马兜铃酸对人肾小管上皮细胞损伤的体外实验研究

目的 研究马兜铃酸(aristoloehie acid,AA)对人近端肾小管上皮细胞(HK-2细胞)的损伤及可能机制.方法 将细胞分为4组:正常对照组与AA30、60、120 μmol/L组(n=6),分别作用于HK-2细胞培养48 h后,倒置相差显微镜观察细胞形态,CCK8试剂盒(Cell Counting Kit-8)检测增殖,流式细胞仪检测凋亡,Western blot分析激活型Caspase-3的表达,全自动生化检测仪测定上清液中乳酸脱氢酶(LDH)和B-N-乙酰氨基匍萄糖苷酶(NAG酶)的含量,激光共聚焦扫描荧光显微镜(Laser scanning eonfocal microscope,LSCM)观察α-平滑肌肌动蛋白(smooth muscle aetin,α-SMA)和E-钙黏连蛋白(E-cadherin)的表达,ELISA测定上清液中转化生长因子-β1(transforming growth factor-β1,TGF-β1)和Ⅲ型胶原的分泌.结果 HK-2分别在30、60、120 μmoi/L AA作用48 h后,出现增殖抑制,凋亡增加,激活型Caspase-3表达增多,LDH和NAG酶升高,均旱剂量依赖性.60μmol/1.浓度的AA还表现E-cadherin表达减弱,α-SMA表达增强,TGF-β1和Ⅲ型胶原分泌明显增加(P<0.05).结论 AA可引起HK-2细胞明显的增殖抑制、凋亡和上皮-间允质转分化呈一定的浓度依赖性.

Mechanism of aristolochic acid induced injury in human renal tubular epithelial cells in vitro

Objective To investigate the possible injury mechanism of human kidney proximal tubular epithelial cell-2(HK-2)induced by aristolochic acid(AA).Methods Cultured HK-2 cells were divided into 4 groups:normal control,treated by AA at the concentration of 30,60 and 1 20μmol/L for 48 h respectively.The morphological changes were observed by inverted phase contract microscopy.The cell viability was measured by the Cell Counting Kit-8 (CCK8) assay.Apoptotic cells were identified by flow cytometry.Expression of active Caspase-3 was measured by Western blot analysis.Automatic biochemical analyzer Was used to detect the contents of LDH and β-N-Acetylglucosaminidase(NAG)in the supematant.The expression of E-eadherin and a-SMA was detected with laser scanning confocal microscope(LSCM).Enzyme linked immunosorbent assay(ELISA)was used to measure the levels of TGF-β1 and collagen Ⅲ in the supernatant quantitatively.Results AA inhibited HK-2 cells proliferation,induced cell apoptosis and activated Caspase-3 expression,and increased the LDH and NAG levels.All of these were in a concentration-dependent manner.AA at the concentration of 60 μmol/L inhibited E-cadherin expression.inereased α-SMA expression and TGF-β1 and collagen III secretion.Conclusion AA inhibits cell proliferation,induces apoptosis and epithelialmesenchymal transition(EMT)in HK-2 cells.AA at relatively low concentration(≤60 ixmol/L)mainly induces EMT in HK-2 cells,while,that at high concentration(≥120μmol/L)causes apoptosis and cytotoxieity.