CAJ | 학술논문

目的研究线粒体靶向性小分子多肽——SS31对肾小管上皮细胞缺氧复氧(HR)损伤的影响及其作用机制.方法以大鼠肾小管上皮细胞株NRK-52E细胞为研究对象,建立细胞HR模型,分为对照组、SS31组、HR组、HR+ SS31组.四甲基偶氮唑盐实验行细胞损伤检测,annexinV/PI双染色法行细胞凋亡检测,荧光显微镜观察活性氧、线粒体膜电位的变化,蛋白质印迹法检测衔接蛋白p66Shc和磷酸化衔接蛋白p-p66Shc表达,比较组间结果差异.结果 HR组细胞活性明显下降,为对照组的(52.2±6.0)％(P＜0.05);HR+ SS31组细胞活性有所提高,呈SS31剂量依赖性.与对照组相比较,HR组细胞凋亡率增加,为(23.03±1.04)％,HR+ SS31组(100 μmol/L)细胞凋亡率较HR组下降,为(10.37±0.80)％(P＜0.05).HR+ SS31组(100 μmol/L)细胞内活性氧的产生明显低于HR组;HR+ SS31组(100 μmol/L)线粒体膜电位下降较HR组有所改善.对照组细胞中p66Shc和p-p66Shc表达很低;在HR组细胞中,p66Shc和p-p66Shc表达都显著升高,分别为0.93±0.05和0.85±0.04;HR+ SS31组(100 μmol/L)两种蛋白的表达有所降低,分别为0.58±0.06和0.45±0.04(P＜0.05).结论 SS31可减轻大鼠肾小管上皮细胞HR损伤,其机制可能是通过p66Shc依赖的通路来抑制HR诱导的细胞凋亡和坏死,从而减少细胞损伤.
목적 연구선립체파향성소분자다태——SS31대신소관상피세포결양복양(HR)손상적영향급기작용궤제.방법 이대서신소관상피세포주NRK-52E세포위연구대상,건립세포HR모형,분위대조조、SS31조、HR조、HR+ SS31조.사갑기우담서염실험행세포손상검측,annexinV/PI쌍염색법행세포조망검측,형광현미경관찰활성양、선립체막전위적변화,단백질인적법검측함접단백p66Shc화린산화함접단백p-p66Shc표체,비교조간결과차이.결과 HR조세포활성명현하강,위대조조적(52.2±6.0)％(P＜0.05);HR+ SS31조세포활성유소제고,정SS31제량의뢰성.여대조조상비교,HR조세포조망솔증가,위(23.03±1.04)％,HR+ SS31조(100 μmol/L)세포조망솔교HR조하강,위(10.37±0.80)％(P＜0.05).HR+ SS31조(100 μmol/L)세포내활성양적산생명현저우HR조;HR+ SS31조(100 μmol/L)선립체막전위하강교HR조유소개선.대조조세포중p66Shc화p-p66Shc표체흔저;재HR조세포중,p66Shc화p-p66Shc표체도현저승고,분별위0.93±0.05화0.85±0.04;HR+ SS31조(100 μmol/L)량충단백적표체유소강저,분별위0.58±0.06화0.45±0.04(P＜0.05).결론 SS31가감경대서신소관상피세포HR손상,기궤제가능시통과p66Shc의뢰적통로래억제HR유도적세포조망화배사,종이감소세포손상.
Objective To investigate whether the pretreatment of SS31 could alleviate hypoxia/reoxygenation (H/R)-induced injury by inhibiting p66Shc.Method The cultured rat renal proximal tubular cell line NRK52E cells were exposed to 24-h hypoxia (5％ CO2,1％ O2,and 94％ N2) followed by 6-h reoxygenation (5％ CO2,21％ O2,and 74％ N2).SS31 was added to the culture medium 4 h prior to the treatment.Then the cell viability,apoptosis,ROS and MTP were determined.In addition,Western blotting was used to detect the expression of p66Shc and p-p66Shc.Result H/R induced apoptotic cell death,accompanied with activation of total and p-p66Shc in NRK52E cells.Total p66Shc and p-p66Shc were detected at low levels in control NRK52E cells,and their levels were dramatically increased in cells after H/R treatment.Pretreatment with 100 μmol/L SS31 significantly prevented cell death and attenuated total p66Shc and p-p66Shc levels after H/R.Conclusion This study revealed that SS31 pretreatment serves a protective role against H/R-induced apoptosis of human renal tubular epithelial cells by suppressing p66Shc.