血管紧张素Ⅱ通过抑制AKT/蛋白激酶B磷酸化诱导肾小管上皮细胞凋亡

Angiotensin n induces apoptosis in tubular epithelial cells through inhibiting the phospho-rylation of serine-threonine kinase Akt/protein kinase B

目的观察血管紧张素Ⅱ(AngⅡ)在诱导肾小管上皮细胞凋亡的信号传导是否有磷脂酰肌醇3激酶(PI3K)-AKT信号系统的参与,及该系统在诱导细胞凋亡中的作用。方法大鼠肾小管上皮细胞株NRK-52E分别与终浓度为0(对照组)、10-9mol/L、10-8mol/L、10-7mol/L、10-5mol/LAngⅡ共培养24h。用流式细胞仪检测细胞凋亡指数,用免疫组化方法检测增殖细胞核抗原(PCNA)的表达。Western印迹检测PI3K及磷酸化AKT和总AKT蛋白表达,AKT的磷酸化水平用473位丝氨酸磷酸化AKT(AKT-ser473)水平与总AKT水平的比值表示。结果随着AngⅡ浓度的增加,10-6mol/LAngⅡ组与对照组相比,凋亡指数显著增加[(22.7±1.41)%比(3.0±0.75)%,P<0.01]。而10-9mol/LAngⅡ组与对照组相比,PCNA指数显著增强[(47.54±2.6)%比(22.63±2.5)%,P<0.01]。与对照组相比,PI3K-p85蛋白的表达随AngⅡ浓度增加表现为先激活后抑制。AKT的磷酸化具有明显的AngⅡ浓度依赖性,随AngⅡ浓度的增加而逐渐受到抑制,并与细胞凋亡指数呈显著负相关(r=-0.90,P<0.01)。结论AngⅡ可以诱导肾小管上皮细胞凋亡并抑制细胞的增殖,可能部分是通过抑制PI3K-AKT信号传导途径实现的。

Objective To investigate whether the signaling pathway of PI3K-AKT is involved in Ang II-induced apoptosis in rat renal tubular epithelial cells (NRK-52E).Methods Cultured cells were incubated in media containing either buffer (control) or different concentrations of Ang II (10-9 to 10-6 mol/L) for 24 h. Cells were stained by Annexin V-F1TC/PI Kit and apoptosis was evaluated by flow cytometer. Western blotting was performed to assess the levels of PI3K, total- and phosphor-AKT. Results Ang II induced tubular epithelial cell apoplosis in a dose dependent manner. Ang II significantly inhibited the phosphorylation of AKT. The level of AKT phosphorylation was negatively correlated with apoptosis in Ang II-stimulated cells(r=-0.90 ,P < 0.01). Conclusion Ang II-induced tubular cells apoptosis is mediated through inhibition of PI3K-AKT signaling pathway.