百草枯诱导A549细胞间质转化及四氢吡咯二硫代氨基甲酸酯的干预作用

Induction of paraquat on epithelial-mesenchymal transition of A549 cells and intervention of pyrrolidine dithiocarbamate( PDTC )

目的探讨体外培养的人肺腺癌细胞(A549)在百草枯(PQ)诱导下能否发生上皮-间质转化(epithelial-mesenchymal transition,EMT)及四氢吡咯二硫代氨基甲酸酯(PDTC)的干预作用和可能机制。方法体外培养A549细胞,实验分为对照A549细胞组(Control)、PQ诱导A549细胞组(PQ)、PDTC 3个浓度干预组(PQ+PDTC1,2,3;终浓度分别为10,20,40μmol/L)。用噻唑蓝(MTT)测定细胞毒性;活性氧荧光定量法(DCFH-DA)测定细胞内活性氧(ROS)水平;酶联免疫吸附试验(ELISA)测定转化生长因子β1(TGF-β1)表达水平;实时定量RCR(Real Time,RT-PCR)测定EMT相关标志物上皮细胞钙粘蛋白(Ecad)、紧密连接蛋白(ZO-1)、N-钙粘蛋白(N-cad)和α-平滑肌肌动蛋白(α-SMA)mRNA表达。结果与对照组比较,PQ(600μmol/L)作用A549细胞24h后,细胞存活率降低,细胞内ROS水平明显升高,TGF-β1表达水平明显升高,上皮细胞标志物E-cad、ZO-1基因表达下调,间质细胞标志物N-cad、α-SMA基因表达明显上调(P<0.05);PDTC干预组能够降低PQ诱导的A549细胞毒性,明显抑制上述作用,其中对TGF-β1表达、E-cad、N-cad基因表达的干预作用呈剂量依赖方式(P<0.05)。结论PQ诱导A549细胞过度表达TGF-β1,继而导致上皮细胞向间质细胞转化,这可能是PQ致肺纤维化的重要机制之一;PDTC的有效干预可能基于其抗氧化和核因子-kappaB(nuclear factor-kappaB,NF-κB)抑制作用。

Objective To investigate the induction of paraquat（PQ）on epithelial-mesenchymal transition of human alveolar epithelial cells type Ⅱ （A549）and intervention of pyrrolidine dithiocarbamate（PDTC）. Methods A549 cells were cultured in vitro ancl divided into 5 groups, i. e. the control group（treated with medium）, PQ group（treated with 600/zmol/L PQ）, and three PQ＋PDTC groups（treated with PDTC 10,20, and 40 μmol/L and followed by treatment of PQ lh later）. The viability of cells was measured by MTT. The reactive oxidative species（ROS）production of cells was measured by DCFH-DA assay. The levels of TGF-β1 were measured by ELISA. The hiomarkers of epithelial-mesenchymal transition were analyzed by real time RT-PCR. Results MTT showed that the cell viability was significantly decreased in PQ group compared with control group（P〈0.05）. Meanwhile, the levels of ROS and TGF-β1 were significantly increased in PQ group compared with control group（P〈0.05）. Real time RT-PCR showed that epithelial marker-associated E-cad and ZO-1 mRNA regulated downward, while mesenchymal-associated N-cad and α-SMA significantly regulated upward（P〈0.05）. PDTC treatment significantly increased cell viability, E-cad and ZO-1 mRNA levels and decreased ROS, TGF-[β1, and N-cad, 〈-SMA mRNA levels compared with PQ group （P 〈 0.05）. Noteworthy, the effect of PDTC on expressions of TGF-β1, E-cad and N-cad showed in a dose-dependentmanner（P〈0.05）. Conclusions PQ could induce EMT of A549, associated with overexpression of TGF-β1. It suggests that EMT could play important role in PQinduced pulmonary fibrosis. PDTC showed effective intervention on A549 cells because of its NF-κB inhibition and anti-oxidation.