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目的观察螺内酯对醛固酮诱导的大鼠肾小球系膜细胞(mesangial cells,MCs)氧化应激的影响,探讨其肾脏保护机制。方法体外培养大鼠MCs,设正常对照组、醛固酮(10-7mol/L)刺激组、不同浓度螺内酯(10-7、10-8、10-9mol/L)干预组。以2’,7’-二氯双氢荧光素二乙酸酯(2’,7-’dichlorofluorescein diacetate,DCFH-DA)为细胞内H2O2的荧光探针标记细胞,采用流式细胞术检测系膜细胞内活性氧(reactive oxygen species,ROS)水平;采用比色法检测培养细胞上清液中丙二醛(malondialdehyde,MDA)的含量和超氧化物歧化酶(superoxide dismutase,SOD)活性。结果与正常对照组比较,醛固酮刺激系膜细胞48 h后,细胞内ROS水平明显增加,上清液中MDA含量增高、SOD活性下降,差异均有统计学意义(均有P<0.05)。螺内酯干预可明显抑制醛固酮刺激下的系膜细胞的上述反应,差异均有统计学意义(均有P<0.05),且呈浓度依赖性。结论螺内酯可呈浓度依赖性地抑制醛固酮诱导的大鼠MCs氧化应激,该作用可能是其肾脏保护机制之一。
Objective To investigate the effects of spironolactone on aldosterone-induced oxidative stress in rat mesangial cells (MCs) and to explore its mechanism of renal protection. Methods Rat MCs were cultured in vitro. The normal control group, aldosterone (10-7mol / L) stimulation group and different concentrations of spironolactone (10-7,10-8,10-9mol / L) intervention group were established. The cells were labeled with 2 ’, 7’-dichlorohydrofluorescein diacetate (DCFH-DA) as fluorescent probe of intracellular H2O2, and the cell membrane was detected by flow cytometry The levels of reactive oxygen species (ROS) in cells were measured. The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in the culture supernatant were measured by colorimetric assay. Results Compared with the normal control group, aldosterone stimulated mesangial cells 48h after intracellular ROS levels increased significantly, the content of MDA in supernatant increased, SOD activity decreased, the difference was statistically significant (both P <0.05). Spironolactone intervention significantly inhibited aldosterone-induced mesangial cells above reaction, the differences were statistically significant (both P <0.05), and in a concentration-dependent manner. Conclusion Spironolactone can inhibit aldosterone-induced oxidative stress in MCs in a concentration-dependent manner, which may be one of the mechanisms of renal protection.