Fangyuan Gong

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita

AIM OF THE STUDY Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita. MATERIALS AND METHODS A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively. RESULTS The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin (1), jaceosidin (2), cirsilineol (3), cirsimaritin (4), hispidulin (5), quercetin (6), 6-methoxytricin (7), acacetin (8), and apigenin (9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol (3), 6-methoxytricin (7) and apigenin (9) significantly inhibited T cell proliferation and activation in the bioassays. CONCLUSION The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.

Erlotinib inhibits T-cell-mediated immune response via down-regulation of the c-Raf/ERK cascade and Akt signaling pathway.

Erlotinib is a potent inhibitor of epidermal growth factor receptor tyrosine kinase and has been demonstrated to treat advanced or metastatic non-small cell lung cancer to prolong survival after failure of first-line or second-line chemotherapy. However, little is known about its effects on immune system. In the present study, we aimed to investigate the immunosuppressive activity of erlotinib on T lymphocytes both in vitro and in vivo, and further explore its potential molecular mechanism. Erlotinib exerted a significant inhibition on the T cell proliferation and activation induced by concanavalin A, anti-CD3 plus anti-CD28, staphylococcal enterotoxin B or phorbol myristate acetate respectively in a concentration-dependent manner and it also inhibited the secretion of the proinflammatory cytokines such as IL-2 and IFN-γ of activated T cells. Further study showed that erlotinib caused G0/G1 arrest and suppressed the phosphorylations of c-Raf, ERK and Akt in activated T cells. Moreover, erlotinib significantly ameliorated picryl chloride-induced ear contact dermatitis in a dose-dependent manner in vivo. In summary, these findings suggest that erlotinib may cause the impairment of T-cell-mediated immune response both in vitro and in vivo through inhibiting T cell proliferation and activation, which is closely associated with its potent down-regulation of the c-Raf/ERK cascade and Akt signaling pathway.

Selective triggering of apoptosis of concanavalin A-activated T cells by fraxinellone for the treatment of T-cell-dependent hepatitis in mice.

Selectively inducing apoptosis of activated T cells is essential for the clearance of pathogenic injurious cells and subsequent efficient resolution of inflammation. However, few chemicals have been reported to trigger apoptosis of activated T cells in the treatment of hepatitis without affecting quiescent T cells. In the present study, we found that fraxinellone, a small natural compound isolated from the root bark of Dictamnus dasycarpus, selectively facilitated apoptosis of concanavalin A (Con A)-activated CD4(+) T cells rather than those non-activated, by disrupting the mitochondrial transmembrane potential, decreasing the ratio of Bcl-2/Bax, and increasing cytochrome c release from the mitochondria to the cytosol. The enhancement in Fas expression and caspase-8 activity, truncation of Bid, and down-regulation of anti-apoptotic cellular FLICE-inhibitory protein expression by fraxinellone also suggested the participation of an extrinsic apoptosis pathway. Furthermore, fraxinellone significantly alleviated Con A-induced T-cell-dependent hepatitis in mice, which was closely associated with reduced serum transaminases, pro-inflammatory cytokines, and pathologic parameters. Consistent with the in vitro results, fraxinellone dramatically induced apoptosis of activated peripheral CD4(+) T cells in vivo, consequently resulting in less CD4(+) T-cell activation and infiltration to the liver. These results strongly suggest fraxinellone might be a potential leading compound useful in treating T-cell-mediated liver disorders in humans.

Mitochondria-dependent apoptosis of activated T lymphocytes induced by astin C, a plant cyclopeptide, for preventing murine experimental colitis.

Facilitating T-cell apoptosis is implicated as an effective therapeutic strategy for treatment of T cell-mediated disease, including inflammatory bowel disease. Here, we report that astin C, a plant cyclopeptide isolated from the roots of Aster tataricus (Compositae), induced apoptosis of activated T cells in a mitochondria-dependent but Fas-independent manner in that such activity was still observed in T cells from Fas-mutated MRLlpr/lpr mice. Although caspase 8 was not activated, astin C treatment led to the cleavage of caspase 9 and caspase 3, the upregulation of Bad protein expression as well as release of cytochrome c in activated T cells. Astin C did not induce the expression of GRP78 and GADD153, excluding involvement of endoplasmic reticulum stress-mediated pathway. Moreover, oral administration of astin C protected mice against TNBS-induced colonic inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Administering astin C significantly decreased serum levels of TNF-α, IL-4 and IL-17, accompanied with the induction of apoptosis in activated T cells in vivo. The results demonstrate, for the first time, the ability of astin C to induce apoptosis in activated T cells and its potential use in the treatment of colonic inflammation.

Novel immunomodulatory properties of cirsilineol through selective inhibition of IFN-γ signaling in a murine model of inflammatory bowel disease☆

Regulation of signal transducer and activator of transcription (STAT) 1 signaling is being explored as a new approach to the treatment of inflammatory bowel diseases. However, few chemicals have been reported to inhibit IFN-gamma/STAT1 signaling for Crohns disease therapy. In the present study, we found that cirsilineol, a small natural compound isolated from Artemisia vestita, significantly ameliorated trinitro-benzene sulfonic acid (TNBS)-induced T-cell-mediated experimental colitis in mice, which was closely associated with reduced autoreactive T-cell proliferation and activation. Moreover, the regulatory action of pro-inflammatory and anti-inflammatory cytokine by cirsilineol treatment was found to decrease the activity of effector Th1 cells but increase the activity of regulatory T cells as characterized by down-regulation of IFN-gamma and corresponding up-regulation of IL-10 and TGF-beta. The therapeutic effect of cirsilineol was attributable to a novel regulatory mechanism with selective inhibiting IFN-gamma signaling in colonic lamina propria CD4(+) T cells, which was mediated through down-regulating STAT1 activation and T-bet expression. Furthermore, cirsilineol was found to down-regulate the activation of JAK2, a critical kinase for IFN-gamma/STAT1 signaling, and abrogate the expression of T-bet, resulting in markedly decreased proliferation and activation of T cells in vitro. Importantly, the inhibition of IFN-gamma/STAT1 signaling by cirsilineol was reversible in the presence of high level of IFN-gamma. These results strongly suggest that cirsilineol might be potentially useful for treating T-cell-mediated human inflammatory bowel diseases.

Jaceosidin inhibits contact hypersensitivity in mice via down-regulating IFN-γ/STAT1/T-bet signaling in T cells

In the present study, we aimed to investigate the immunosuppressive activity of jaceosidin, a flavone isolated from Artemisia vestita, on T lymphocytes both in vitro and in vivo, and further explore its potential molecular mechanism. Jaceosidin exerted a significant inhibition on the T cell proliferation and activation induced by concanavalin A (Con A) in a concentration-dependent manner and it also inhibited the secretion of the proinflammatory cytokines such as IL-2, TNF-α and IFN-γ of activated T cells. Further study showed that jaceosidin down-regulated STAT1 activation and T-bet expression in activated T cells. Moreover, in order to investigate the immunosuppressive effect of jaceosidin in vivo, the picryl chloride (PCl)-induced ear contact dermatitis model was performed on BALB/c mice. Jaceosidin significantly ameliorated PCl-induced ear swelling in a dose-dependent manner, which was due to its inhibition of the STAT1/T-bet signaling pathway. In summary, these findings suggest that jaceosidin exerts its immunosuppressive effect both in vitro and in vivo through inhibiting T cell proliferation and activation, which is closely associated with its potent down-regulation of the IFN-γ/STAT1/T-bet signaling pathway.

Involvement of Na+, K+-ATPase and its inhibitors in HuR-mediated cytokine mRNA stabilization in lung epithelial cells

Increasing evidence demonstrates that Na+, K+-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na+, K+-ATPase inhibitors to explore the possible involvement of Na+, K+-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na+, K+-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na+, K+-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3′-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na+, K+-ATPase, as well as its inhibitors in inflammation.

Obaculactone suppresses Th1 effector cell function through down-regulation of T-bet and prolongs skin graft survival in mice.

Allograft rejection is a predominantly Th1 immune response. In this study, we showed that obaculactone, a natural compound derived from citrus fruit, prolonged skin graft survival in mice when treated after but not before transplantation. Furthermore, obaculactone inhibited alloantigen-specific production of Th1 cytokine IFN-gamma as well as proinflammatory cytokine IL-2, TNFalpha and IL-6. In parallel, IL-10 production was markedly up-regulated. Obaculactone significantly enhanced the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in the CD4(+) splenocytes without any effect on their inhibitory function. In vitro and in vivo tests showed obaculactone down-regulated T-bet expression in Th1 effector cells. Taken together, the unique immunomodulatory properties might qualify obaculactone as a putative, therapeutic compound for the treatment of Th1-driven diseases, including transplant rejection.

Blocking initial infiltration of pioneer CD8+ T‐cells into the CNS via inhibition of SHP‐2 ameliorates experimental autoimmune encephalomyelitis in mice

In contrast to T‐cell priming in the periphery, therapeutic strategies targeting the initiation step of T‐cell trafficking into the CNS have not been extensively investigated. In this study, we examined the effect of NSC‐87877, a potent Src homology 2‐containing protein tyrosine phosphatase 2 (SHP‐2) inhibitor, on experimental autoimmune encephalomyelitis (EAE) and elucidated its unique mechanism of action.

Dregea volubilis ameliorates concanavalin A-induced liver injury by facilitating apoptosis of activated T cells.

Activation of T cells is a critical event in the pathogenesis of concanavalin A (Con A)-induced liver injury, and facilitating apoptosis of activated T cells may provide a strategy for the treatment. Here, we found that the ethanol extract from the stem parts of Dregea volubilis (DVE) inhibited cell proliferation and induced apoptosis, which was selective for Con A-activated, rather than nonactivated, lymph node cells. Administration of DVE prevented mice from Con A-induced elevation of serum transaminases, liver necrosis and increased TNF-α, IFN-γ, IL-2 and IL-4 in mice sera. DVE also caused apoptosis of in vivo activated T cells. In addition, increased active fragments of caspase-3 were found in the DVE-treated cells. But DVE-induced apoptosis was Fas-independent, as it was still observed in T cells from Fas ligand-mutated gld/gld mice. These results suggest that DVE may have great potential to treat T cell-mediated diseases through facilitating apoptosis of activated T cells.