论文部分内容阅读
目的研究马兜铃酸(aristolochic 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、120μmol/L组(n=6),分别作用于HK-2细胞培养48h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β-N-乙酰氨基葡萄糖苷酶(NAG酶)的含量,激光共聚焦扫描荧光显微镜(laser scanning confocal microscope,LSCM)观察α-平滑肌肌动蛋白(smooth muscle actin,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、120μmol/L AA作用48h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,均呈剂量依赖性。60μmol/L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-间充质转分化呈一定的浓度依赖性。
Objective To study the effect of aristolochic acid (AA) on human proximal tubular epithelial cells (HK-2 cells) and its possible mechanism. Methods The cells were divided into 4 groups: normal control group and AA 30, 60, 120 μmol/L group (n=6), which were treated with HK-2 cells for 48 h, and then inverted by phase contrast microscopy to observe the cell morphology. CCK8 kit (Cell Counting) Kit-8) proliferation was detected, apoptosis was detected by flow cytometry, expression of activated Caspase-3 was analyzed by Western blot, and lactate dehydrogenase (LDH) and β-N-acetylamino were measured by an automatic biochemical analyzer. The content of glucosidase (NAG enzyme) was observed by a laser scanning confocal microscope (LSCM) to observe the expression of α-smooth muscle actin (α-SMA) and E-cadherin (E- Expression of cadherin) and ELISA were used to determine the secretion of transforming growth factor-β1 (TGF-β1) and type III collagen in the supernatant. Results After HK-2 was treated with 30, 60, and 120 μmol/L AA for 48 h, proliferation was inhibited, apoptosis was increased, expression of activated Caspase-3 was increased, and LDH and NAG enzymes were increased in a dose-dependent manner. 60μmol/L AA also showed decreased expression of E-cadherin, increased expression of α-SMA, and increased secretion of TGF-β1 and type III collagen (P<0.05). Conclusion AA can induce significant inhibition of proliferation, apoptosis and epithelial-mesenchymal transdifferentiation in HK-2 cells in a concentration-dependent manner.