马兜铃酸对体外培养大鼠近端肾小管上皮细胞的损伤作用

Damage Effects of Aristolochic Acid on Proximal Tubular Epithelial Cells of Rats in Vitro

目的：观察马兜铃酸（aristolochic acid,AA）对体外培养的大鼠近端肾小管上皮细胞（NRK-52E）的损伤作用。方法：体外培养NRK-52E,分别以不同浓度的AA-Na刺激细胞,每组设6个复孔,孵育24 h。透射电镜观察NRK-52E细胞的超微结构,MTT法检测不同浓度AA对NRK-52E细胞增殖的影响,流式细胞仪检测NRK-52E细胞的凋亡,免疫细胞化学法检测AA对NRK-52E细胞α-平滑肌肌动蛋白（α-SMA）表达的影响;检测不同时间段（12、24、48 h）马兜铃酸对NRK-52E细胞转化生长因子-β1（TGF-β1）及内皮素-1（ET-1）mRNA和Ⅰ型胶原（ColⅠ）表达的影响。结果：①AA浓度低于10μg/ml时对NRK-52E的增殖无明显影响,20~40μg/ml浓度的AA可明显抑制NRK-52E细胞的增殖;②较高浓度的AA（40μg/ml）可明显刺激细胞凋亡;③AA5、10、20、40μg/ml均可刺激NRK-52E表达α-SMA,其中AA10μg/ml对α-SMA表达较强;④AA刺激NRK-52E细胞24 h后TGF-β1mRNA、ET-1 mRNA表达最多,48 h时表达水平下降,而COL-I的表达水平随时间的延长而上调,呈时间依赖性。结论：AA可直接损伤肾小管上皮细胞,促进其表达肌成纤维细胞标志蛋白α-SMA,即有促转分化作用,且其对NRK-52E细胞的损伤作用呈剂量及时间依赖性。

Objective： To observe the damage effects of aristolochic acid（AA） on proximal tubular epithelial cells（NRK-52E） of rats in vitro.Methods： NRK-52E were cultured in vitro with different concentrations of AA-Na stimulating these cells for 24 hours and 6 duplicated pores were set in each group.The ultrastructure of NRK-52E was observed by transmission electron microscope.The proliferation and apoptosis of NRK-52E were tested by MTT assay and flow cytometry respectively.The expression of α-smooth muscle actin（α-SMA） of NRK-52E was detected by immunocyto-chemical method.The expressions of transforming growth factor-β1（TGF-β1）,endothelin-1（ET-1） mRNA and collagenⅠof NRK-52E were measured in different periods（12,24,48 h）.Results： ① The AA-Na had no significant effect on proliferation of NRK-52E below the concentration of 10 μg/ml and the AA-Na inhibited the proliferation of NRK-52E obviously at the concentration of 20～40 μg/ml.② The AA-Na with high concentration（40 μg/ml） could stimulate the apoptosis of NRK-52E.③ The AA-Na could stimulate the expression of α-SMA of NRK-52E at the concentrations of 5,10,20 and 40 μg/ml especially at 10 μg/ml.④ The expressions of TGF-β1 mRNA and ET-1 mRNA of NRK-52E were highest after 24 hours stimulated by AA-Na and the expressions decreased after 48 hours,while the expression of collagenⅠincreased with time.Conclusion： AA-Na can directly damage renal tubular epithelial cells and promote the expression of α-SMA to promote transdifferentiation,and the damage effects of AA-Na on NRK-52E have dose and time dependent manner.