氰酸盐诱导氧化应激促进肾小管上皮细胞上皮–间充质转化

Cyanate Induces Oxidative Stress and Epithelia-Mesenchymal Transition on Renal Tubular Epithelial Cells

该研究探讨氰酸盐(cyanate)诱导肾小管上皮细胞氧化应激损伤和促进肾纤维化的作用。氰酸盐作用HK-2肾小管上皮细胞后, CCK8法检测其对细胞活力的影响;倒置显微镜观察细胞形态的改变; DCFH-DA法检测细胞ROS水平;细胞免疫荧光和Western blot分别检测E-cadherin、Fibronectin、α-SMA的表达; Western blot检测TGF-β的表达水平。结果显示, 2 mmol/L氰酸盐明显下调HK-2细胞的活力(P<0.05),细胞形态变为长梭形。氰酸盐作用24 h后, HK-2细胞内ROS水平呈浓度依赖性升高。免疫荧光和Western blot结果均显示,氰酸盐作用24 h后, HK-2的Fibronectin、α-SMA表达升高, E-cadherin表达下降; TGF-β的表达水平随氰酸盐浓度升高而上调(P<0.05)。以上结果表明,氰酸盐诱导肾小管上皮细胞产生过量ROS,上调TGF-β水平促进细胞上皮–间充质细胞转化(epithelia-mesenchymal transition, EMT)。

The study was to explore the pathologic role of cyanate inducing oxidative stress and epithelia-mesenchymal transition(EMT) on renal tubular epithelial cells. After treating with cyanate on different concentrations, the viability of HK-2 cells was measured by CCK8 assay. The morphologic changes were observed with the inverted microscope. DCFH-DA assay was utilized to detect ROS level. The distributions of E-cadherin, Fibronectin,α-SMA were detected by immunofluorescence assay, and the expression levels of E-cadherin,Fibronectin,α-SMA, TGF-β were detected by Western blot. After treating with 2 mmol/L cyanate for 24 h, the viability of HK-2 cells was significantly decreased(P<0.05), with morphologic changes as long-spindled cell. ROS levels were greatly increased on a dose dependent manner. Western blot and Immunofluorescence results showed the expression levels of Fibronectin and α-SMA were significance increased. However, the expression of E-cadherin was significantly decreased. The expression level of TGF-β was increased after treating with cyanate in a does dependent manner(P<0.05). Cyanate could directly generate excess ROS, which induce the expression of TGF-β increased to promote epithelia-mesenchymal transition.