馬兜鈴酸誘發人類腎臟近曲小管細胞之氧化壓力和 DNA 損壞   全文下載

Induction of Oxidative Stress and DNA Damage in Human Renal Proximal Tubular Cells by Aristolochic Acid

馬兜鈴酸(aristolochic acid i，aai)主要是馬兜鈴酸屬的植物萃取物，服用aai會造成永久性的腎臟傷害及泌尿道細胞癌。本研究利用腎臟近曲小管上皮細胞株（HK-2）為模式，以此檢驗aai所誘發之細胞內氧化壓力和dna損傷以及maPK訊息路徑活化之間的關連性。高濃度的aai會導致HK-2細胞的存活率下降以及Caspase 3活性的上升，aai處理亦導致細胞內活性氧分子（ROS）的含量顯著提高，藉由抗氧化劑麩胱甘?（gSH）添加可抑制 ROS的增加；使用單細胞凝膠電泳分析證實 aai 使 HK-2 細胞中 dna 斷裂的情形加劇，此外經 aai亦可活化細胞內 nrf2 結合序列並提高下游 luciferase 的酵素活性 。進一步探討ROS生成與maPKs訊息傳遞路徑之關聯。aai處理HK-2細胞會令細胞內eRK1/2和p38磷酸化的訊息增強，若以meK1/2抑制劑U0126前處理，不但會抑制aai所活化的eRK1/2磷酸化，也會同時降低aai所誘發的ROS生成量。此外，不論是U0126 或是p38抑制劑SB202190皆能顯著降低aai所引起的dna損傷。由此得知aai主要藉由meK/eRK1/2的訊息傳遞途徑促使ROS產生，而aai所造成的dna傷害則同時與eRK1/2和p38途徑的活化有關。

Aristolochic acid I (AAI) is found primarily in the plant Aristolochia. Consumption of products containing AAI has been linked with permanent kidney damage and urothelial carcinoma. This study applied human proximal tubule epithelial cell line (HK-2) to examine the relationship among AAI-induced intracellular oxidative stress, DNA damage and MAP kinase activation. High concentrations of AAI caused a decrease in cell viability and an increase in the activity of caspase 3. AAI treatment also led to a dose-dependent increase of reactive oxygen species (ROS) in HK-2 cells, and the presence of antioxidant glutathione (GSH) effectively inhibited ROS generation. Stimulating HK-2 cultures with AAI also led to GSH depletion. Results from single cell gel electrophoresis (SCGE) assays demonstrated that AAI showed the ability to increase the levels of DNA strand breaks in HK-2 cells. Up-regulation of luciferase activity driven by the Nrf2 binding element was also observed after 200 µM AAI treatment. Exposure of HK-2 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only the MEK1/2 inhibitor, U0126, significantly decreased the levels of AAI-mediated ROS. In addition, both U0126 and SB202190 effectively reversed the levels of DNA damage triggered by AAI. This suggests that AAI treatment of HK-2 results in ROS formation and DNA damage. Furthermore, ROS generation occurs via the MEK/ERK1/2 signaling pathway, whereas DNA damage occurs via both the ERK1/2 and p38 pathways.