| Summary: | 博士 === 中國醫藥大學 === 中醫學系 === 98 === Plant species of the genus Aristolochia are known for many therapeutic effects in past centuries. For example, there are many formulas containing
various species of the genus Aristolochia that are commonly used in traditional medicine in Taiwan, China, Japan, and Singapore. Beside in East Asia, several species of the genus Aristolochia have been used to regulate menstruation, induce labor, expel parasites and treat arthritis, cancer and snake bites in India, West Africa, the Mediterranean, and South America. Many plant species of the genus Aristolochia were commonly used as folk medicine until 1990’ s when studies showed that aristolochic acid (AA), the major active component of the genus Aristolochia induces aristolochic acid nephropathy (AAN) and urothelial cancer in AAN patients. In this study, we used microarray analysis to clarify the pharmacological and toxicological mechanism of AA (AA I：AA II= 41 % : 56 %) in normal human kidney proximal tubular (HK-2) cells, the target cells of AA.
HK-2 cells were treated with AA (0, 10, 30, 90 μM) for 24 hours and 30 μM for 1, 3, 6, 12, and 24 hours. 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 and the results of this microarray study were in triplicate. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was used to verify the microarray data for selected genes. The results showed that AA induced cytotoxic effects in a dose-dependent manner (10, 30, 90 μM for 24 h) and a time-dependent manner (30 μM for 1, 3, 6, 12, and 24 h). Microarray analysis showed that AA can induce alterations in expression profiles of genes related to DNA damage response, apoptosis, regulation of cell cycle, and stress response. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. 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 real-time RT-PCR. Inhibition of NF-κB activity by AA was confirmed by immunofluorescence confocal microscopy and by NF-κB luciferase reporter assay.
Moreover, among the differentially expressed genes analyzed by Limma and Ingenuity Pathway Analysis software, we found that genes related to DNA repair processes were the most significantly regulated by all AA treatments. Among the differentially expressed genes found in the dose-response and time-course studies that were involved in those biological processes, two up-regulated genes (GADD45B, NAIP), and six down-regulated genes (TP53, PARP1, OGG1, ERCC1, ERCC2, and MGMT) were confirmed by qRT-PCR. AA exposure also caused a down-regulation of the gene expression of antioxidant enzymes, such as superoxide dismutase, glutathione reductase, and glutathione peroxidase. AA treatment led to increased frequency of DNA strand breaks, 8-hydroxydeoxyguanosine-positive nuclei, and micronuclei in a dose-dependent manner in HK-2 cells, possibly as a result of the inhibition of DNA repair. These data suggest that oxidative stress plays a role in the cytotoxicity of AA.
In 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. In addition, our results provide insight into the involvement of suppression of DNA repair genes, and trigger DNA strand breaks, oxidative DNA damages, and chromosome damages in normal human kidney proximal tubular cells. These data may provide some guidance to TCM doctors on how to use AA-containing herbs safely.
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