Kozo Kayano

Inhibitory effects of the herbal medicine Sho-saiko-to (TJ-9) on cell proliferation and procollagen gene expressions in cultured rat hepatic stellate cells

BACKGROUND/AIMS It is of extreme importance to prevent liver fibrosis and subsequent progression to liver cirrhosis. The aim of our study was to elucidate in vitro whether Sho-saiko-to exerted inhibitory effects on hepatic stellate cells. METHODS Hepatic stellate cells were isolated from male Wistar rats. Water-soluble ingredients of Sho-saiko-to were obtained at concentrations of 10, 100, 250, 500 and 1000 microg/ml. Morphological transformation was observed under a phase-contrast microscope. Flow cytometric analysis was performed on day 4 after culture to evaluate the potential to proliferate of the stellate cells by analyzing cell cycles. Northern blot analysis was carried out on day 3 after culture to determine the expressions of type I and type III procollagen mRNAs. RESULTS (i) Sho-saiko-to 500 and 1000 microg/ml inhibited morphological transformation of the stellate cells to myofibroblast-like cells. (ii) Sho-saiko-to 500 and 1000 microg/ml significantly (p<0.0001) accumulated the cells in the G0/G1 phase (118.8+/-0.7%, 119.2+/-0.5%, respectively as compared with control) and significantly (p<0.0001) decreased cell numbers subsequently in G2/M phase (47.5+/-8.1%, 48.9+/-2.0%, respectively). (iii) Sho-saiko-to 500 and 1000 microg/ml also significantly (p<0.05 or p<0.0001) suppressed procollagen mRNA expression of type I to 51.5+/-6.4%, 34.9+/-3.7%, respectively, and type III to 51.3+/-12.3%, 46.7+/-11.4%, respectively. CONCLUSIONS We have clarified the inhibitory effects of Sho-saiko-to on hepatic stellate cells in vitro. Sho-saiko-to could be a potent inhibitor in the pathogenesis of liver fibrosis.

Protection against Acetaminophen-Induced Liver Injury in Vivo by an Iron Chelator, Deferoxamine

BACKGROUND Recent data indicate that iron ions play a major role in lipid peroxidation, a hepatotoxic effect of acetaminophen (APAP). METHODS We investigated whether an iron chelator, deferoxamine (DFO), can protect against APAP-induced liver injury in vivo in rats. RESULTS DFO diminished the increase in serum alanine aminotransferase (ALAT) in a dose-dependent manner after APAP administration and also reduced mortality. Administration of 750 mg/kg APAP resulted in an increased ALAT (11,666 +/- 4633) after 8 h, and the mortality at 24 h was 88%. Pretreatment with 200 mg/kg DFO for 1 h significantly reduced ALAT (to 3406 +/- 894) and mortality (38%). DFO also attenuated histopathologic changes. Treatment with DFO depressed malondialdehyde formation by APAP without inhibiting glutathione depletion in the liver or reducing covalent binding of [3H]APAP to liver proteins. CONCLUSIONS These results indicate that the protective effect of DFO against APAP-induced liver injury may be attributable not to changes in APAP metabolism but to the chelation of iron, which can catalyze the generation of active oxygen species, in hepatocytes.

Interferon γ- but not α-treatment prevents fibrosis by inhibiting procollagen and tissue inhibitor of metalloproteinase-1 gene expression in pig serum-induced rat liver fibrosis in vivo

Abstract The aim of this study was to investigate the effect of interferons- γ (IFN- γ ) and - α (IFN- α ) on the synthesis of matrix proteins such as collagens as well as the gene expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in vivo. We investigated the effects of IFN- γ and - α in a model of liver fibrosis induced by pig serum in male Wistar rats, which develop fibrosis without an increase in serum ALT (i.e. without hepatocyte injury). Rats were injected with 0.5 ml of pig serum twice a week for 8 weeks with or without 50 000 U of IFN- γ or 100 000 U of IFN- α . IFN- γ at the dose of 50 000 U day −1 prevented fibrosis, as indicated by reduced hydroxyproline content in the liver. IFN- γ at 50 000 U day −1 also reduced expression of type I procollagen as well as TIMP-1 in the liver. However, fibrosis was not reduced by IFN- α . Histologically, IFN- γ at 50 000 U day −1 also reduced the number of myofibroblast-like cells (activated stellate cells). These results indicate that IFN- γ , but not IFN- α , can prevent fibrosis by inhibiting the activation and proliferation of stellate cells, resulting in reduced expression of procollagen and TIMP-1 mRNA in pig serum-induced rat liver fibrosis in vivo.

Th1 down-regulation at the single-lymphocyte level in HCV-related liver cirrhosis and the effect of TGF-β on Th1 response: possible implications for the development of hepatoma

Patients with hepatitis C virus (HCV) related-liver cirrhosis (LC) often develop hepatoma. The type 1 helper T cell (Th1) response presents an antitumor effect. We evaluated the Th1 response in patients with HCV-related LC at the single-cell level and examined the influence of transforming growth factor (TGF)-beta, an immunosuppressive cytokine, on the Th1 response. We determined the ratios of Th1-type cytokine (IFN-gamma, IL-2)-producing cells to CD3-positive cells in 14 patients (LC group) and in 16 normal controls using flow cytometry and measured serum TGF-beta(1) and TGF-beta(2) levels by ELISA. We then incubated, peripheral blood mononuclear cells from seven healthy volunteers with recombinant TGF-beta(1) or TGF-beta(2) for 48 h, and determined the ratio of IFN-gamma producing cells to CD3-positive cells. The IFN-gamma ratio was significantly lower in the LC group (29.7+/-0.3 vs. 44.2+/-15.0%, P<0.01). The serum TGF-beta(2) level was significantly increased in the LC group (601+/-232 vs. 329+/-118 pg/ml, P<0.001). TGF-beta(2) significantly suppressed IFN-gamma production at the single-cell level (10.0+/-4.3 vs. 7.3+/-2.0%, P<0.05). These findings indicated that the enhanced down-regulation of Th1 by TGF-beta(2) in patients with HCV-related LC might be effective against hepatoma.

Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity.

Purpose. Alterations in the kinetics of hepatic stellate cells (HSCs) after the cells are activated once have not been well documented. We investigated the characteristic profiles of cell proliferation of once-activated HSCs in contrast to the in fibrogenesis activity. Methods. HSCs from male Wistar rats were submitted to primary culture for 14 days and to secondary culture for 7 days. The potential for cell proliferation was evaluated by the number of the cells in G2/M phase, based on flow cytometric analysis of the cell cycle. The fibrogenesis activity was assessed by Northern blot analysis of the expression of type I and type III procollagen mRNA. Results. The number of HSCs in G2/M phase was maintained at a low level in primary culture after 6 days, while a significantly (P < 0.05) elevated number of HSCs in G2/M phase was observed on days 3 to 4. In secondary culture, the number of HSCs in G2/M phase was also consecutively maintained at a decreased level. By contrast, HSCs showed progressively increased type I and type III procollagen mRNA expression during the experimental periods of primary culture. Conclusions. These results clearly demonstrated consecutively decreased proliferative activity, evaluated by the potential for cell mitogenesis, in once-activated HSCs, in contrast to their progressively increased fibrogenesis activity.

Preventive effect of FK 506 (tacrolimus hydrate) on experimentally induced acute liver injury in rats.

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 μg/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) -α mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF-α production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 ± 401 in the saline group versus 119 ± 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF-α concentration was lower in the FK 506-treated group (1419 ± 957 pg/ml) than in the saline group (9205 ± 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF-α production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF-α production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.

HBV-related fulminant hepatic failure: successful intensive medical therapy in a candidate for liver transplantation

Fulminant hepatic failure (FHF) usually has a fatal prognosis without liver transplantation. We describe the case of a woman who developed FHF, and was evaluated as a candidate for liver transplantation, but who was cured without transplantation through intensive medical care that included glucagon-insulin therapy, methylprednisolone pulse therapy, interferon beta and lamivudine administration, cyclosporine administration, and high-volume hemodiafiltration and plasma exchange. In a patient with FHF who is a candidate for liver transplantation but for whom the transplantation cannot be performed for some reason, intensive medical therapy, including regeneration-promoting therapy, immunosuppressive therapy, antiviral therapy, and vigorous hepatic support, should be carried out.

Protective effect of deferoxamine for acetaminophen induced liver injury

Acetaminophen is widely prescribed analgesic and antipyretic drug, which is known to cause hepatic necrosis in both humans and experimental animals at high doses. Accumulated data implicated a metabolism-dependent (cytochrome p-450) generation of partially reduced oxygen species which can initiate the peroxidation of membrane lipids related to loss of cell viability in this compound. Iron ions play a major role in lipid peroxidation, which is a effect of several agents including acetaminophen (APAP)[I]. Deferoxamine (DFO) can prevent lipid peroxidation leading to cell death by trapping iron ions. Therefore, it is of interest to determine whether DFO can protect against APAP induced liver injury. Male Sprague-Dawley rats were treated with 25 mg/kg of 3-methylcholanthrene (Sigma) as a i0 ml solution in corn oil by intraperitoneal injection 16 h prior to use and then fasted overnight. This treatment makes rats more susceptible to acetaminophen by the induction of cytochrome p-450. In the experiments, rats were divided into three groups as follows : (A) rats treated with 750 mg/kg of APAP alone ; (B) rats pretreated with 200 mg/kg of DFO 60 min before APAP injection ; (C) rats treated with 200 mg/kg of DFO 60 min before and 120 min after APAP injection. All rats were treated by intraperitoneal injection. Serum ALT was examined 8 h after APAP treatment and mortality was investigated 24 h later. Figure indicates that APAP produced an increased ALT level (11666 • within 8 h and mortality was 87.5 %. By contrast, pretreatment with DFO significantly reduced ALT to 3406• 894 concomitant with reduced mortality (37.5%). This protection by DFO was dose-dependent up to 400 mg/kg (data not shown). Additional DFO treatment showed more protective effect. Histology showed massive liver cell necrosis within 8 h with the treatment of APAP (data not shown). DFO attenuated the impairment of liver histology as well as the increase in ALT level (data not shown). These results indicate that DFO is a strong protective agent against APAP induced liver injury. Figure : Serum ALT level and mortality by either APAP alone (A) ALT or with pretreatment of DFO (B) or additional treatment of DFO (C) Reference : I. Sakaida I., et al : J. Biol. Chem. 1991 ; 266 : 717-722 i0000

Inhibitory Effects of the Herbal Medicine Sho-saiko-to on Liver Fibrosis

It is of extreme importance to prevent liver fibrosis and subsequent progression to liver cirrhosis. The aim of our study was to elucidate whether the herbal medicine Sho-saiko-to exerted inhibitory effects on liver fibrosis in vivo as well as in vitro. In vivo, liver fibrosis was induced in rats by a choline-deficient L-amino acid-defined (CDAA) diet. The effects of Sho-saiko-to on various markers of liver fibrosis were investigated. In vitro, hepatic stellate cells (HSCs) were isolated from male Wistar rats. Water-soluble ingredients of Sho-saiko-to were obtained at concentrations of 10, 100, 250, 500, and 1000μg/ml. Morphological transformation was observed under a phase-contrast microscope. Flow cytometric analysis was performed on day 4 after culture to evaluate the potential of HSCs to proliferate by analyzing cell cycles. Northern blot analysis was carried out on day 3 after culture to determine the expression of type I and type III procollagen mRNAs. The results showed that Sho-saiko-to (1% w/w) clearly suppressed serum levels of hyarulonic acid, hydroxyproline content in the liver, and expression of α-smooth muscle actin-positive cells. Sho-saiko-to at 500 and 1000μg/ml inhibited morphological transformation of HSCs to myofibroblastlike cells, cell proliferation by inducing arrest at the G0/G1 phase, and mRNA expressions of type I and type III procollagen.