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Aim: (5R)-5-hydroxytriptolide (LLDT-8) displayed anti-arthritis and anti-allogenic transplantation rejection activities in our previous studies. Here, we aim to further clarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-gamma., Methods: T cells were activated with anti-CD3 antibody or concanavalin A (ConA). The expression of cell surface molecules was detected with flow cytometry. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to test cell division. IFN-gamma production was determined by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by [3H]-thymidine uptake. Mice were immunized with ovalbumin to assess the in vivo immune response. RT-PCR and Real-time PCR were applied to determine the mRNA expression. The protein phosphorylation levels were detected by Western immunoblot assay., Results: LLDT-8 at 100 nmol/L did not change the CD25, CD69, and CD154 expressions in anti-CD3-stimulated T cells. LLDT-8 markedly blocked the cell division of CD4 and CD8 T cells after ConA stimulation. LLDT-8 inhibited T cell-derived IFN-gamma production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell proliferation and IFN-gamma generation. In anti-CD3-activated T cells, LLDT-8 abrogated the mRNA expression of signal transducer and activator of transcription1 (STAT1), T-box transcription factor, IL-12 receptor beta2, STAT4, and interferon regulatory factor 1 in the IFN-gamma expression pathway. Western blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T cells. In addition, LLDT-8 reduced the transcripts of macrophage inflammatory protein (Mip)-1alpha, Mip-1beta, regulated upon activation normally T-cell expressed and secreted, inducible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced by IFN-gamma in IFN-gamma-stimulated murine macrophage cell line Raw 264.7 cells., Conclusion: LLDT-8 was a potential inhibitor for IFN-gamma-associated signaling.

Aim: To investigate the global gene expression profile changes in Jurkat cells after triptolide treatment in order to find the possible triptolide targets., Methods: Jurkat cells were treated with or without triptolide 10 microg/L for 2 h. Total RNA were isolated and used as templates for reverse transcriptional labeling of fluorescent cDNA probes. High density DNA microarray chips with a set of 13,872 human genes/Ests were used to generate the expression profile of triptolide-treated or untreated control Jurkat cells by hybridizing with fluorescent labeled probes. Array image was acquired and analyzed with array analyzing software GeneSpring., Results: Triptolide significantly suppressed expression of 117 genes in Jurkat cells. Among these 117 genes, 30 % were Ests or genes without known functions, 13 % were transcription factors, 9 % were signal transduction pathway regulators, and 9 % were DNA binding proteins. Notably, the expression of mitogen-activated protein kinase kinase kinase kinase 5 (MAP kinase 5) and phosphoinositide-3-kinase (PI-3 kinase) was inhibited more than 100-fold. Moreover, the expression of genes involved in lipid transportation and metabolism was down-regulated by triptolide., Conclusion: High-density microarray provided an effective approach to identify drug targeting molecules. It is suggested that the widely known immune suppressive and antitumor effects of triptolide were mediated at least in part by suppression of MAP kinase and PI-3 kinase gene expression.

Aim: To study the interaction of resveratrol and ethanol on the production of pro-inflammatory factors from activated murine peritoneal macrophages (MPM)., Methods: NO production was measured with Griess assay; IL-1 production was measured through thymocyte co-stimulating assay; IL-6 and TNF-alpha were detected by ELISA method., Results: Resveratrol (6.25, 12.5, 25 micromol/L) and ethanol (0.2 %, 0.8 %) synergistically inhibited the 24 h production of NO from lipopolysaccharide (LPS, 1 mg/L) and IFN-gamma (5 kU/L) stimulated MPM; resveratrol at higher dose (25 micromol/L) also inhibited IL-6 production. Ethanol additively strengthened this effect. Ethanol had no significant influence on 24 h MPM IL-1 production, but it promoted the ability of resveratrol on enhancing the IL-1 release from activated MPM. Low doses of ethanol inhibited 24 h production of TNF-alpha, however, both dose of ethanol enhanced the promoting effect of resveratrol on TNF-alpha production., Conclusion: Resveratrol and ethanol can interact to influence the production of macrophage function molecules, which is noteworthy in evaluating the health-care effect of wine consumption.

Aim: To study the immune modulating effect of low dose of resveratrol., Methods: Concanavalin A (ConA) and Staphylococcus aureus Cowan (Sac) were used to induce the activation of T lymphocyte and antigen presenting cell and cytokine production. [3H]-Thymidine incorporation was used to evaluate the proliferation of lymphocyte. Cytokine production was detected by ELISA method. Dinitrofluorobenzene (DNFB) was used to induce mice delayed type hypersensitivity (DTH, delayed hypersensitivity) response and ear swelling was used as an evaluating indicator. Changes of lymphocyte subtypes were detected by flow cytometry., Results: Resveratrol (0.75-6 micromol/L) concentration-dependently promoted lymphocyte proliferation and IL-2 production induced by ConA. Sac induced IL-12 and IFN-gamma (interferon type II) production were also concentration-dependently enhanced by resveratrol, while IL-10 production was inhibited. Resveratrol (4 mg/kg, ig) promoted DTH response of mouse, which was suppressed by ethanol (16 %, w/v) consumption. Resveratrol treatment had no significant influence on lymphocyte subtypes in mice, however it could reverse the suppressive effect of ethanol both on macrophage percentage and on macrophage MHC-II molecule expression., Conclusion: Low dose resveratrol enhanced cell-mediate immune response. Promoting Th1 cytokine production and influencing on macrophage function might be its mechanisms.

Aim: To investigate the effect of astragaloside IV (ASI) on T, B lymphocyte proliferation, antibody production, and cytokines produced by murine peritoneal macrophages., Methods: MTT assay was used to determine T, B lymphocyte proliferation and quantitative hemolysin spectrophotometry (QHS) assay was applied to test antibody production; IL-1 production was measured by thymocyte proliferation assay; TNF-alpha production was determined by the cytotoxicity assay against L929 cells., Results: 1) In vivo, ASI 50-200 mg/kg ig for 7 d increased T lymphocyte proliferation and antibody production, and ASI 50-100 mg/kg ig for 7 d increased B lymphocyte proliferation but ASI 200 mg/kg had no effect on B lymphocyte proliferation; 2) In vitro, ASI increased T, B lymphocyte proliferation only at 100 nmol/L; 3) ASI increased IL-1 activity at 1 nmol/L in vitro, but decreased it at 100 and 1000 nmol/L; 4) ASI inhibited TNF-alpha activity with or without LPS-stimulation in vitro., Conclusion: ASI increased T, B lymphocyte proliferation and antibody production in vivo and in vitro; but inhibited productions of IL-1 and TNF-alpha from peritoneal macrophages in vitro.

Aim: To study the chemical characterization and some biological activities of an acidic polysaccharide, named SC4 from Salvia chinensis., Methods: Chemical and spectral methods were employed to identify the structural characterization of SC4. Effect of SC4 on the proliferation of T- and B-lymphocytes both in vivo and in vitro was measured at various concentrations, and the protective effects of SC4 on PC12 cells against H2O2-induced injury was observed., Results: SC4 was a highly branched polysaccharide with mean molecular weight of 4.5 10(5), composed of Rha, Xyl, Ser, Gal, and GalA in the molar ratio of 1.0 7.0 5.3 1.2 4.2. Methylation analysis and NMR identified the linkages of the residues of SC4. SC4 increased the B-lymphocyte proliferation and spleen weight remarkably while no obvious changes were examined in T-lymphocyte proliferation, thymus weight, and the level of IgG and C3 in blood of mice. Moreover, SC4 enhanced the PC12 cells viability after H2O2 treatment., Conclusion: SC4, the acidic polysaccharide with complicated structure, was a B-lymphocyte stimulator and protected PC12 cells at the concentration of 20 mg/L against H2O2-induced injury.