Effects Of Shosaikoto On Hepatic Fibrosis

In the injured liver, HSCs in the space of Disse are generally thought to be the primary target cells for inflammatory stimuli (32), and to produce ECM components, while in intact liver lobules they function as the primary storage area for retinoids (33). It has been shown that the activation of HSCs in injured livers leads to their proliferation and transformation into myofibro-

blast-like cells. This is accompanied by a loss of cellular retinoid, and the synthesis of a-SMA and large quantities of the major components of the ECM, including collagen types I, III, and IV, fibronectin, laminin, and proteoglycans. a-SMA is an activation marker of HSCs. It has been shown that, in vivo, HSCs express the genes that encode for enzymes such as matrix metalloproteinase (MMP)-1, which catalyzes the digestion of native fibrillar collagen types I and III, and MMP-2, which acts on denatured collagen types I and III and native collagen type IV, as well as a tissue inhibitor of metalloproteinase (TIMP)-1 (34). The net effect of the production of proteins involved in matrix synthesis and degradation could be reduced matrix degradation, which could account for the marked increases in matrix deposition and nodule formation observed during hepatic fibrosis and cirrhosis (9,35).

Several reports concerning the role of Sho-saiko-to with respect to the prevention and treatment of experimental liver damage induced in rats by D-galactosamine (36), carbon tetrachloride (37), dimethylnitrosamine (DMN) (38), and pig serum (PS) (38) have appeared. Although the mechanism by which Sho-saiko-to prevents hepatic fibrosis is not at present clear, it has been reported that the preadministration of this herbal medicine protects the liver plasma membrane and HSCs against injury (31). Moreover, Sho-saiko-to was reported to prevent the development of hepatic fibrosis by the inhibition of HSC activation in a different animal model, the choline-deficient rat (39). We confirmed the preventive and therapeutic effects of Sho-saiko-to on rat experimental hepatic fibrosis induced by DMN and PS (7). The rats were fed a basic diet that contained Sho-saiko-to for 2 weeks prior to the induction of hepatic fibrosis, or during the last 2 weeks of treatment. Sho-saiko-to suppressed the induction of hepatic fibrosis, maintained hepatic retinoid stores, and reduced hepatic collagen levels and the hepatic expression of aSMA and type I collagen. In addition, when incubated with cultured rat HSCs, the presence of Sho-saiko-to led to an increase in lipid droplets, which include retinoid and occupy the cytoplasmic space, and inhibit type I collagen production, a-SMA expression, cell spreading, and DNA synthesis. Furthermore, Sho-saiko-to was reported to induce the arrest at the G0/G1 phase in the cell cycle of HSCs (40). These findings suggest that the antifibrogenic activities of Sho-saiko-to are associated with the regulation of ECM proteins including type I collagen and a-SMA expression, retinoid disappearance, as well as HSC proliferation.

In investigating the mechanism by which Sho-saiko-to inactivates HSCs, Kakumu et al. showed that Sho-saiko-to enhanced the in vitro production of interferon (IFN)-g and antibodies to the hepatitis B core and e antigens, produced by peripheral blood mononuclear cells from patients with chronic hepatitis (41). IFN-g is a potent cytokine with immunomodu-

latory and antiproliferative properties, which inhibits HSC activation and ECM production in in vivo models of hepatic schistosomiasis and carbon tetrachloride-, DMN-, and PS-induced hepatic fibrosis (42-46). Oxidative stress (10,47), including the generation of ROS, has also been implicated as a cause of hepatic fibrosis (Fig. 2). There is evidence that the products of lipid peroxidation modulate collagen gene expression (34,48), suggesting that lipid peroxidation is a link between liver tissue injury and fibrosis (22,49). It has been reported that paracrine stimuli derived from hepatocytes undergoing oxidative stress induce HSC proliferation and collagen synthesis (50). HSCs have also been shown to be activated by the generation of free radicals with Fe2+/ascorbate (29) and by MDA and 4-hydroxynonenal (47,51), aldehydic products of lipid peroxidation, and antioxidants such as a-tocopherol were observed to inhibit HSC activation (29). We also reported that Sho-saiko-to supplementation led to a dose-dependent suppression in oxidative stress in cultured rat HSCs in parallel with the inhibition of the type I collagen production (7). In hepatic fibrosis, Sho-saiko-to may exert its suppressive effects, at least in part, by acting as an antioxidant, and/or by stimulating IFN-g.

Inflammatory cells, such as Kupffer cells and invading mononuclear cells, which release cytokines, transforming growth factor-h (TGF-h1), and platelet-derived growth factor (PDGF), may also contribute to the fibrogenic response to liver injury. Although the precise nature of the Kupffer-cell-derived factors that induce HSC activation is currently poorly understood, it has been shown, for example, that TGF-h and PDGF activate cultured HSCs (52,53). HSCs also produce and respond to TGF-h in an autocrine manner with increased collagen expression. These findings suggest that these growth factors may act as paracrine and autocrine (i.e., from HSCs) mediators that trigger the transformation of HSCs in vivo. PDGF is a major mitogen that drives HSC proliferation (54). Importantly, TGF-h is a key fibrogenic mediator that is capable of enhancing ECM deposition and inhibiting MMP activity (55). It is also noteworthy that TGF-h is an inhibitor of the proliferation of hepatocytes (56), and that, at higher concentrations, TGF-h induces oxidative stress leading to hepatocyte apoptosis (56). A preliminary report concluded that Sho-saiko-to inhibited the PDGF-induced proliferation of HSCs (40) (Fig. 2).

Because of their anatomical location, their ultrastructural features, and similarities with pericytes that regulate blood flow in other organs, it has been proposed that HSCs function as liver-specific pericytes (57). Previous studies have shown that the contraction and relaxation of HSCs regulate hepatic sinusoidal blood flow (57,58). Two vasoregulatory compounds with obvious effects on HSCs include endothelin (ET)-1 and nitric oxide (NO) (59-61). Experimental evidence suggests that ET-1 is a potent vasoconstrictor in the liver microcirculation in vivo, acting at both the sinusoidal and extrasinusoi-

Figure 4 Comparison of the chemical structure of baicalin, baicalein, silybinin, and quercetin. Each of these molecules contain a 2-phenyl-1-benzopyrane-4-one (flavone) structure.

dal sites (62), and that exogenous NO prevents ET-induced contraction as well as causing precontracted cells to relax (63). HSCs and sinusoidal endothelial cells produce NO in response to various stimuli in the presence and absence of endotoxins (64 65). It should be pointed out that Sho-saiko-to upregulates the inducible NO synthase in hepatocytes, when cultured in the presence of IFN-g (66).

In our study, among the herbal components of Sho-saiko-to, the Scutellaria root extract actively inhibited superoxide anion production (7). Superoxide anion is generated in reperfusion-induced oxidative stress and liver inflammation, and is closely related to membrane injury by lipid peroxidation (67). The antioxidant activity of Scutellaria root was due to the action of baicalin, baicalein, and viscidulin III, all of which showed antiox-idative effects. These findings are consistent with previously reported data (68). However, because an ethanol extract of Sho-saiko-to contains approximately 3.5% baicalin, 0.3% baicalein, and <0.1% viscidulin III (Table 1), the antioxidant action of Sho-saiko-to may be largely dependent on the action of the two flavonoids, baicalin and baicalein (Fig. 4). These flavonoids have been shown to suppress the proliferation of HSCs (69) and vascular smooth muscle cells (70,71). It is noteworthy that the chemical structures of the flavonoids are very similar to those of silybinin and quercetin (72). Each molecule contains a 2-phenyl-1-benzophyrane-4-one (flavone) structure (72). Silybinin (73) and quercetin (74) have been reported to have antifibrogenic properties; silybinin acts as an oxygen radical scavenger (73), affecting the lipid profile of hepatocyte membranes (75), inhibits the proliferation of HSCs in vitro, and retards collagen accumulation in chronic bile duct ligated rats (73); and quercetin suppresses the proliferation and a-SMA expression of HSCs and interferes with PDGF-induced signal transduction (74). In addition, some positive clinical trials involving silymarin have been reported. The latter is prepared as a standardized extract from milk thistle, the principle active component of which is the silybinin, comprising 60-70% of the silymarin (76,77). However, the latest results of a randomized controlled trial indicate that silymarin had no effect on survival and the clinical course in alcoholic patients with cirrhosis (78).

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