CAJ | 학술논문

目的研究线粒体呼吸链在高糖腹膜透析液(PDS)诱导人腹膜间皮细胞(HPMC)通透性升高中的作用,探讨高糖PDS导致腹膜高通透状态的分子机制.方法体外培养HPMC,用不同浓度葡萄糖PDS加或不加抗氧化剂谷胱甘肽,培养24 h.采用跨细胞电阻(TER)技术测定HPMC通透性的变化;免疫荧光及Western印迹观察claudin-1的表达;线粒体活性氧(ROS)荧光探针检测细胞线粒体ROS的生成;线粒体呼吸链酶复合物活性检测试剂盒检测复合物Ⅰ～Ⅳ的活性变化.结果各组细胞的TER值均随时间的延长而下降,4.25%PDS组TER值24 h内从(34.7±1.5)Ω·cm2下降至(4.3±1.4)Ω·cm2;高糖PDS使细胞间连接蛋白claudin-1的表达显著下调,葡萄糖浓度越高作用越明显;同时高糖PDS导致线粒体呼吸链酶复合物Ⅲ活性较对照组下降89.2%(P＜0.01),进而使线粒体ROS产生增加.谷胱甘肽可逆转上述指标的变化.结论高浓度葡萄糖PDS通过抑制线粒体呼吸链酶复合物Ⅲ的活性,引起HPMC的氧化损伤,进而导致腹膜间皮细胞层的高通透性增高.
목적 연구선립체호흡련재고당복막투석액(PDS)유도인복막간피세포(HPMC)통투성승고중적작용,탐토고당PDS도치복막고통투상태적분자궤제.방법 체외배양HPMC,용불동농도포도당PDS가혹불가항양화제곡광감태,배양24 h.채용과세포전조(TER)기술측정HPMC통투성적변화;면역형광급Western인적관찰claudin-1적표체;선립체활성양(ROS)형광탐침검측세포선립체ROS적생성;선립체호흡련매복합물활성검측시제합검측복합물Ⅰ～Ⅳ적활성변화.결과 각조세포적TER치균수시간적연장이하강,4.25%PDS조TER치24 h내종(34.7±1.5)Ω·cm2하강지(4.3±1.4)Ω·cm2;고당PDS사세포간련접단백claudin-1적표체현저하조,포도당농도월고작용월명현;동시고당PDS도치선립체호흡련매복합물Ⅲ활성교대조조하강89.2%(P＜0.01),진이사선립체ROS산생증가.곡광감태가역전상술지표적변화.결론 고농도포도당PDS통과억제선립체호흡련매복합물Ⅲ적활성,인기HPMC적양화손상,진이도치복막간피세포층적고통투성증고.
Objective To investigate the role of mitochondrial respiratory chain in the hyperpermeability of human peritoneal mesothelial cells (HPMCs) induced by high glucose peritoneal glucose PDS was also added. Transmesothelial electrical resistance (TER) measurement was examined for detection of permeability damage in HPMCs. Immunostaining and Western blotting analysis were used to detect claudin-1 expression. Mitochondrial superoxide (MitoSOX) Red staining and respiratory chain complexes activities were determined for detection of mitochondrial reactive oxygen species (ROS) production and mitochondrial complexes activities. Results TER was decreased in a time- and concentration-dependent manner after culture with high glucose PDS for was also down-regulated significantly by high glucose PDS (P＜0.01). Complex Ⅲ activity was inhibited (10.8% of control, P＜0.01) accompanied with increased mitochondrial ROS generation.These changes were partially prevented by glutathione. Conclusion Mitochondrial respiratory complex Ⅲ pathway has crucial importance in maintaining TER of HPMCs, which may reveal a valuable target for novel therapies to fight hyperpermeability of peritoneum during the prolonged PD treatment.