Oligonucleotide microarray analysis of apoptosis induced by 15‐methoxypinusolidic acid in microglial BV2 cells

Abstract

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.

Mice Homozygous for a Deletion in the Glaucoma Susceptibility Locus INK4 Show Increased Vulnerability of Retinal Ganglion Cells to Elevated Intraocular Pressure

“…95 CDKN2B was described as one of those genes that were up-regulated in microglia after treatment with the plant compound 15-methoxypinusolidic acid that is under investigation as a potential inhibitor of neuroinflammation and microglial activation. 96 The expression levels of CDKN2B are reduced significantly in the MUT mice, which may have the opposite effect of inducing microglial activation even without IOP elevation. Microglial activation in the retina and the optic nerve is an early indicator of glaucoma that precedes ganglion cell loss, 84,90,93 and neuroinflammation plays a role in the pathogenesis of glaucoma.…”

Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

“…Microarray analyses have matured sufficiently in terms of accessibility and cost to make their application to this question tractable. We are not the first to make use of microarray technology to examine mRNA expression levels in HEK293 cells [ 26 ], however, most studies that have used microarray analyses on these cell lines have examined specific gene products of interest and reported data only for those gene products [ 20 , 26 - 28 ]. Their full data sets are available through NCBI, but are difficult for the non-specialist user to access and collate.…”

Recent progress on the traditional Chinese medicines that regulate the blood

“…15-MPA, not pinusolide, suppressed adipocyte differentiation in a dose-dependent manner (10–200 μM), as revealed by lipid droplet formation and expression of adipogenic genes dependent on peroxisome proliferator-activated receptor-γ, such as adiponectin and adipocyte protein 2 (aP2) [ 29 ]. In addition, 15-MPA induced apoptosis in murine microglial cells (12.5–50 μM), presumably via inhibition of cell cycle progression [ 30 ]. Moreover, 15-MPA can inhibit LPS-induced iNOS expression and NO production, independent of mitogen-activated protein kinase (MAPK) and NF-κB in microglial cells [ 31 ].…”