醛固酮在乳鼠心肌细胞表达肿瘤坏死因子-α中的作用

目的:观察醛固酮对培养心肌细胞表达肿瘤坏死因子(TNF-α)的影响。方法:在原代培养的新生大鼠心肌细胞,采用免疫组化和RT-PCR,评价10-6M、10-5M醛固酮及10-5M醛固酮拮抗剂螺内酯对心肌细胞表达TNF-α的影响。用凝胶滞留实验(EMSA)验证NF-κB是否参与TNF-α的转录调控,筛选TNF-α可能启动子的位点。结果:TNF-α的免疫组化染色在对照组心肌细胞呈阴性,10-5和10-6M醛固酮作用心肌细胞48h,心肌细胞胞浆中出现棕色颗粒,10-5M醛固酮与螺内酯共同孵育的心肌细胞内TNF-α染色呈阴性。与单纯培养的对照组心肌细胞相比,10-5M,10-6醛固酮刺激心肌细胞24h致心肌细胞TNF-α表达的mRNA显著增高,与螺内酯共同培养的心肌细胞中TNF-αmRNA水平与对照组相比没有明显差异。醛固酮组心肌细胞的核蛋白与TNF-α上游含有潜在NF-κB结合位点的2段寡核苷酸(-619~-591 and-508~-481)的结合强于对照组,螺内酯能阻断这一效应。结论:正常情况下培养的新生鼠心肌细胞内无TNF-α合成,醛固酮通过醛固酮受体促使培养的心肌细胞表达TNF-α,NF-κB核转移是醛固酮激活心肌细胞表达TNF-α的一条通路。

The role of Aldosterone in the production of Tumor Necrosis Factor-α in cultured

Objective： To observe the production of Tumor Necrosis Factor-α （TNF-α） in cardiac myocytes stimulated with aldosterone. Method： To examine the contribution of aldosterone to TNF-α production, primary cultured rat myocytes was incubated in 10^-6 M aldosterone, 10^-5 M aldosterone and 10^-5 M aldosterone with 10^-5 M spironolactone （an antagonist of aldosterone） for 48 hours. Then TNF-α protein and mRNA was detected by immunohistochemistry and RT-PCR respectively. Electrophoretic motif shift assay （EMSA）was performed to detect wheather there is NF-κB translocation to the nuclear and to screen for the probable promoter of TNF-α. Resuit：TNF-α immunohistochemistry staining was negative in eontrol myocytes, but it was positive in myocytes treated with aldosterone for 48 hours and was abolished by spironolactone. TNF-α mRNA level was detected by RT-PCR in control cardiomyocytes, the optical dense ratio of TNF-α PCR product was obviously inereased in myocytes treated with aldosterone for 24 hours. EMSA results showed that the binding between nuclear extract of myocytes treated with aldosterone and the potential NF-κB binding sites in the upstream oligonucleotides of TNF-α （ - 619 - - 591 and - 508 - - 481 ） was increased. Spironolactone could abolish the adverse effect of aldosterone. The translocation of NF-κB to the nuclear was elevated in myocytes treated with aldosterone. Conclusion： The TNF-α protein level was negative in control cardiomyocytes, and the mRNA level was very low, but it was obviously increased in cardiomyocytes treated with aldosterone, with NF-κB activation and translocation to the nuclear, which may be a pathway leading to the expression of TNF-α.