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

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 μmol/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-κB)-regulated genes. Furthermore, the subcellular localization of NF-κB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. The NF-κB activity was examined by a luciferase reporter assay in HK-2/NF-κB 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 downregulated in AA-treated HK-2 cells. A network analysis revealed that NF-κB played a central role in the network topology. Among NF-κB-regulated genes, 8 differentially expressed genes were verified by qRT-PCR. The inhibition of NF-κB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-κB luciferase reporter assay. Conclusion: Our data revealed that AA could suppress NF-κB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia.Keywords: aristolochic acid; microarray analysis; nuclear factor-kappa B; human kidney HK-2 cells; confocal microscopy; luciferase reporter assay Acta Pharmacologica Sinica (2010) 31: 227-236; doi: 10.1038/aps.2009 Original Article # These authors contributed equally to this work. * To whom correspondence should be addressed. [7,8] . AA was found to possess anti-inflammation effects as demonstrated by its ability to inhibit phospholipase A 2 (PLA 2 ) when administered by intramuscular or intraperitoneal injection [9,10] . Furthermore, AA was also reported to inhibit Group I PLA 2 in humans with sepsis [11] . From in vitro studies, AA has been shown to suppress phospholipohydration of PLA 2 derived from human synovial fluid, cobra venom, porcine pancreas, and human platelets [12] . The anti-inflammatory activities of AA in different models of inflammation have promoted its use in many countries in herbal formulations for arthritis, rheumatism, gout and chronic inflammatory skin diseases [13,14] . Moreover, double-blind studies in healthy volunteers show that AA increased the phagocytic activity of peripheral granulocytes after treatment with AA 0.9 mg/d for three to ten consecutive days [...

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