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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。
Objective: To investigate the effect of rosmarinic acid (RA) on transdifferentiation of human renal tubular epithelial cells induced by aldosterone (ALD) and its mechanism. Methods: Human renal tubular epithelial cells (HK-2) cultured in vitro were divided into 5 groups: normal control group, ALD group (100nmol / L), ALD group , ALD (100nmol / L) + RA (25μg / ml) and ALD (100nmol / L) + mitochondrial respiratory chain enzyme inhibitor rotenone (ROT, 10μmol / L) The morphological changes of cells were observed by inverted phase contrast microscope. The expression of vimentin, α-SMA and E-cadherin were detected by RT-PCR and Western blot. The level of phosphorylation of ERK1 / 2 was detected by flow cytometry. The expression of reactive oxygen species (ROS) was detected by DCFDA. Results: ① Compared with the control group, aldosterone induced tubular epithelial cells from the original epithelial cell morphology to long spindle myofibroblast-like morphology; Vimentin and α-SMA expression was significantly increased, E-cadherin expression decreased; ERK1 / 2 phosphorylation increased; reactive oxygen species (ROS) release was significantly increased. ② The expressions of Vimentin mRNA and α-SMA protein were down-regulated in the groups of RA (5μg / ml, 25μg / ml) and ROT (10μmol / L) The expression of phosphorylation of ERK1 / 2 decreased and the expression of reactive oxygen species (ROS) decreased significantly. CONCLUSION: RA can inhibit aldosterone-induced renal tubular epithelial-mesenchymal transition (EMT). The negative regulation of EM on EM may be through inhibiting the ERK1 / 2 signal transduction pathway induced by ROS.