Carbon tetrachloride (CCl₄)-induced liver injury in a range of laboratory animals is regarded as an analogue of the liver damage caused by various hepatotoxins in humans (Muriel, 1998). Involvement of bioactivation by a microsomal cytochrome P450-dependent monooxygenase system, resulting in formation of trichloromethyl free radicals, leading to hepatocellular damage in hepatic necrosis caused by CCl₄ has been suggested (Morio et al., 2001). D-Galactosamine (GalN) is a well-established hepatotoxicant that induces a diffuse type of liver injury closely resembling human viral hepatitis (Aristatile et al., 2009). GalN-induced liver injury reduces the intracellular pool of uracil nucleotides in hepatocytes, thus inhibiting synthesis of RNA and proteins (Siendones et al., 2004). α-Naphthylisothiocyanate (ANIT) is a hepatotoxicant that damages biliary cells and hepatocytes; therefore, it is used experimentally in rodents as a model of intrahepatic cholestasis (Xu et al., 2004). ANIT is detoxified by glutathione conjugation within hepatocytes and the complex of ANIT-glutathione is secreted into bile. However, the ANIT-glutathione conjugate dissociates rapidly in bile, leading to release of the parent compound, and exposes biliary epithelial cells to toxic concentrations of ANIT, thus causing hepatobiliary toxicity (Ramaiah and Jaeschke, 2007).

Moutan Cortex Radicis (MCR), the root cortex of Paeonia suffruticosa Andrews (Paeoniaceae), is a well-known traditional herbal medicine used in treatment of cardiovascular and female genital diseases (Hirai et al., 1983; Sakamoto et al., 1992). MCR removes heat from the blood, promotes blood circulation, and removes blood stasis (Chun et al., 2007). MCR has potent analgesic, sedative, anti-inflammatory, and anti-microbial properties (Jiang et al., 2007). Furthermore, MCR has a protective effect on acetaminophen-induced hepatotoxicity in mice (Shon and Nam, 2004). Paeonol, a major phenolic component of MCR, has recently been shown to alleviate alcoholic liver injury via inhibition of hepatic steatosis and inflammation (Hu et al., 2010).

As shown in Table 1, 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 Figure 1A 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 (Figure 1B). These alterations were significantly attenuated by 300 mg/kg of MCR, showing mild hepatocellular necrosis and inflammation (Figure 1C). These results suggest that MCR may have potential clinical applications for treatment of liver disorders.

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 (Table 2). The histological features shown in Figure 1D 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 (Figure 1E). These pathological changes were attenuated by 300 mg/kg of MCR (Figure 1F). Taken together, our results suggest that MCR has a potential for use as a therapeutic agent for treatment of acute hepatitis.

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 (Figure 2A). 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 (Figure 2B). These results indicate that MCR may offer protection to hepatocytes and bile duct epithelial cells against acute liver cholestasis.