普罗布考抑制氧化低密度脂蛋白诱导的人肾近曲小管上皮细胞转分化的作用及机制研究

Inhibitory effects of probucol on the transition of human renal proximal tubular epithelial cells induced by Ox-LDL and exploration of its related mechanisms

目的研究普罗布考对氧化低密度脂蛋白（oxidized low-density lipoprotein，Ox-LDL）诱导的人肾近曲小管上皮细胞HK-2转分化的保护作用，并探讨凝集素样氧化低密度脂蛋白受体1（lectin-like oxidized low-density lipoprotein receptor-1，LOX-1）及氧化应激在其中的作用和相互关系。方法采用不同浓度Ox-LDL（0-100ug／ml）孵育HK-2细胞，并予普罗布考（20umol／L）和LOX-1抑制剂多聚肌苷酸（250ug／m1）干预。采用四甲基偶氮唑盐微量酶反应比色法检测细胞增殖情况，用DCFH—DA法检测活性氧（reactive oxygen species，ROS）活性，生化比色法检测一氧化氮（nitric oxide，N0）浓度，Western blot检测E-钙粘素（cadherin）、a平滑肌动蛋白（a-smooth muscle actin，a-SMA）、LOX-1、还原型烟酰胺腺嘌呤二核苷酸磷酸（Nicotinamide adenine dinucleotide 2＇-phosphate reduced tetrasodium salt，NADPH）氧化酶4（NOX4）表达。结果12．5～200ug／ml浓度范围内的Ox-LDL对HK-2细胞的增殖无明显影响，25-100ug／ml的Ox-LDL以浓度依赖方式促进HK-2细胞LOX-1、a-SMA蛋白表达上升，E-cadherin下降。而多聚肌苷酸、普罗布考能使LOX-1、a-SMA蛋白表达抑制，E-cadherin表达上调。50ug／ml的Ox-LDL能促进NOX4表达上调，ROS活性增加（451．5ug／ml kg839．8ug／ml，P〈0．05），NO含量略增加（46．0umol／L比46．2umol／L），但无统计学意义（P〉0．05）。而多聚肌苷酸、普罗布考能使NOX4表达下调，ROS活性下降，但对NO浓度无明显影响。结论Ox-LDL可诱导HK-2细胞转分化，其机制可能与其结合LOX-1促进氧化应激有关，而普罗布考可以通过降低LOX-1，抑制氧化应激损伤，从而延缓HK-2细胞转分化的进程。

Objective To study the inhibitory effects of probueol on the transition of human renal proximal tubular epithelial cells （HK-2） induced by oxidized low-density lipoprotein （Ox-LDL）, and to explore the role and interrelation of lectiwlike oxidized low-density lipoprotein receptor-1 （LOX-1） and oxidative stress. Methods HK-2 cells were cultured and pretreated by different dosages of Ox-LDL （0-100ug/ml） in vitro with probucol （20 umol/L） and LOX-1 inhibitor polyinosinic acid （250ug/ml）. Cell proliferation was measured using MTT assay. Activity of reactive oxygen species （ROS） was evaluated using DCFH-DA method. Concentration of nitric oxide （NO） was determined u- sing biochemical colorimetric method. The expression levels of protein E-cadherin, a-SMA, LOX-1 and NADPH oxidase 4 （NOX-4） were examined using Western blotting. Results There was no significant influence of Ox-LDL （12. 5-200 ug/ml） on the proliferation of HK-2 cells, however, OxLDL （25-100 ug/ml） could induce the expression of LOX-1 and a-SMA proteins, while E-cadherin declined simultaneously in a dose-dependent manner. Polyinosinic acid and probucol could inhibit the expression of LOX-1 protein as well as a-SMA protein, while the expression of E-cadherin increased. 50 ug/mL Ox-LDL could induce the expression of NOX4 protein as well as intracellular ROS activity （451.5ug/ml vs. 839. ug/ml, P〈0. 05）, while the concentration of cell supernatant NO increased slightly with no significant influence （46 umol/L vs 46. umol/L）. However, the increases of NOX4 protein and ROS activity were inhibited by polyinosinic acid and probucol, while the concentration of NO barely changed. Conclusions Ox-LDL could induce the transition of human renal proximal tubular epithelial cells probably through the oxidative stress by the receptor LOX-1. However, probucol and the LOX-1 inhibitor polyinosinic acid could inhibit the injury of oxidative stress by reducing the expression of LOX-1, and delay the process of epithelial to mesenchymal transition.