Yuan-chao Li

The suppressive effect of triptolide on experimental autoimmune uveoretinitis by down-regulating Th1-type response

We investigated the suppressive effect of triptolide (TRD), a purified component from a traditional Chinese herb, Tripterygium wilfordii Hook F. (TWHf), on uveitogenic peptide (K2)-induced experimental autoimmune uveoretinitis (EAU). K2-peptide immunized B10.A mice were divided into four groups. One group was EAU control which was treated with PBS. The other two groups were treated with TRD with different time courses (from day 0 to day 28 and from day 14 to day 28). The last group was treated with Cyclosporin A (CsA) as a positive control of the treatment. TRD was administered at dose of 0.1 mg/kg/day (i.p.). CsA was administered at dose of 20 mg/kg/day (i.p.) from day 0 to day 28 during whole period of EAU induction. The data showed that the EAU was suppressed in the whole period of TRD-treated mice, but was not in TRD-treated mice from day 14 to day 28 following immunization. The inhibition of EAU induced by TRD treatment was comparable to CsA-treated mice. The K2-specific lymphocyte proliferation and mRNA expressions of Th1-type cytokines (IL-12p40, IFN-gamma and TNF-alpha) in draining lymph node and inflamed eyes were reduced in TRD-treated mice. The K2-specific IFN-gamma production in the draining lymph node cells (LNC) of TRD-treated mice (whole period) was significantly inhibited. This effect was not related to an apoptotic effect of TRD on CD4+ T cells. Our results suggested that TRD suppressed the induction of EAU by down-regulating Th1-type response in B10.A mice. This preventive effect on EAU induction may be related to the inhibition of TRD on T cell priming and activation.

(5R)-5-Hydroxytriptolide (LLDT-8), a novel triptolide derivative, prevents experimental autoimmune encephalomyelitis via inhibiting T cell activation

A novel triptolide derivative (5R)-5-hydroxytriptolide (LLDT-8) has been shown to have potent immunosuppressive activities. Here LLDT-8 was evaluated in experimental autoimmune encephalomyelitis (EAE), the model of multiple sclerosis (MS). LLDT-8 reduced the incidence and severity of EAE, which was associated with the inhibition of the MOG 35-55 lymphocyte recall response, anti-MOG 35-55 T cell responses, interleukin (IL)-2 and interferon (IFN)-gamma production. In vitro, LLDT-8 inhibited primary T cells proliferation, division, IL-2 and IFN-gamma production stimulated with anti-CD3/28. These findings highlight the fact that LLDT-8 prevents EAE by suppressing T cell proliferation and activation, with a potential for treatment of MS.

(5R)-5-Hydroxytriptolide Attenuated Collagen-Induced Arthritis in DBA/1 Mice via Suppressing Interferon-gamma Production and Its Related Signaling

(5R)-5-Hydroxytriptolide (LLDT-8) displays strong immunosuppressive activities both in vitro and in vivo in our previous studies. This study aims to investigate whether LLDT-8 has antiarthritic potential in a murine model of type II bovine collagen (CII)-induced arthritis (CIA) and to show the mechanism(s) of LLDT-8 action. DBA/1 mice were immunized with CII to induce arthritis and administered with LLDT-8. The severity of arthritis was evaluated according to the clinical score and joint damage. The effects of LLDT-8 on immune responses were determined by measurement of serum antibody levels, lymphocyte proliferation assay, cytokine assay, nitric oxide (NO) production, arginase activity assays, fluorescence-activated cell sorting analysis of splenic Mac-1+ cells, as well as polymerase chain reaction analysis for interferon-γ (IFN-γ)-related gene expression. We showed that LLDT-8 treatment significantly reduced the incidence and severity of CIA. The preventive and therapeutic effects of LLDT-8 are associated with 1) reduction of serum anti-CII immunoglobulin (Ig) G, IgG2a, and IgG1 levels; 2) inhibition of CII-specific lymphocyte proliferation, IFN-γ and interleukin-2 production; 3) blockade of gene expressions in IFN-γ signaling, including IFN-γ production pathways [signal transducer and activator of transcription (STAT) 1, T-box transcription factor, interleukin 12Rβ2, and STAT4] and IFN-γ-induced chemokine transcription [macrophage inflammatory protein (Mip)-1α, Mip-1β, regulated on activation normally T cell expressed and secreted, and inducible protein 10]; and 4) retardation of the abnormal increase of NO via IFN-γ/STAT1/interferon regulatory factor 1/inducible nitric-oxide synthase pathway and arginase activity. Moreover, the mRNA transcription of chemokine receptors was also suppressed [including C-C chemokine receptor (CCR) 1, CCR5, and C-X-C chemokine receptor 3]. In conclusion, our data suggest that the antiarthritic effect of LLDT-8 is closely related to the blockade of IFN-γ signaling. LLDT-8 may have a therapeutic value in the treatment of rheumatoid arthritis.

(5R)-5-hydroxytriptolide (LLDT-8) protects against bleomycin-induced lung fibrosis in mice

AbstractAim:To study the protective effects of a triptolide-derived, novel compound, (5R)-5-hydroxytriptolide (LLDT-8), on bleomycin-induced lung fibrosis.Methods:C57BL/6 mice received an intratracheal injection of bleomycin and were then treated with LLDT-8 (0.5, 1, 2 mg/kg, ip) once daily for 7 or 14 consecutive days. The body weight loss and lung index augmentation was observed; the inflammatory response including differential cells counts of neutrophils, macrophages, and lymphocytes in the bronchoalveolar lavage fluid (BALF), superoxide dismutase (SOD), and malondialdehyde (MDA) level in the lung homogenates was detected, and the fibrosis extent was evaluated by hydroxyproline content and histopathological changes in the lungs. In addition, the pro-inflammatory and pro-fibrotic cytokines, tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and transforming growth factor-α (TGF-α) production in the lungs were measured.Results:LLDT-8 alleviated the body weight loss and lung index increase caused by bleomycin, reduced neutrophils and lymphocytes in the BALF, promoted SOD activity, decreased MDA production, and inhibited the hydroxyproline level and the amelioration of lung tissue histological damage. Moreover, LLDT-8 suppressed TNF-α, IL-4, and TGF-β production in the lung homogenates.Conclusion:LLDT-8 showed protective effects against bleomycin-induced lung fibrosis, and the results suggested the potential role of LLDT-8 in the treatment of this disease.

Suppression of (5R)-5-hydroxytriptolide (LLDT-8) on allograft rejection in full MHC-mismatched mouse cardiac transplantation

Background. (5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). Studies in vitro and in vivo have demonstrated that LLDT-8 had potent immunosuppressive activities. Here we tested LLDT-8 in major histocompatibility complex (MHC)-mismatched cardiac transplantation and investigated the mechanisms underlying the prevention of transplant rejection. Methods. LLDT-8 was administered orally to recipients in Balb/c to C57BL/6 murine cardiac transplantation model. Allograft survival after transplantation was recorded in recipients. The T cell immunity and cytokine production were observed. Histological analysis was performed. The chemokine and its receptor were analyzed by reverse transcriptase-polymerase chain reaction on cardiac graft RNA. Results. LLDT-8 administered orally significantly induced the survival prolongation of allogeneic cardiac graft. Histological results showed that LLDT-8 well preserved myocardium and significantly reduced infiltration of the graft with inflammatory cells. LLDT-8 decreased IL-2 production in recipient splenocytes stimulated by concanavalin A (ConA) ex vivo. LLDT-8 significantly inhibited the immunoreactivity of recipient to specific donor alloantigens, but preserved immunity to third-party alloantigens and mitogen. However, the flow cytometry analysis of the proportion of CD4+, CD8+ T cell subgroup in recipient spleens showed LLDT-8 had a normalizing effect on the splenic lymphocytes population. LLDT-8 decreased CC chemokine receptor 5 (CCR5) and their ligands macrophage inflammatory protein 1 alpha (MIP-1α) and beta (MIP-1β) mRNA expressions in allografts. Conclusion. The results outline the great potential of LLDT-8 as a therapeutic tool in transplant rejection.

Discovering novel anti-HCV compounds with inhibitory activities toward HCV NS3/4A protease.

Aim:To discover novel hepatitis C virus (HCV) inhibitors and elucidate the mechanism of action of the active compounds.Methods:HCV subgenomic replicon-based luciferase reporter cell line was used to screen 1200 synthetic compounds with novel structures. Huh7.5.1 cell line stably transfected with HCV NS3/4A protease reporter was established to investigate the anti-HCV mechanism of the active compounds. The active compounds were further examined in an in vitro HCV infection assay to confirm their anti-HCV activity.Results:After two-round screening in the anti-HCV replicon assay, some 2,4-diaminoquinazoline derivatives and carboxamide analogues were found to possess anti-HCV replicon activities (the IC50 values were less than 5 μmol/L). Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 μmol/L, respectively. Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 μmol/L, respectively.Conclusion:Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

(5 R )-5-hydroxytriptolide inhibits IFN-γ-related signaling

AbstractAim:(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-γ.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-γ 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-γ production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell proliferation and IFN-γ generation. In anti-CD3-activated T cells, LLDT-8 abrogated the mRNA expression of signal transducer and activator of transcription 1 (STAT1), T-box transcription factor, IL-12 receptor β2, STAT4, and interferon regulatory factor 1 in the IFN-γ 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)-1α, Mip-1β, regulated upon activation normally T-cell expressed and secreted, induc-ible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced by IFN-γ in IFN-γ-stimulated murine macrophage cell line Raw 264.7 cells.Conclusion:LLDT-8 was a potential inhibitor for IFN-γ-associated signaling.

(5R)-5-hydroxytriptolide inhibits the immune response of human peripheral blood mononuclear cells ☆

AIM (5R)-5-hydroxytriptolide (LLDT-8) displayed immunosuppressive activities both in vitro and in autoimmune disease models. Here, we aim to further clarify the effect of LLDT-8 on the immune responses of human peripheral blood mononuclear cells (PBMC). METHOD Cell proliferation of human PBMC from healthy donors was evaluated by [3H]-thymidine uptake. NK cell cytotoxicity was assayed using K562 cells in a [3H] lysis assay. Cytokine production was determined by enzyme-linked immunosorbent assay. The expression of cell surface molecules was detected with flow cytometry. The mRNA expression and the protein phosphorylation levels were detected by RT-PCR and Western immunoblot assay. RESULTS LLDT-8 at 25 and 50 nM significantly inhibited the PHA- and recall antigens-induced T cell proliferation, and suppressed mixed lymphocyte reaction. LLDT-8 reduced cytokines production (IFN-gamma, IL-2, TNF-alpha) in PHA- and Sac-activated PBMC. LLDT-8 did not alter the increased expression of MHC class I/II and B7.1, but reduced B7.2 by approximately 30%. No effect of LLDT-8 was observed for the expression of T cell activation markers (CD69, CD154). However, LLDT-8 significantly reduced IFN-gamma-expressing T cell percentages and IFN-gamma mRNA transcription in PHA-activated T cells. It also inhibited the phosphorylation levels of JNK and p38. LLDT-8 did not affect NK cytotoxic activity against K562 cells. CONCLUSION LLDT-8 was a promising immunosuppressant for human immune-related diseases.

Synthesis and Hepatitis C Antiviral Activity of 1-Aminobenzyl-1H-indazole-3-carboxamide Analogues

FIGHTING HCV: Two potent antiviral analogues were developed from a previously identified lead as novel agents against hepatitis C virus. Their potency and selectivity (5 n: IC50 =0.013 μM and EC50 =0.018 μM; 5 t: IC50 =0.007 μM and EC50 =0.024 μM) make them good candidates for further development as antiviral agents.