Xingxin Wu

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 Sequestration of STAT1 in the Cytoplasm via Phosphorylated SHP-2 Ameliorates Murine Experimental Colitis

The side effects of current immunosuppressive drugs have impeded the development of therapies for immune diseases. Selective regulation of STAT signaling is an attractive strategy for treating immune disorders. In this study, we used a small-molecule compound to explore possible means of targeting STAT1 for the treatment of Th1-mediated inflammation. Selective regulation of STAT1 signaling in T cells from C57BL/6 mice was accomplished using fusaruside, a small-molecule compound that triggers the tyrosine phosphorylation of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2). The interaction of tyrosine phosphorylated SHP-2 (pY-SHP-2) with cytosolic STAT1 prevented the recruitment of STAT1 to IFN-γR and specifically inhibited STAT1 signaling, resulting in a reduction in Th1 cytokine production and an improvement in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice. Blocking the pY-SHP-2–STAT1 interaction, with SHP-2 inhibitor NSC-87877 or using T cells from conditional SHP-2 knockout mice, reversed the effects of fusaruside, resulting in STAT1 activation and worsened colitis. The fusaruside-induced ability of pY-SHP-2 to selectively sequestrate STAT1 from recruitment to the receptor is independent of its function as a phosphatase, demonstrating a novel role for SHP-2 in regulating both STAT1 signaling and Th1-type immune responses. These findings could lead to increased options for the treatment of Crohn’s disease and other Th1-mediated inflammatory diseases.

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.

Sorafenib induces apoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3 phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is constitutively active in approximately 50% of acute myeloid leukemia (AML) cases and mediates multiple cellular processes including cell resistance to apoptosis. Inhibition of constitutively active STAT3 has been shown to induce AML cell apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit STAT3 signaling and, therefore, be efficacious for AML. We found that sorafenib inhibited proliferation and induced apoptosis in human AML cell line (HL60) cells. In addition, sorafenib exposure reduced constitutive STAT3 phosphorylation in HL60 cells and repressed STAT3 DNA-binding activity and Mcl-1 and Bcl-2 expression. Similar results were obtained with the Src kinase inhibitor I, suggesting that sorafenib suppresses STAT3 phosphorylation by inhibiting Src-kinase activity. Furthermore, significant inhibition of Src kinase activity by sorafenib was observed in the kinase assay. In addition, Src could be coimmunoprecipitated with STAT3, and the phosphorylation of STAT3 was significantly inhibited by sorafenib only in cell lines in which phosphorylated Src is highly expressed. Taken together, our study indicates that sorafenib blocks Src kinase-mediated STAT3 phosphorylation and decreases the expression of apoptosis regulatory proteins Mcl-1 and Bcl-2, which are associated with increased apoptosis in HL60 cells. These findings provide a rationale for the treatment of human AML.

CUG-binding protein 1 regulates HSC activation and liver fibrogenesis

Excessive activation of hepatic stellate cells (HSCs) is a key step in liver fibrogenesis. Here we report that CUG-binding protein 1 (CUGBP1) expression is elevated in HSCs and positively correlates with liver fibrosis severity in human liver biopsies. Transforming growth factor-beta (TGF-β) selectively increases CUGBP1 expression in cultured HSCs in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Knockdown of CUGBP1 inhibits alpha smooth muscle actin (α-SMA) expression and promotes interferon gamma (IFN-γ) production in HSCs in vitro. We further show that CUGBP1 specifically binds to the 3′ untranslated region (UTR) of human IFN-γ mRNA and promotes its decay. In mice, knockdown of CUGBP1 alleviates, whereas its overexpression exacerbates, bile duct ligation (BDL)-induced hepatic fibrosis. Therefore, CUGBP1-mediated IFN-γ mRNA decay is a key event for profibrotic TGF-β-dependent activation of HSCs, and inhibiting CUGBP1 to promote IFN-γ signalling in activated HSCs could be a novel strategy to treat liver fibrosis.

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.

Cerebroside D, a glycoceramide compound, improves experimental colitis in mice with multiple targets against activated T lymphocytes

In the present paper, we aimed to examine the novel effects of cerebroside D, a glycoceramide compound, on murine experimental colitis. Cerebroside D significantly reduced the weight loss, mortality rate and alleviated the macroscopic and microscopic appearances of colitis induced by dexran sulfate sodium. This compound also decreased the levels of TNF-α, IFN-γ and IL-1β in intestinal tissue of mice with experimental colitis in a concentration-dependent manner, accompanied with markedly increased serum level of IL-10. Cerebroside D inhibited proliferation and induced apoptosis of T cells activated by concanavalin A or anti-CD3 plus anti-CD28 antibodies. The compound did not show an effect on naive lymphocytes but prevented cells from entering S phase and G2/M phase during T cells activation. Moreover, the treatment of cerebroside D led to apoptosis of activated T cells with the cleavage of caspase 3, 9, 12 and PARP. These results showed multiple effects of cerebroside D against activated T cells for a novel approach to treatment of colonic inflammation.

Cortex Dictamni extract induces apoptosis of activated hepatic stellate cells via STAT1 and attenuates liver fibrosis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE In traditional Chinese medicines, Cortex Dictamni is prescribed for the treatment of a variety of inflammatory diseases such as acute rheumatoid arthritis, skin inflammation and jaundice. AIM OF THE STUDY This study was designed to investigate the effect of ethanol extract of Cortex Dictamni on treatment of hepatic fibrosis and its possible mechanisms. MATERIALS AND METHODS The in vivo effect of Cortex Dictamni extract (CDE) was evaluated by measuring histological changes and collagen content in CCl(4)-indcued hepatic fibrosis mice. Viability, apoptosis and protein expression of hepatic stellate cells (HSC) were analyzed by MTT, Annexin V staining and Western blot respectively. RESULTS CDE alleviated CCl(4)-induced hepatic fibrosis in mice and showed a much stronger inhibition of cell viability in activated HSC cell line HSC-T6 than that in normal hepatocyte L02 cells. Furthermore, CDE induced apoptosis of HSC-T6 cells associated with increased expressions of cleaved PARP and cleaved caspase-3. Interestingly, CDE activated STAT1 in HSC-T6 cells and the effect of CDE on apoptosis of HSC-T6 cells could be neutralized using JAK/STAT1 signaling inhibitor AG490. CONCLUSIONS These findings suggest that CDE possesses anti-fibrosis activity with selectively induction of activated HSC apoptosis via activating STAT1, which might be a novel strategy for hepatic fibrosis therapy.

Andrographolide alleviates imiquimod-induced psoriasis in mice via inducing autophagic proteolysis of MyD88

Psoriasis is a chronic inflammatory skin disease with excessive activation of toll-like receptors (TLRs), which play important roles in developing psoriasis. Targeting TLR signaling remains a challenge for treating psoriasis. Here, we found that andrographolide (Andro), a small-molecule natural product, alleviated imiquimod- but not interleukin 23 (IL-23)-induced psoriasis in mice with reducing expressions of IL-23 and IL-1β in the skin. The improvement in imiquimod-induced psoriasis by Andro was not observed in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) knockout mice. Furthermore, Andro inhibited mRNA expressions of IL-23, IL-6 and IL-1β but not CD80 and CD86 in bone-marrow derived dendritic cells (BMDCs) treated with lipopolysaccharide (LPS) in a MAP1LC3B-dependent manner. In addition, Andro inhibited imiquimod-induced mRNA expressions of IL-23, IL-6, IL-1β, CD80 and CD86 in BMDCs from mice. Interestingly, Andro induced a degradation of myeloid differentiation factor 88 (MyD88) and blocked the recruitment of TNF receptor-associated factor 6 (TRAF6) to MyD88 upon LPS stimulation in BMDCs from mice. Blockade of autophagic proteolysis using NH4Cl or MAP1LC3B(-/-) BMDCs abolished the Andro-induced MyD88 degradation. In conclusion, Andro controls activation of MyD88-dependent cytokines and alleviates psoriasis in mice via inducing autophagic proteolysis of MyD88, which could be a novel strategy to treat psoriasis.