马兜铃酸Ⅰ致肾小管上皮细胞DNA损伤的实验研究

Aristolochic acidⅠinduce DNA damage and cell cycle arrest in proximal tubule epithelium cell

目的:研究马兜铃酸Ⅰ(AAⅠ)致肾小管上皮细胞(LLC-PK1细胞)DNA损伤和对细胞周期的影响,并探讨其与P53途径的关系。 方法:不同浓度的AAⅠ纯品(80、160、320、640和1280ng/ml)体外刺激LLC-PK1细胞24h,采用单细胞凝胶电泳和流式细胞仪观察AAⅠ对LLC-PK1细胞DNA损伤和细胞周期的影响。用免疫荧光染色和流式细胞仪检测小管上皮细胞P53蛋白的表达。 结果:单细胞凝胶电泳实验发现 AAⅠ浓度为160,320,640和1280ng/ml时能导致LLC-PK1细胞产生彗星拖尾现象,且剂量越大,拖尾越明显,尾长和尾部荧光值与对照组比较有明显差异(P<0.05或P<0.01)。此剂量的AAI使LLC-PK1细胞在G2/M期的比例明显增高,且剂量越大,增高越明显,与对照组比较有明显差异(P<0.05或P<0.01)。各组小管上皮细胞P53蛋白的表达无明显差异(P>0.05)。 结论:AAⅠ可导致LLC-PK1细胞DNA损伤,使细胞周期阻滞在G2/M期,这可能是马兜铃酸肾毒性损伤后肾小管上皮细胞再生修复能力差和形成泌尿道肿瘤的机制之一。AAⅠ对LLC-PK1细胞DNA损伤和细胞周期阻滞作用是非P53依赖的,具体机制有待进一步研究。

Objective: Aristolochic acid I (AAI) has been proved responsible for the acellular tubulointerstitial damage in Chinese herb nephropathy and carcinogenic property in rodents. The mechanism has been seldom probed into so far. In this study, porcine proximal tubule epithelial cells (LLC-PK1) were used to examine AAI associated DNA damage and cell cycle arrest with respect to the involvement of P53 pathway activation. Methodology: LLC-PK1 cells were treated by adding AAI at different concentrations (80, 160, 320, 640 and 1280ng/ml) for 24 hours and a control group was set up. DNA damage and cell cycle arrest of LLC-PK1 cells were examined by single-cell gel electrophoresis (or comet assay) and flow cytometry (FCM). Expression of P53 protein in LLC-PK1 cells was detected with immunofluorescence staining and FCM. Results: DNA damage was not detected in control cells and LLC-PK1 cells treated with AAI (80ng/ml) by using comet assay. However, comet assay was positive ( with the appearance of a tail of damaged DNA migrating away from the nucleus) in LLC-PK1 cells exposed to AAI (160~1280ng/ml). A dose-dependant effect of AAI was detected by measuring the tail length, the ratio of tail length to full length, and the tail fluorescence intensity in these groups(P<0.01). Necrosis and apoptosis were not found in LLC-PK1 cells treated with the range of AAI dosage (160~1280ng/ml). By using FCM, the percentage of cells in G2/M phase increased markedly and dose- dependantly in AAI treated (160-1280ng/ml) LLC-PK1 cells (P<0.05 or P<0.01). No difference was found between the treatment groups and the control group in the expression of P53 protein by detecting the intensity of immunofluorescence staining and mean fluorescence (P>0.05 ). Conclusions: For cultured LLC-PK1 cells, AAI treatment can induce DNA damage and cell cycle arrest in G2/M phase in a dose-dependant manner. These effects of AAI in LLC-PK1 cells shed light on the mechanism of acellular and irreversible injury in tubulointerstitium of AAI nephropathy, and oncogenicity in urinary tract in AAI treated rodents.