Hepatocyte Injury Induced By Hcv Infection

During the course of a chronic HCV infection, hepatocytes are continuously damaged and replicated, and hepatic fibrosis appears to progress, whereas the frequencies of genetic alteration also probably increase. It is generally accepted that multiple genetic alteration, induced by mutations, is an important factor in carcinogenesis. Therefore, continuous cell death and replication and multiple genetic alteration may lead to the development of cirrhosis and HCC (Fig. 2). However, the mechanisms by which HCV induces liver injury and hepatocyte death remain, to a great extent, ambiguous.

Cytotoxic T lymphocytes (CTLs) are not only thought to be a major host defense against viral infection, but have been implicated in immuno-pathogenesis as well. Two pathways, the perforin and Fas/Fas ligand pathways, have been proposed to account for all cytolytic activity of CTLs (11), although tumor necrosis factor-a (TNF-a), a proinflammatory cytokine, is released by all HCV-specific CTL clones studied to date (12) and may also contribute to the cytotoxicity of CTLs (13). The core protein of HCV has been recognized as a target for CTLs (14-16). Viral proteins have various accessory functions that target host proteins and thus alter normal cellular growth properties, affecting many signal pathways. A few examples of signaling molecules, such as the nuclear factor-nB (NF-kB) and activator protein 1 (AP-1), have been reported to be activated by HCV proteins. The NF-kB pathway plays an important role in the cellular response to a variety of extracellular stimuli, including TNF-k and interleukin-1 (IL-1). Because the HCV core protein binds both the TNF receptor and p53 (17,18), induction of NF-kB by HCV proteins may either cause or suppress hepatocyte death and, on other occasions, may promote cellular proliferation, thus contributing to liver injury or tumorigenesis induced by HCV. Likewise, the activation of AP-1, a transcription factor that regulates a number of genes involved in the control of cellular growth, by viral proteins presumably would promote oncogenesis and a myriad of other effects. Our preliminary study demonstrated that prooxidants induce the activation both of AP-1 and NF-kB with

Figure 2 A working hypothesis regarding the role of ROS generation in stimulating hepatic fibrogenesis and carcinogenesis. Black boxes indicate the putative suppression site by Sho-saiko-to. ROS, reactive oxygen species; NO, nitric oxide; MDA, malondialdehyde; TGF, transforming growth factor; PDGF, platelet-derived growth factor; HSC, hepatic stellate cell.

Figure 2 A working hypothesis regarding the role of ROS generation in stimulating hepatic fibrogenesis and carcinogenesis. Black boxes indicate the putative suppression site by Sho-saiko-to. ROS, reactive oxygen species; NO, nitric oxide; MDA, malondialdehyde; TGF, transforming growth factor; PDGF, platelet-derived growth factor; HSC, hepatic stellate cell.

degradation of InB-n, an inhibitory subunit of NF-nB, in cultured rat hepatocytes, and that Sho-saiko-to inhibited the oxidative-stress-induced activation of NF-nB and AP-1 in a dose-dependent manner (Fig. 3). In addition, oxidative stress was observed to induce early apoptosis, by decreasing the expression of antiapoptotic proteins Bcl-2 and Bcl-XL, and by increasing the expression of proapoptotic protein Bad in cultured rat hepa-tocytes, whereas Sho-saiko-to was found to suppress oxidative-stress-induced

Figure 3 Sho-saiko-to prevents oxidative stress-induced activation of AP-1 (a) and NF-nB (b) by blocking the degradation of InB-a (c) in cultured rat hepatocytes in a dose-dependent manner. Cells were incubated with 100 Amol/L of ferric nitrilotriacetate solution (oxidative stress) in the presence or absence of Sho-saiko-to at the indicated dose. Whole-cell extracts were measured for DNA-binding activities of AP-1 and NF-nB by electrophoretic mobility shift assay, and analyzed by Western blotting using the antibody of InB-a, an inhibitory subunit of NF-nB.

Figure 3 Sho-saiko-to prevents oxidative stress-induced activation of AP-1 (a) and NF-nB (b) by blocking the degradation of InB-a (c) in cultured rat hepatocytes in a dose-dependent manner. Cells were incubated with 100 Amol/L of ferric nitrilotriacetate solution (oxidative stress) in the presence or absence of Sho-saiko-to at the indicated dose. Whole-cell extracts were measured for DNA-binding activities of AP-1 and NF-nB by electrophoretic mobility shift assay, and analyzed by Western blotting using the antibody of InB-a, an inhibitory subunit of NF-nB.

early apoptosis by modulating Bcl-2 family protein expression (I. Shimizu, unpublished data, 2000).

Sho-saiko-to has also been reported to modulate in vitro cytokine production in peripheral blood mononuclear cells, downregulating the synthesis of IL-4 and -5 in favor of IL-10 in patients with chronic hepatitis C. IL-10 production in mononuclear cells of hepatitis C patients was reported to be lower than that of healthy subjects (19). IL-10 would be expected to have an effective activity for inflammatory bowel disease (20), endotoxin shock (21), and experimental liver injury (M. Tanaka, unpublished data, presented at the 37th annual meeting of the Japanese Society of Gastroenterology, 1995).

These findings suggest that Sho-saiko-to may play a role in the repair of abnormalities in the cytokine production system in patients with chronic HCV infection.

Parenchymal cell membrane damage could result in the release of oxygen-derived free radicals and other reactive oxygen species (ROS) derived from lipid peroxidative processes, which represent a general feature of sustained inflammatory response and liver injury, once the antioxidant mechanisms have been depleted (22). Many cells have their own unique enzymatic defense systems against oxidative stress, including the production of superoxide dismutase (SOD) and glutathione peroxidase (GPx). In addition, histopathological studies associated with chronic HCV infection showed fatty changes in 31-72% of patients (23-25), indicating that hepatic steatosis is a characteristic feature of chronic HCV infection. It has been suggested that hepatic steatosis may reflect a direct cytopathic effect of HCV and may play a role in the progression of the disease. In support of these proposals, a transgenic mouse model, which expresses the HCV core gene, was observed to develop progressive hepatic steatosis and HCC (26,27). It is conceivable that followig hepatocyte injury, hepatic steatosis leads to an increase in lipid peroxidation, which might contribute to HSC proliferation and transformation by releasing soluble mediators (28,29), and, thus, induce hepatic fibrosis.

We previously reported that, in two rat models of hepatic fibrosis, hepatic concentrations of malondialdehyde (MDA), an end product of lipid peroxidation, in Sho-saiko-to-fed animals were significantly lower than in those fed a basal diet, and that Sho-saiko-to inhibited the prooxidant-enhanced lipid peroxidation in cultured rat hepatocytes, and inhibited lipid peroxidation induced in rat liver mitochondria by Fe2+/adenosine 5'-diphosphate (7). In addition, we observed that Sho-saiko-to scavenges the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) (7). Free radicals, generated mainly by Kupffer cells, are thought to cause tissue injury by initiating lipid peroxidation and inducing irreversible modifications of cell membrane structure and function (30). In an animal model of endotoxemia, preadministra-tion of Sho-saiko-to was found to significantly increase the activity of SOD and GPx, suggesting that Sho-saiko-to acts by protecting against plasma membrane damage by ROS such as free radicals (31).

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