Tatsuya Ishihara

Tryptanthrin inhibits nitric oxide and prostaglandin E2 synthesis by murine macrophages

Nitric oxide (NO) and prostaglandins have been implicated in the pathogenesis of several inflammatory diseases. In this study, we investigated the effect of tryptanthrin (6,12-dihydro-6, 12-dioxoindolo-(2,1-b)-quinazoline), an antimicrobial and antitumoral plant compound isolated from Porigonum tinctorium, on NO and prostaglandin E(2) production by interferon-gamma and lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells. Tryptanthrin markedly inhibited both NO and prostaglandin E(2) production in a dose-dependent manner. Tryptanthrin at 20 microM fully inhibited expression of inducible NO synthase, suggesting that the inhibitory effect on NO synthesis was mediated by inhibited expression of the enzyme. On the other hand, tryptanthrin had no effect on the levels of cyclooxygenase-2 protein, but inhibited cyclooxygenase enzyme activity with a ICM(50) value of 1.5 microM. Thus, tryptanthrin has the dual functions of inhibiting both NO and prostaglandin E(2) production by activated macrophages, suggesting that tryptanthrin exhibits anti-inflammatory properties.

IL-18 Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 × DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-γ production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.

The natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice.

The therapeutic effects of tryptanthrin (TRYP), a natural product from the medicinal plant Polygonum tinctorium, were examined in a murine model of inflammatory bowel disease (IBD). Colitis was induced by 5% dextran sodium sulfate (DSS) in drinking water for 7 days from day 0. TRYP (100 mg/kg) was administered orally suspended in 5% arabia gum everyday from day 3 for 5 days. Histopathological analysis showed reduced colon damage in TRYP-treated mice on day 6; however, colon injury resumed after treatment was stopped. The production of prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) by untreated and treated mouse colon tissues cultured in vitro were mostly unchanged by TRYP treatment. However, mitogen-stimulated spleen cells from TRYP-treated colitic mice produced less interleukin 2 (IL-2) and less interferon-gamma (IFN-gamma) than untreated colitic mouse spleen cells, early after induction of colitis. When colitis was induced with 5% DSS for 7 days and TRYP was given to the mice for 8 days from day 3, TRYP enhanced the survival of the mice but results were not significant. A significant reduction of weight loss was observed in TRYP-treated mice with colitis induced by 5% DSS for 4 days as compared to control mice. Remarkably, whereas 90% of the vehicle-treated mice died from wasting disease, all the TRYP-treated mice survived, suggesting that TRYP may have a therapeutic effect on colitis.

Immuno-potentiating Effects of the Antler-shaped Fruiting Body of Ganoderma lucidum (Rokkaku-Reishi)

The immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi, RR), which has been used as a traditional supplement for human health, were investigated in mice. BALB/c mice were administered orally with RR for 3 days at a dose of 50 mg/kg or 500 mg/kg, and interferon-gamma (IFN-γ) production by splenocytes in response to lipopolysaccharide (LPS) was examined on day 4. The oral administration of 500 mg/kg of RR resulted in a significant increase (p<0.05) in IFN-γ production. Stimulation of splenic adherent cells from these mice with LPS also resulted in a significant increase (p<0.05) in interleukin-12 (IL-12) production compared with that from the control mice, suggesting that splenic macrophages were activated by RR administration. Furthermore, 500 mg/kg of RR administered for 14 days resulted in a significant increase (p<0.05) in IFN-γ production by splenocytes in response to both LPS and concanavalin A (Con A). These results suggest that not only splenic macrophages but also T cells were activated by the long-term treatment with RR in vivo. On the other hand, the production of interleukin-4 (IL-4), which is known as an allergic disease-related cytokine, was not affected by the long-term treatment with RR. Our results suggest that the oral administration of RR resulted in Th1-associated immuno-potentiating activities in vivo.

Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression.

Despite its beneficial role in host defense mechanisms, excessive nitric oxide (NO) production by activated macrophages has been implicated in several inflammatory diseases. To clarify the mechanisms of anti-inflammatory activities of Polygonum tinctorium, we evaluated whether extracts of P. tinctorium could modulate the production of NO by activated macrophages. An AcOEt extract of P. tinctorium markedly inhibited NO synthesis by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages and the macrophage-like cell line RAW 264.7 in a dose-dependent manner. Inhibition of NO synthesis was achieved by reducing inducible NO synthase (iNOS) expression at protein and mRNA levels. However, the AcOEt extract of P. tinctorium failed to inhibit NO synthesis when iNOS was already expressed following stimulation with IFN-gamma and LPS. The AcOEt extract also exhibited inhibitory activity on iNOS expression in human lung epithelial A549 cells stimulated with a combination of IFN-gamma, TNF-alpha and IL-1 beta without affecting the expression of constitutive isoforms of NOS. Furthermore, in vivo injection of the AcOEt extract of P. tinctorium into LPS-treated mice significantly reduced NO synthesis by peritoneal exudate cells under ex vivo conditions. These results suggest that P. tinctorium extract may be a potential therapeutic modulator of NO synthesis in various pathological conditions.