Protective Effect Of JBP485 On Concanavalin A-induced Hepatocyte Toxcity In Primary Cultured Rat Hepatocytes

Objective: Hepatitis, an inflammatory liver disease, seriously harms human's health, among which, viral hepatitis is most frequently happened. Recently, autoimmune hepatitis tends to gain more attention. The common characteristic of viral hepatitis and autoimmune hepatitis is that immune factors play a key role. Because of the occurrence of hepatitis viral protein or autoantibody, hepatocytes are easily recognized by lymphocytes and then be killed. 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.Methods: Two in vitro models were established. Model I: primary cultured female rat hepatocytes were only incubated with Con A (50μg/ml); model II: co-culture system of hepatocytes and autologous splenic lymphocytes, both were stimulated with Con A (20μg/ml). JBP485 (25μM) was pre-incubated with the two models. The enzyme levels or cytokines in the supernatant were determined. DNA fragmentations were assayed by agarose electrophoresis and the expressions of caspase-3 and ICAM-1 were determined by RT-PCR and immunocytochemistry. Lymphocytes prolife- ration experiment was carried out to determine whether JBP485 can suppress lymphocytes proliferation.Results: 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, induced either by Con A directly or by the interaction between hepatocytes and lymphocytes, were attenuated in the JBP485 pre-incubated groups, and at the same time, immunocyto- chemistry 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), which is related to the interaction between hepatocytes and lymphocytes, was also down-regulated by this dipeptide. However, JBP485 showed no significant inhibitory effect on the lymphocytes proliferation.Conclusions: (1) JBP485 decreased the enzyme leakage and cytokines release induced by either Con A only or interaction between hepatocytes and lymphocytes; (2) JBP485 reduced DNA fragmentation and inhibited the expression and activity of caspase-3; (3) JBP485 down-regulated the expression of ICAM-1 induced by inflammatory cytokines but can not suppress lymphocytes proliferation;(4) JBP485 exhibited hepatoprotective effect through inhibition of hepatocytes apoptosis and ICAM-1 expression and may be useful for the therapeutic treatment of immune-mediated hepatitis.