【摘要】 目的:探讨迷迭香酸(rosmarinic acid,RA)对醛固酮(aldosterone,ALD)诱导的人肾小管上皮细胞转分化的影响及其作用机制。方法:将体外培养的人肾小管上皮细胞(HK-2),分为5组:正常对照组、ALD(100nmol/L)诱导组、ALD(100nmol/L)+RA(5μg/ml)干预组、ALD(100nmol/L)+RA(25μg/ml)干预组、ALD(100nmol/L)+线粒体呼吸链酶抑制剂rotenone(ROT,10μmol/L)干预组。应用倒置相差显微镜观察细胞形态学的变化;应用RT-PCR、Western blot检测波形蛋白(Vimentin)、平滑肌肌动蛋白(α-SMA)及钙粘蛋白(E-cadherin)的表达水平;采用Western blot检测细胞胞外信号调节激酶(ERK1/2)的磷酸化水平;DCFDA荧光法定量检测活性氧(ROS)表达情况。结果:①与对照组相比,醛固酮诱导肾小管上皮细胞从原有典型的上皮细胞形态转变为长梭形肌成纤维细胞样形态;Vimentin和α-SMA表达显著上调,E-cadherin表达下降;ERK1/2磷酸化水平增高;活性氧族(ROS)释放显著增高。②不同浓度的RA(5μg/ml,25μg/ml)及ROT(10μmol/L)干预组与ALD(100nmol/L)诱导组相比,Vimentin mRNA和α-SMA蛋白表达下调,E-cadherin mRNA及蛋白表达均上调;ERK1/2磷酸化水平下降;活性氧族(ROS)表达显著下降。结论:RA能够抑制醛固酮诱导的肾小管上皮细胞-间充质转分化(EMT),RA对EMT的负性调节作用可能是通过抑制ROS引起的ERK1/2信号转导途径实现的。更多还原

【Abstract】 Objective:To explore the effects of rosmarinic acid (RA) on the tubular epithelial-mesenchymal transition by aldosterone (ALD) and its possible mechanisms in vitro. Methods:HK-2 cells were divided into five groups:normal group,HK-2 cells induced by ALD,ALD+RA (5 μg / ml),ALD+RA (25 μg / ml),ALD+rotenone (ROT,10 μmol / L). The morphology of transdifferentiate tubular cells was observed using phase-contrast-microscopy. E-cadherin,Vimentin,α-SMA and ERK1 / 2 were determined by semi-quantitative RT-PCR and Western blot;Reactive oxygen species(ROS) was detected by fluorescence microscopic and fluorescence elisa. Results: HK-2 cells induced by ALD converted into spindle shape from typical epithelium shape,the expression of Vimentin,α-SMA,phosphorERK1 / 2 and ROS production was significantly increased,the expression of E-cadherin was significantly decreased;compared with ALD treated groups,RA (5 μg / ml,25 μg / ml) and mitochondrial respiratory chain complex I inhibitor rotenone (ROT,10μmol / L) significantly inhibited ROS generation,vimentin,α-SMA and phosphor-ERK1 / 2 expression,and E-cadherin expression was restored. Conclusion:RA inhibited ALD-induced EMT via blocking mitochondrial-originated,ROS-dependent ERK1 / 2 activation.更多还原