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目的研究高糖记忆对人系膜细胞(HMCs)表达局部醛固酮系统、活性氧(ROS)及癌胚纤维连接蛋白(oncofe-tal FN)mRNA水平的影响,并进一步探讨局部醛固酮系统在其中的作用。方法实验细胞分组如下:正糖组(NG,5mmol/LD-葡萄糖培养2d)、高糖组(HG,25mmol/L D-葡萄糖培养2d)、记忆组(M,25mmol/L D-葡萄糖培养2d→5mmol/L D-葡萄糖培养4d)、记忆+依普利酮组(MY,25mmol/L D-葡萄糖培养2d→5mmol/L D-葡萄糖+10μmol/L依普利酮培养4d)、正糖+依普利酮组(NY,5mmol/L D-葡萄糖培养2d→5mmol/L D-葡萄糖+10μmol/L依普利酮培养4d)、持续正糖组(SN,5mmol/L D-葡萄糖培养6d)。荧光显微镜及酶标仪检测细胞内ROS水平,蛋白质印迹法检测醛固酮合酶蛋白(CYP11B2)水平,RT-PCR检测11β-羟基类固醇脱氢酶Ⅱ、CYP11B2、oncofetal FN mRNA表达水平,激光共聚焦显微镜观察盐皮质激素受体(MR)表达及转位,放射免疫法测定上清液醛固酮水平。结果 (1)HG组和M组诱导人系膜细胞CYP11B2mRNA及蛋白分别为NG组的3.45、2.09倍和3.14、2.06倍,HG组和M组细胞上清液醛固酮含量分别为NG组的2.01、1.81倍(P均<0.05),HG组和M组均可促进MR蛋白发生核转位,定量分析示HG组和M组胞质/胞核荧光强度较NG组分别降低30%、21%(P均<0.05)。(2)HG组和M组oncofetal FN mRNA、ROS表达增加,分别为NG组的2.23、1.99倍和2.16、1.90倍(P均<0.05),MY组ROS、oncofetal FN mRNA表达分别较M组降低35%、51%(P均<0.05)。结论 HMCs存在高糖记忆效应,局部醛固酮系统可能介导了高糖致系膜细胞损伤的记忆作用。
Objective To investigate the effect of high glucose on the expression of local aldosterone system, reactive oxygen species (ROS) and oncofe-tal FN mRNA in human mesangial cells (HMCs) and further explore the role of local aldosterone system in them . Methods The experimental cells were divided into the following groups: normal glucose group (NG, 5 mmol / L glucose for 2 days), high glucose group (HG, 25 mmol / L D-glucose for 2 days) (4 days after 5 mmol / L D-glucose incubation), memory + eplerenone (MY, 25 mmol / L D-glucose for 2 days → 5 mmol / L D-glucose + 10 μmol / + Eplerenone group (NY, 5mmol / L D-glucose culture for 2d → 5mmol / L D-glucose + 10μmol / L eplerenone for 4 days) 6d). Fluorescence microscopy and microplate reader were used to detect intracellular ROS levels. Western blotting was used to detect the level of aldosterone synthase protein (CYP11B2). The expression of 11β-hydroxysteroid dehydrogenase Ⅱ, CYP11B2 and oncofetal FN mRNA was detected by RT-PCR. The expression and translocation of mineralocorticoid receptor (MR) were observed and the level of aldosterone in supernatant was determined by radioimmunoassay. Results (1) CYP11B2 mRNA and protein of human mesangial cells were 3.45, 2.09 times and 3.14 times, 2.06 times respectively in HG group and M group. The content of aldosterone in supernatant of HG group and M group were 2.01, 1.81-fold higher than that of NG group (all P <0.05). HG group and M group could promote the nuclear translocation of MR protein. Quantitative analysis showed that the fluorescence intensity of cytoplasm / nucleus in HG group and M group decreased by 30% and 21% P <0.05). (2) The expressions of oncofetal FN mRNA and ROS in HG group and M group were increased by 2.23, 1.99 and 2.16, 1.90 times respectively in NG group (P <0.05), while the expression of ROS and oncofetal FN mRNA in MY group was lower than that in M group 35%, 51% (all P <0.05). Conclusions HMCs have high glucose memory effect. Local aldosterone system may mediate the memory function of mesangial cells injured by high glucose.