College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Korea.
Br J Pharmacol. 2009 Jul;157(6):1053-64. doi: 10.1111/j.1476-5381.2009.00247.x. Epub 2009 May 19.
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.
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.
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 [gadd45alpha, gadd45gamma 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.
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.
我们进行了全基因组基因表达分析，以探索从东方生物中分离出的 15-甲氧基胡椒酸（15-MPA）的生物学方面，并试图确认 15-MPA 作为治疗中枢神经系统损伤的候选药物的适用性，重点是小胶质细胞。
用 15-MPA 处理鼠小胶质细胞 BV2 细胞，并通过寡核苷酸微阵列分析其转录组。选择经 15-MPA 处理后差异表达的基因进行 RT-PCR（逆转录-聚合酶链反应）分析，以确认基因表达。通过溴脱氧尿苷测定、多聚 ADP-核糖聚合酶的 Western blot 分析和流式细胞术检测 15-MPA 对细胞增殖的抑制和诱导凋亡作用。
15-MPA 处理共诱导 514 个基因差异表达。生物途径分析表明，15-MPA 诱导细胞周期途径中基因表达发生显著变化。参与生长停滞和 DNA 损伤的基因[gadd45alpha、gadd45gamma 和 ddit3（DNA 损伤诱导转录 3）]和细胞周期蛋白依赖性激酶抑制剂（cdkn2b）上调，而参与细胞周期进程的基因（ccnd1、ccnd3 和 ccne1）、DNA 复制（mcm4、orc1l 和 cdc6）和细胞增殖（fos 和 jun）下调。代表性基因的 RT-PCR 分析证实了表达水平。15-MPA 显著减少溴脱氧尿苷掺入，增加多聚 ADP-核糖聚合酶裂解和凋亡细胞数量，表明 15-MPA 诱导 BV2 细胞凋亡。
15-MPA 诱导鼠小胶质细胞凋亡，可能通过抑制细胞周期进程。由于小胶质细胞激活在中枢神经系统损伤中是有害的，这些数据表明 15-MPA 具有很强的治疗潜力。
