摘要
目的:研究马兜铃酸(从)对人近端肾小管上皮细胞(HK-2)毒性作用中p53和Cagpase-3活性的改变,对其在从肾毒性作用中的意义进行探讨.方法:用乳酸脱氢酶(LDH)释放试验测AA对HK-2的直接毒性作用,相差显微镜观察细胞形态学改变,HoechsG3258染色法观察细胞凋亡形态的改变,流式细胞技术测细胞凋亡百分率,RT-PCR法测p53 mRNA的表达水平,分光光度法测细胞Caspase-3活性改变.结果:从浓度为80,160 mg/L时,LDH释放率显著增高(P＜0.01);10 mg/L从可诱导HK-2细胞凋亡百分率显著增高,AA为40 mg/L时,细胞凋亡形态改变最明显和细胞凋亡比例最高(P＜0.01);各组HK-2细胞p53 mRNA表达水平有统计学差异;40 mg/L从诱导HK-2细胞凋亡时,细胞内Caspase-3活性显著升高(与正常组比较,P＜0.01).结论:从能诱导HK-2细胞凋亡,其凋亡途径可能是非p53依赖途径;凋亡过程中存在Caspase-3酶的激活.
Abstract
AIM: To investigate the features of p53 expression and Caspase-3 activity assay in aristolochic acid (AA) induced renal tubular epithelial cell apoptosis. METHODS: The direct toxic effect of AA on HK-2 cells was evaluated by Lactate dehydrogenase(LDH) releasing essay, Apoptosis was observed under fluorescence microscope after the cells were stained by Hoechst 33258. The apoptosis rate of HK-2 cells induced by AA was estimated with flow cytometric analysis and the expression of wildtype p53 mRNA was measured by RT-PCR. Caspase-3 activity assay was performed by spectrophotometric method. RESULTS: The LDH release rate significantly increased after stimulation with AA at the concentrations of 80 and 160 mg/L(compared with norreal control, P＜0.01). A.A(at the concentrations of 20 and 40 rag/L) inhibited HK-2 cells growth and induced cell apoptesis. The apoptotic index in AA groups markedly increased compared with that in normal control(P＜0.01). The expressions of wildtype p53 mRNA in HK-2 cells after AA treatment(at the concentrations of 10, 20 and 40 mg/L) were similar to these in control group. The activity of Caspase-3 markedly increased after AA treatment(at the concentrations of 20 and 40 mg/L) compared with that in normal control(P＜0.01 ). CONCLUSION: AA is cytotoxic to renal tubular epithelial cells by inhibiting renal tubular epithelial cells growth and inducing apeptosis. Our results demonstrate that AA may induce apoptosis not through p53-independent pathway in renal tubular epithelial cells. Activation of Caspases-3 occurs in AA-induced apoptosis pathway.
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维普
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