-hepatitis activity. JBP485, a dipeptide, was first isolated from Laennec (a trade name for the hydrolyzate of human placenta), as mitogens for a baby hamster kidney cell line, and was synthesized by chemical means (Yagi et al., 1998). Laennec injection has been clinically used to treat chronic hepatic injuries for over 40 years in Japan. Earlier results showed that Laennec stimulated liver regeneration and decreased cytosolic enzyme activities in serum in α-naphthylisothiocyanate (ANIT)-intoxicated rats (Liu et al., 1995). It is noteworthy that JBP485 was well absorbed after oral administration. Liu et al. (2000) suggested that it was possible that JBP485 was recognized by the peptide transporter system in the gastrointestinal tract. Therefore, it is valuable to clarify the anti-hepatitis molecular mechanism of JBP485 to develop a new oral anti-hepatitis drug.

Con A is a powerful immunostimulant (Taniguchi et al., 1989) and inflammogen (Shier, 1976) in vivo, when given intravenously to mice without pre-treatment by a specific hepatotoxic agent such as d-galactosamine. Con A-induced selective liver failure, which is characterized by polyclonally activating T cells, and follows the systemic release of cytokines (Tiegs et al., 1992, Gantner et al., 1995, Küsters et al., 1996). As previously reported, Con A bound strongly to the hepatocyte plasma membrane, which correlated well with the degree of hepatotoxicity induced by Con A (Leist and Wendel, 1996), resulting from the fact that Con A-binding not only induced a direct toxic effect on primary cultured hepatocytes independent of the presence of T cells but enhanced the susceptibility of hepatocytes to activated autologous lymphocytes (Yoshifumi et al., 1996). Both activation of lymphocytes and Con A-binding to hepatocytes are essential for hepatic cytotoxicity. Hepatocytes are first sensitized by Con A or even killed by Con A at a high concentration, and then interact with polyclonally activated T cells, which results in cell apoptosis or even necrosis. The interaction between hepatocytes and lymphocytes is mainly mediated by ICAM-1/lymphocyte function associated antigen (LFA-1) interaction (Yoshifumi et al., 1996). To a certain extent, the pathogenesis of this hepatitis model is similar to human immune-mediated hepatitis, such as autoimmune and viral hepatitis. Therefore, the hepatitis model induced by Con A may have implications for the development of new treatment options.

Here we studied the effect of JBP485 on Con A-induced hepatic cytotoxicity and its possible mechanism in established in vitro models. Our findings demonstrated that JBP485 showed a potent hepatoprotective effect through inhibition of hepatocyte apoptosis and ICAM-1 expression.

Section snippets

Animals

Wistar rats (from the Experimental Animal Center of Dalian Medical University, Dalian, China) were treated in accordance with local institutional guidelines for the care and use of laboratory animals.

Reagents

Con A and collagenase were purchased from Wako (Japan); JBP485 was provided by Japan Bioproducts Industry Co. Ltd (Tokyo, Japan). Before use, Con A and JBP485 were dissolved and adjusted to the required concentration with William's E medium (Hyclon, USA) free of serum. Anti-rat caspase-3 antibody

Concentration-dependent hepatocyte toxicity induced by Con A

To examine whether cytotoxicity induced by Con A in primary cultured rat hepatocytes showed a concentration-dependent effect, the cell survival rate was measured by MTT assay after 24 h exposure to Con A. Con A in itself did not have any cytotoxic effect at a concentration of 20 mg/ml within 24 h (Fig. 2A). The threshold concentration of Con A-induced toxicity was 25 μg/ml (≈ 0.1 μM) (Fig. 2A). The IC50 value of Con A was 60.79 μg/ml, and the concentration of Con A was 50 μg/ml when the cell

Discussion

We examined the effect of JBP485 on hepatic cytotoxicity and the possible molecular mechanisms. Two cytotoxicity models were used in our experiments in order to evaluate their hepatoprotective activities in vitro. When cultured hepatocytes were pre-treated with JBP485, both inflammatory reaction and apoptosis-associated events were significantly suppressed. Furthermore, our results also indicated that JBP485 inhibits ICAM-1 expression in hepatocytes, which provided a hint as to the involvement

Acknowledgments

The work was supported by a grant from the National Natural Science Foundation of China (No. 30672498) and the Liaoning Natural Science Fund (No. 2004225003-6). The authors would like to thank the entire staff of the Department of Clinical Pharmacology in Dalian Medical University for their kind help. Special thanks go to Professor Sun Changkai from the Department of Physiology in Dalian Medical University for providing the fluorescence micro-digital imaging system.

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