不同浓度马兜铃酸对肾小管上皮细胞的毒性作用以及骨形成蛋白-7的抗凋亡作用

目的 探讨不同浓度马兜铃酸（AA）对肾小管上皮细胞（RTECs）毒性作用的差异，以及BMP-7对AA所诱导RTECs凋亡的保护作用。 方法 （1）用浓度分别为5、10、20、40、80、160µg/ml AA刺激体外培养人近端肾小管上皮细胞株（HK-2细胞），应用乳酸脱氢酶（LDH）释放试验检测不同浓度AA对HK-2细胞的直接毒性作用，相差显微镜观察细胞形态学改变，用Hoechst33258染色法观察不同浓度AA诱导的HK-2细胞凋亡核形态的改变，计算细胞凋亡百分率；（2）用300ng/ml BMP-7处理经AA（20µg/ml和40µg/ml）诱导的HK-2细胞48h，然后Hoechst 33258荧光染色检测细胞凋亡百分率，分光光度法检测Caspase-3蛋白酶活性。 结果 当AA浓度达到80μg/ml时，细胞LDH释放率明显升高（P<0.001）；而AA浓度为10μg/ml时，细胞凋亡率开始明显升高（P<0.001），AA浓度为40μg/ml时，细胞凋亡率最高；BMP-7处理后，细胞凋亡率和Caspase-3蛋白酶活性明显下降（P<0.01）。 结论 大剂量AA致RTECs坏死，中小剂量AA致RTECs凋亡；BMP-7可减轻AA诱导的RTECs凋亡，其作用可能部分通过抑制Caspase-3酶活性而实现。

Protective effects of BMP-7 against aristolochic acid-induced apoptosis of renal tubular epithelial cells

0bjective To investigate the difference of aristolochic acid (AA) toxicity in renal tubular epithelial cells(RTECs) with different concentration, and the effect of BMP-7 on apoptosis of RTECs induced by AA. Methods (1) The human proximal tubular epithelial cells (HK-2 cells) were stimulated with AA of 5, 10, 20, 40, 80, 160 μg/ml in vitro. The direct toxic effects of AA on HK-2 cells with different concentrations were detected by lactate dehydrogenase (LDH) release assay, and morphological changes were observed by inverted phase contrast microscope, the nuclear morphological changes of apoptotic HK-2 cells induced by AA with different concentrations were observed by Hoechst33258 staining, and the percentage of apoptotic cells was calculated. (2) HK-2 cells induced by AA (20μg/ml and 40μg/ml) were treated with 300ng/ml BMP-7 for 48h. The cell apoptotic percentage was detected by Hoechst 33258 staining, and caspase-3 activity was detected by Spectrophotometric Determination. Results The LDH release rate was increased statistically(P<0.001) with 80, 160μg/ml AA. The apoptotic rate was statistically increased with 10μg/ml AA (P<0.001), and the most remarkable with 40μg/ml AA. Treating with BMP-7, HK-2 cells apoptotic rate and caspase-3 activity was statistically decreased (P<0.01). Conclution High-dose AA can induce RTECs necrosis, small dose of AA can induce RTECs apoptosis. BMP-7 can reduce the apoptosis partly by inhibition of caspase-3 activity.