Background and purpose: Traditionally, the stem and root bark of Ulmus davidiana var. japonica (Ulmaceae) have been known to be anti-inflammatory in Korea. Anti-inflammatory effects of torilin, isolated from this plant and the underlying mechanisms were examined by using lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Experimental approach: The cells were treated with torilin prior to LPS exposure and the effects on pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and a pro-inflammatory cytokine, interleukin-1b (IL-1b) were analysed by RT-PCR, Western blot or ELISA. To reveal the mechanism of action of torilin we investigated the involvement of mitogen-activated protein kinase (MAPK) cascades and their downstream transcription factors, nuclear factor-kB (NF-kB) and cyclic AMP-responsive element (CRE)-binding protein (CREB). Key results: Torilin significantly reduced the LPS-induced expression of iNOS, COX-2 and IL-1b, and the subsequent release of NO, prostaglandin E2 and IL-1b into culture medium. LPS stimulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK was inhibited by torilin. In addition, the inhibitory effect of torilin on NF-kB and CREB was shown by torilin-mediated recovery of LPS-induced degradation of inhibitor kB-a and suppression of LPS-induced phosphorylation of CREB respectively. Conclusion and implications:This study indicates that torilin inhibited LPS-induced iNOS, COX-2 and IL-1b via downregulation of ERK1/2, p38 MAPK, NF-kB and CREB and suggests that torilin has a potential as an anti-inflammatory drug candidate.
Background and purpose: Apoptosis is a fundamental process required for neuronal development but also occurs in most of the common neurodegenerative disorders. In an attempt to obtain an anti-apoptotic neuroprotective compound from natural products, we isolated the diterpenoids, pinusolide and 15-MPA, from B. orientalis and investigated their neuroprotective activity against staurosporine (STS) -induced neuronal apoptosis. In addition, we determined the anti-apoptotic mechanism of these compounds in rat cortical cells. Experimental approach: Primary cultures of rat cortical cells injured by STS were used as an in vitro assay system. Cells were pretreated with pinusolide or 15-MPA before exposure to STS. Anti-apoptotic activities were evaluated by the measurement of cytoplasmic condensation and nuclear fragmentation. The levels of cellular peroxide, malondialdehyde (MDA) and [Ca 2 þ ] i , as well as the activities of superoxide dismutase (SOD) and caspase-3/7, were measured. Key results: Pinusolide and 15-MPA, at a concentration of 5.0 ìM, reduced the condensed nuclei and rise in [Ca 2 þ ] i that accompanies apoptosis induced by 100 nM STS. Pinusolide and 15-MPA also protected the cellular activity of SOD, an antioxidative enzyme reduced by STS insult. Furthermore, the overproduction of reactive oxygen species and lipid peroxidation induced by STS was significantly reduced in pinusolide and 15-MPA treated cells. In addition, pinusolide and 15-MPA inhibited STS-induced caspase-3/7 activation. Conclusions and Implications: These results show that pinusolide and 15-MPA protect neuronal cells from STS-induced apoptosis, probably by preventing the increase in [Ca 2 þ ] i and cellular oxidation caused by STS, and indicate that they could be used to treat neurodegenerative diseases.
Background and purpose:We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45a, gadd45g and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA.
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