Effect of echinalkamide identified from Echinacea purpurea (L.) Moench on the inhibition of osteoclastogenesis and bone resorption.

Plant cell cultures have been exploited to provide stable production and new secondary metabolites for better pharmacological activity. Fractionation of adventitious root cultures of Echinacea purpurea resulted in the isolation of eleven constituents, including three new compounds. The structures of the three new compounds were determined to be an alkylamide (1), a polyacetylene (2) and a lignan (3) on the basis of combined spectroscopic analysis. To discover new types of antiresorptive agents, we screened for new compounds that regulate osteoclast differentiation, and survival. Among three new compounds, echinalkamide (compound 1) had considerably inhibitory effects on RANKL-induced osteoclast differentiation, and on proliferation of osteoclasts and efficiently attenuated osteoclastic bone resorption without toxicity. In addition, echinalamide treatment inhibited the osteoclast-specific gene expression level. Echinalkamide achieved this inhibitory effect by disturbing phosphorylation of MAPK and activation of osteoclast transcription factors c-Fos and NFATc1. Conclusionally, our study investigated that echinalkamide remarkably inhibited osteoclast differentiation and osteoclast specific gene expression through repression of the MAPK-c-Fos-NFATC1 cascade.

Neogambogic Acid Suppresses Receptor Activator of Nuclear Factor κB Ligand (RANKL)-Induced Osteoclastogenesis by Inhibiting the JNK and NF-κB Pathways in Mouse Bone Marrow-Derived Monocyte/Macrophages.

BACKGROUND Neogambogic acid (NGA) is used in traditional Chinese medicine. The aim of this study was to investigate the effects of NGA on gene signaling pathways involved in osteoclastogenesis in mouse bone marrow-derived monocyte/macrophages (BMMs) and on bone resorption in vitro. MATERIAL AND METHODS Primary mouse BMMs were cultured with increasing concentrations of NGA. Real-time polymerase chain reaction was used to study the expression of mRNAs corresponding to gene products specific to receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, including tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), cathepsin K (CTSK), and nuclear factor of activated T cells c1 (NFATc1). A cell counting kit-8 assay was used to evaluate cell proliferation. Western blotting and confocal immunofluorescence microscopy were used to investigate the signaling pathways. A bone resorption model was used to quantify bone resorption. RESULTS An NGA dose of ≤0.4 µg/ml had no significant effect on the proliferation of mouse BMMs in vitro (P>0.05); concentrations of between 0.1-0.4 µg/ml significantly inhibited RANKL-induced osteoclastogenesis (P<0.01) in a dose-dependent manner. Compared with the control group, NGA significantly reduced RANKL-induced bone resorption in vitro (P <0.01), and downregulated the expression of osteoclast-related mRNAs of TRAP, CTR, CTSK, and NFATc1. NGA suppressed the activation of JNK but not the p38 signaling pathway and significantly reduced NF-κB p65 phosphorylation and the nuclear transport of NF-κB molecules, which inhibited NFATc1 expression. CONCLUSIONS NGA suppressed RANKL-induced osteoclastogenesis by inhibiting the JNK and NF-κB pathways in mouse BMMs in vitro and reduced osteoclastic bone resorption.

Glechoma hederacea Suppresses RANKL-mediated Osteoclastogenesis.

Glechoma hederacea (GH), commonly known as ground-ivy or gill-over-the-ground, has been extensively used in folk remedies for relieving symptoms of inflammatory disorders. However, the molecular mechanisms underlying the therapeutic action of GH are poorly understood. Here, we demonstrate that GH constituents inhibit osteoclastogenesis by abrogating receptor activator of nuclear κ-B ligand (RANKL)-induced free cytosolic Ca(2+) ([Ca(2+)]i) oscillations. To evaluate the effect of GH on osteoclastogenesis, we assessed the formation of multi-nucleated cells (MNCs), enzymatic activity of tartrate-resistant acidic phosphatase (TRAP), expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and [Ca(2+)]i alterations in response to treatment with GH ethanol extract (GHE) in primarily cultured bone marrow-derived macrophages (BMMs). Treatment of RANKL-stimulated or non-stimulated BMMs with GHE markedly suppressed MNC formation, TRAP activity, and NFATc1 expression in a dose-dependent manner. Additionally, GHE treatment induced a large transient elevation in [Ca(2+)]i while suppressing RANKL-induced [Ca(2+)]i oscillations, which are essential for NFATc1 activation. GHE-evoked increase in [Ca(2+)]i was dependent on extracellular Ca(2+) and was inhibited by 1,4-dihydropyridine (DHP), inhibitor of voltage-gated Ca(2+) channels (VGCCs), but was independent of store-operated Ca(2+) channels. Notably, after transient [Ca(2+)] elevation, treatment with GHE desensitized the VGCCs, resulting in an abrogation of RANKL-induced [Ca(2+)]i oscillations and MNC formation. These findings demonstrate that treatment of BMMs with GHE suppresses RANKL-mediated osteoclastogenesis by activating and then desensitizing DHP-sensitive VGCCs, suggesting potential applications of GH in the treatment of bone disorders, such as periodontitis, osteoporosis, and rheumatoid arthritis.

NF-kappaB inhibitors from Brucea javanica exhibiting intracellular effects on reactive oxygen species.

AIM: Brucea javanica was studied to identify nuclear factor kappaB (NF-κB) inhibitors exhibiting reactive oxygen species (ROS) intracellular amplification. MATERIAL AND METHODS: Eight compounds were evaluated for selective cytotoxicity using HT-29, HeLa, and HL-60 cells, and in a NF-κB assay. Active compounds were then tested using ROS and mitochondria transmembrane potential (MTP) assays. NF-κB and nuclear factor activated T-cell (NFAT) translocation were also assessed using their respective whole cell assays. RESULTS: Bruceajavanone B, bruceantin, bruceine A, (-)-hydnocarpin, and chrysoeriol exhibited cytotoxic potential and NF-κB p65 inhibition. Chrysoeriol exhibited selective cytotoxicity against leukemia cells with greater potency and also showed an ability to up-regulate NFAT transcriptional pathways through the amplification of intracellular ROS, in the presence of H2O2, to a greater degree than bruceantin and bruceine. CONCLUSION: Chrysoeriol selectively kills leukemic cells and potentiates the amplification of ROS levels. Therefore, chrysoeriol could serve as a potential chemotherapeutic modifier for leukemia chemotherapy since leukemia cells have a higher susceptibility to elevated ROS levels.

Immunopotentiation on murine spleen lymphocytes induced by polysaccharide fraction of Panax ginseng via upregulating calcineurin activity.

Calcineurin (CN), a unique Ca2+/calmodulin (CaM)-dependent serine/threonine protein phosphatase, plays a pivotal role in the activation and proliferation of T lymphocytes. Based on the effective molecular screening model established in our laboratory, we found that a part of polysaccharides from the stem and leaves of Panax ginseng, termed PGP-SL, could activate CN activity. Subsequently, we investigated whether PGP-SL also has immunological competence on murine spleen lymphocytes. In the present study, we demonstrated that PGP-SL could significantly promote in vitro spleen lymphocyte proliferation in the absence of either concanavalin A or LPS in a concentration-dependent manner at concentrations ranging from 100 to 500 microg/ml (p<0.001). In addition, the proliferation of cyclosporin A (CsA)-treated spleen lymphocytes was also significantly promoted in the same pattern (p<0.001); the production of IL-2 was elevated and the effect appeared as early as 24 h after PGP-SL treatment. The results of RT-PCR also indicated that the IL-2 mRNA level was markedly enhanced, particularly at PGP-SL concentrations of 300 and 500 microg/ml, and Fura-2/AM fluorescence probe analysis showed that PGP-SL could dramatically increase the intracellular free calcium concentration of spleen lymphocytes, i.e. [Ca2+]i was significantly increased by approximately 181 and 107% at 300 and 500 microg/ml of PGP-SL, respectively. However, this effect could be totally inhibited by verapamil treatment. Taking our results together, we suggest that PGP-SL exhibits immunopotentiation effects on murine spleen lymphocytes by the Ca2+-CN-NFAT-IL-2 signaling pathway.

Mechanistic study of saikosaponin-d (Ssd) on suppression of murine T lymphocyte activation.

Saikosaponin-d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti-inflammatory, anti-bacterial, anti-viral and anti-cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF-kappaB, NF-AT and AP-1 signaling pathways, cytokine secretion, and IL-2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28-costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A-induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA-induced T cell activation was associated with down-regulation of NF-kappaB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF-AT and activator protein 1 (AP-1) of the PMA/Ionomycin-stimulated T cells. The cell surface markers like IL-2 receptor (CD25) were also down-regulated together with decreased production of pro-inflammatory cytokines of IL-6, TNF-alpha and IFN-gamma. These results indicate that the NF-kappaB, NF-AT and AP-1 (c-Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell-mediated autoimmune conditions.

Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

BACKGROUND AND PURPOSE: Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. We purified suberosin from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). EXPERIMENTAL APPROACH: Proliferation of PBMC in culture was measured by uptake of 3H-thymidine; production of cytokines and cyclins by Western blotting and RT-PCR. Transcription factors NF-AT and NF-kappaB were assayed by immunocytochemistry and EMSA. KEY RESULTS: Suberosin suppressed PHA-induced PBMC proliferation and arrested cell cycle progression from the G1 transition to the S phase. Suberosin suppressed, in activated PBMC, transcripts of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and cyclins D3, E, A, and B. DNA binding activity and nuclear translocation of NF-AT and NF-kappaB induced by PHA were blocked by suberosin. Suberosin decreased the rise in intracellular Ca2+ concentration ([Ca2+]i) in PBMC stimulated with PHA. Suberosin did not affect phosphorylation of p38 and JNK but did reduce activation of ERK in PHA-treated PBMC. Pharmacological inhibitors of NF-kappaB, NF-AT, and ERK decreased expression of mRNA for the cyclins, IL-2, and IFN-gamma and cell proliferation in PBMC activated by PHA. CONCLUSIONS AND IMPLICATIONS: The inhibitory effects of suberosin on PHA-induced PBMC proliferation, were mediated, at least in part, through reduction of [Ca2+]i, ERK, NF-AT, and NF-kappaB activation, and early gene expression in PBMC including cyclins and cytokines, and arrest of cell cycle progression in the cells. Our observations provide an explanation for the anti-inflammatory activity of P. zeylanica.