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. 2010 Feb;31(2):227-36.
doi: 10.1038/aps.2009.197.

Microarray analysis reveals the inhibition of nuclear factor-kappa B signaling by aristolochic acid in normal human kidney (HK-2) cells

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Microarray analysis reveals the inhibition of nuclear factor-kappa B signaling by aristolochic acid in normal human kidney (HK-2) cells

Ya-yin Chen et al. Acta Pharmacol Sin. 2010 Feb.

Abstract

Aim: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
The cytotoxicity of various doses of AA in HK-2 cells. HK-2 cells were treated with 0, 10, 30, and 90 μmol/L of AA. The cell viability was measured by MTT assay. Values are mean±SD of three independent experiments. cP<0.01 vs control group.
Figure 2
Figure 2
Network analysis of expression profiles of 10 μmol/L AA in HK-2 cells. We constructed the interaction network by BiblioSphere Pathway Edition software which was selected the genes with fold changes >2.0 and false discovery rates <0.05 after 10 μmol/L AA treatment. Intensity of nodes for regulated genes and NFKB1 are color-coded according to their log2 expression values in gradient with red and green indicating the degree of gene upregulation or downregulation, respectively. As shown, NF-κB played a central role in the network.
Figure 3
Figure 3
Cellular localization of the p65 subunit of NF-κB before and after AA treatment in HK-2 cells. NF-κB localization was visualized in panel D–F (green) and PI was used for nuclear staining (red) (panel G–I) by immunofluorescence and confocal microscopy. The nuclear distribution of NF-κB in AA-treated cells was less apparent than that in normal HK-2 cells (A–F).
Figure 4
Figure 4
The inhibitory effect of 10 μmol/L AA on NF-κB activity in HK-2 cells. HK-2 cells were transfected with NF-κB luciferase gene and were subsequently incubated with 10 μmol/L AA for 24 h. Experiments were done in triplicate and all results of luciferase assay expressed as relative luciferase units (RLU). Relative luciferase activity was calculated by dividing RLU of non-transfected cells, empty vector (pcDNA 3.1)-transfected cells, and AA-treated NF-κB-transfected cells by the RLU of untreated NF-κB-transfected cells. Error bars represent mean±SD of relative luciferase activity. cP<0.01 vs untreated NF-κB-transfected cells.

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References

    1. Rosenmund H, Reichstein T. Zur Kenntnis der Aristolochiasäure. Pharm Acta Helv. 1943;18:243–61.
    1. Debelle FD, Vanherweghem JL, Nortier JL. Aristolochic acid nephropathy: a worldwide problem. Kidney Int. 2008;74:158–69. - PubMed
    1. Zhang G, Shimokawa S, Mochizuki M, Kumamoto T, Nakanishi W, Watanabe T, et al. Chemical constituents of Aristolochia constricta: antispasmodic effects of its constituents in guinea-pig ileum and isolation of a diterpeno-lignan hybrid. J Nat Prod. 2008;71:1167–72. - PubMed
    1. Messiano GB, Vieira L, Machado MB, Lopes LM, de Bortoli SA, Zukerman-Schpector J. Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis. J Agric Food Chem. 2008;56:2655–9. - PubMed
    1. Hinou J, Demetzos C, Harvala C, Roussakis C. Cytotoxic and antimicrobial principles from the roots of Aristolochia longa. Int J Crude Drug Res. 1990;28:149–51.

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