高糖诱导的大鼠肾小球系膜细胞中eNOS/NO的变化及调节机制研究

Mechanism of glucose-mediated induction of endothelial nitric oxide synthase expression in rat glomerular mesangial cells

目的:探讨高糖条件下大鼠肾小球系膜细胞(GMCs)中内皮一氧化氮合酶/一氧化氮(eNOS/NO)的变化及可能的调节机制。方法:将大鼠GMCs常规培养在含5.5 mmol/L葡萄糖的RPMI1640培养液中,用胰蛋白酶-乙二胺四乙酸(EDTA)混合消化酶传代。用RT、实时-PCR和Western blot测定GMCs中eNOS蛋白和其mRNA的相对含量。用NO显示剂DAF-2 DA(0.5 nmol/L)标记,NO的变化用QM-6荧光计测定。结果:在GMCs中,高糖可明显增加eNOS的表达(P<0.05),并有一定的量效、时效关系,同时可促进NO产生;放线菌酮可以抑制eNOS蛋白的合成。高糖对eNOS表达的调节与eNOS mRNA的稳定性没有明显关系。结论:高糖可以上调eNOS mRNA和其蛋白的表达,并可促进NO产生。该效应可能与eNOS蛋白的合成相关,而与eNOS mRNA的稳定性没有明显关系。本实验的结果为eNOS介导的NO产生和由此所致肾小球的超滤过提供了新的思路。

AIM： To explore the high glucose-induced changes of eNOS expression and NO generation in rat glomerular mesangial cells（GMCs） and the mechanism of glucose-mediated eNOS expression in GMCs.METHODS： Rat glomerular mesangial cells were cultured in RPMI1640 medium containing 1000 mg/L（5.5 mmol/L） glucose unless otherwise specified and supplemented with 14% fetal bovine serum and penicillin（50 U/ml）/streptomycin（50 mg/L） at 37℃ in a humidified 5% CO2 atmosphere incubator.They were harvested with trypsin（0.05%）-EDTA（0.02%） when culture reached confluence（usually 72 h culture following passage）.NO production was measured using the QM-6 fluorometer.mRNA and protein levels of eNOS were analyzed by RT-real time-PCR method and immunoblot analysis,respectively.RESULTS： Steady-state mRNA and protein levels of eNOS increased significantly in GMCs cultured in high concentrations of glucose（11 and 30mmol/L,72h） compared with glucose（5.5mmol/L,72h）（P〈0.05）.The parallel increases in eNOS mRNA and protein by high glucose（11 mmol/L） were cells grown in control concentration of glucose（5.5 mmol/L,72 h）（P〈0.05）.The parallel increases in eNOS mRNA and protein by high glucose（11 mmol/L） were time dependent and at least 24 h treatment was required for the induction of eNOS mRNA and protein.Cyclohexamide,an inhibitor of protein translation,inhibited high glucose（11 mmol/L）-induced eNOS protein expression and high glucose did not affect the stability of eNOS mRNA.CONCLUSION： High glucose enhances the expression of eNOS protein and NO generation in GMCs.Upregulation of eNOS protein by high glucose requires novel protein synthesis and the expression of eNOS protein under high glucose is regulated through protein translation.The enhanced eNOS expression and NO generation in GMCs under hyperglycemic condition may be responsible for the glomerular hyperfiltration in diabetes.