China Medical University, Taichung, Taiwan, China.
Acta Pharmacol Sin. 2010 Feb;31(2):227-36. doi: 10.1038/aps.2009.197.
To study the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family using microarray analysis.
Human kidney (HK-2) cells were treated with AA (0, 10, 30, and 90 micromol/L) for 24 h, and the cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA in triplicate. A quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay was used to verify the microarray data for selected nuclear factor kappa B (NF-kappaB)-regulated genes. Furthermore, the subcellular localization of NF-kappaB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. The NF-kappaB activity was examined by a luciferase reporter assay in HK-2/NF-kappaB transgenic cells.
AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in the gene expression profiles related to the DNA damage response, DNA repair, macromolecule metabolic process, carbohydrate metabolic process, DNA metabolic process, apoptosis, cell cycle, and transcription. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. A network analysis revealed that NF-kappaB played a central role in the network topology. Among NF-kappaB-regulated genes, 8 differentially expressed genes were verified by qRT-PCR. The inhibition of NF-kappaB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-kappaB luciferase reporter assay.
Our data revealed that AA could suppress NF-kappaB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia.
使用基因芯片分析研究马兜铃酸（Aristolochic acid，AA）作为马兜铃科植物的主要活性成分的作用机制。
将人肾（HK-2）细胞用 AA（0、10、30 和 90 μmol/L）处理 24 小时，用 3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四氮唑溴盐测定细胞活力。用 cDNA 微阵列分析重复检测暴露于 AA 的 HK-2 细胞的基因表达模式。用定量逆转录聚合酶链反应（qRT-PCR）检测选择的核因子 kappa B（NF-kappaB）调控基因的微阵列数据。此外，通过免疫荧光共聚焦显微镜观察 HK-2 细胞中 NF-kappaB p65 的亚细胞定位。在 HK-2/NF-kappaB 转基因细胞中通过荧光素酶报告基因检测 NF-kappaB 活性。
AA 在 HK-2 细胞中表现出剂量依赖性细胞毒性作用，并诱导与 DNA 损伤反应、DNA 修复、大分子代谢过程、碳水化合物代谢过程、DNA 代谢过程、细胞凋亡、细胞周期和转录相关的基因表达谱发生改变。此外，在 AA 处理的 HK-2 细胞中下调了 9 个与免疫调节功能相关的生物途径。网络分析表明，NF-kappaB 在网络拓扑中起核心作用。在 NF-kappaB 调控的基因中，通过 qRT-PCR 验证了 8 个差异表达基因。通过免疫荧光共聚焦显微镜和 NF-kappaB 荧光素酶报告基因检测进一步证实了 AA 对 NF-kappaB 活性的抑制作用。
我们的数据表明，AA 可以抑制正常人类细胞中的 NF-kappaB 活性，这可能部分解释了一些马兜铃属植物的抗炎作用。
