In this study, the protective effect of MCR was examined using a model of CCl4
-, GalN, and ANIT-induced hepatotoxicity. CCl4
, a classic hepatotoxin, caused acute, reversible liver injury characterized by centrilobular necrosis (Son et al., 2007
). Hepatotoxicity of CCl4
is thought to involve two phases. The first phase consists of activation by cytochrome P450 2E1, 2B1, or 2B2 in the liver to form a highly reactive trichloromethyl radical and then a trichloromethyl peroxy radical (Yamaji et al., 2008
). These radicals can attack membrane phospholipid stimulating lipid peroxidation and cell lysis. This damage to the structural integrity of the liver was observed from elevated serum levels of hepatospecific enzymes, i.e. AST and ALT (Aktay et al., 2000
). The second phase is caused by the inflammatory response, which involves activation of Kupffer cells, probably by free radicals. Activation of Kupffer cells is accompanied by production of proinflammatory mediators (Chen et al., 2004
As shown in , serum ALT activity in the control group averaged 25.5 ± 1.2 U/L. However, the value in the vehicle-treated CCl4 group showed a dramatic increase (1983.0 ± 28.6 U/L, P < 0.01), indicating severe hepatocellular damage. Treatment with MCR, at doses of 30, 100, and 300 mg/kg resulted in a markedly attenuated increase in ALT activity to approximately 66.3%, 54.3%, and 37.3% of that in the vehicle-treated CCl4 group, respectively. Consistent with the ALT data, the serum level of AST increased significantly from 46.4 ± 6.7 U/L to 1764.0 ± 158.1 U/L, and this increase was reduced by 30, 100, and 300 mg/kg of MCR. Histological observation of liver samples strongly supported the release of aminotransferases by damaged hepatocytes, as well as the protective effect of MCR. The histological features shown in demonstrated normal liver lobular architecture and cell structure in the control group. In contrast, the vehicle-treated CCl4 group exhibited various histological changes to the liver, including cell necrosis, fatty metamorphosis in adjacent hepatocytes, ballooning degeneration, cell inflammation, and infiltration of lymphocytes and Kupffer cells (). These alterations were significantly attenuated by 300 mg/kg of MCR, showing mild hepatocellular necrosis and inflammation (). These results suggest that MCR may have potential clinical applications for treatment of liver disorders.
Effect of MCR on serum aminotransferase activities in mice after CCl4 administration
Figure 1 Histological features of liver sections stained with hematoxylin and eosin at 24 h after CCl4 (A–C) or GalN (D–F) exposure. Typical images were chosen from each experimental group (original magnification ×400). (A): The control (more ...)
In terms of both morphological and functional aspects, GalN-induced acute injury in rat livers is well-established and recognized as resembling viral hepatitis in humans (Decker and Keppler, 1972
). The toxic effect of GalN is connected with an insufficiency of UDP-glucose and UDP-galactose, as well as loss of intracellular calcium homeostasis. These changes affect cell membranes and organelles, as well as synthesis of proteins and nucleic acids (Devaki et al., 2009
). Recent studies have demonstrated that GalN can induce hepatic hypoxia/hypoperfusion and trigger production of reactive oxygen species from affected hepatocytes, infiltrated leukocytes, and activated Kupffer cells and enhance mitochondrial apoptosis-and the proinflammatory cytokine-signaling pathway, contributing to oxidative stress and inflammation in the liver (Lin et al., 2009
In the present study, serum ALT activity in the control group was 44.4 ± 1.0 U/L. The vehicle-treated GalN group showed a significant increase in serum ALT activity at 24 h after GalN injection (235.7 ± 28.6 U/L, P < 0.01). In contrast, treatment with MCR at doses of 30, 100, and 300 mg/kg attenuated the increase in ALT activity to approximately 71.6%, 66.1%, and 62.9% of that in the vehicle-treated GalN group, respectively. The serum level of AST also showed a significant increase from 81.5 ± 0.8 U/L to 664.6 ± 41.7 U/L, and this increase was reduced by 30, 100, and 300 mg/kg of MCR (). The histological features shown in demonstrate normal liver lobular architecture and cell structure in the control group. However, livers exposed to GalN showed multiple and extensive areas of portal inflammation and hepatocellular necrosis, randomly distributed throughout the parenchyma, as well as a moderate increase in inflammatory cell infiltration (). These pathological changes were attenuated by 300 mg/kg of MCR (). Taken together, our results suggest that MCR has a potential for use as a therapeutic agent for treatment of acute hepatitis.
Effect of MCR on serum aminotransferase activities in rats after GalN administration
The key function of the liver is to synthesize, concentrate, and secrete bile acids and to excrete other toxicants, such as bilirubin. ANIT induces bile duct epithelial cell necrosis, followed by cessation of bile flow, and consequent hyperbilirubinemia (Hasegawa et al., 2008
). Due to its dose-dependent effects and high reproducibility between studies, ANIT is a typical hepatotoxin used in the study of intrahepatic cholestasis (Hasegawa et al., 2008
). This drug provokes an acute cholestatic hepatitis, due in part to its recycling through repeated rounds of glutathione conjugation and biliary excretion (Kodali et al., 2006
). The drug is initially detoxified in hepatocytes by conjugation with glutathione (Xu et al., 2004
). Instability of the ANIT-glutathione conjugate and recycling rounds of ANIT metabolism result in high ANIT concentration in bile (Luyendyk et al., 2009
). This causes bile duct epithelial cell damage and formation of foci of hepatic necrosis characterized by dead hepatocytes (Luyendyk et al., 2009
According to our results, serum TBIL concentration was 0.077 ± 0.027 mg/dL in the control group. However, the vehicle-treated ANIT group showed significantly increased serum TBIL concentration by approximately 8.6 times that of the control group. Treatment with MCR at doses of 100 and 300 mg/kg attenuated an increase in TBIL concentration to approximately 62.5% and 36.1% of that in the vehicle-treated ANIT group, respectively (). Bile flow in the control group was 2.249 ± 0.252 mL/min/g liver. In the vehicle-treated ANIT group, bile flow showed a significant decrease (0.379 ± 0.023 mL/min/g liver, P < 0.01); however, treatment with MCR, at doses of 30, 100 and 300 mg/kg attenuated this decrease in bile flow (). These results indicate that MCR may offer protection to hepatocytes and bile duct epithelial cells against acute liver cholestasis.
Figure 2 Effect of MCR on serum total bilirubin (A) and bile flow (B) in ANIT-induced acute hepatotoxicity. The values are represented as mean ± S.E.M. for 8–10 rats per group. ** Denotes significant differences (P < 0.01) compared with (more ...)