AngⅡ激活p53/SAT1信号通路诱导白色脂肪细胞铁死亡

Angiotensin Ⅱ activates p53/SAT1 signaling pathway to induce ferroptosis in white adipocytes

摘要:

目的]探讨血管紧张素Ⅱ(AngⅡ)对白色脂肪细胞铁死亡的影响及其机制。 [方法]通过诱导物刺激的方法使3T3-L1脂肪前体细胞向白色脂肪细胞分化,实验分为对照组、AngⅡ组、AngⅡ+Fer-1(铁死亡抑制剂)组和AngⅡ+PFT-α(p53抑制剂)组。使用AngⅡ处理细胞,利用RT-qPCR和Western blot检测细胞铁死亡因子和脂肪因子表达水平,JC-1试剂盒检测线粒体膜电位(MMP)水平,铁离子试剂盒检测细胞内铁含量,谷胱甘肽(GSH)试剂盒检测GSH含量。加入Fer-1和AngⅡ处理细胞,检测细胞铁死亡水平变化。检测p53和亚精胺/精胺N1-乙酰转移酶1(SAT1)蛋白表达。随后加入PFT-α和AngⅡ共处理细胞,检测p53和SAT1蛋白表达变化,观察抑制p53表达对细胞铁死亡因子和脂肪因子表达水平的影响。 [结果]通过刺激物诱导分化法成功将3T3-L1细胞分化为白色脂肪细胞。AngⅡ诱导白色脂肪细胞发生铁死亡。RT-qPCR结果显示,与对照组相比,AngⅡ组的抗铁死亡因子谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)和铁调节蛋白1(IRP-1)的mRNA表达均下调,促铁死亡因子酯酰辅酶A合成酶长链家族成员4(ACSL4)的mRNA表达上调。Western blot结果显示,与对照组相比,AngⅡ组的SLC7A11和GPX4的蛋白表达均下调,ACSL4的蛋白表达上调。AngⅡ处理使细胞内铁离子的含量增加,GSH和MMP水平下降。与AngⅡ组相比,AngⅡ+Fer-1组的IRP-1和SLC7A11的mRNA表达均上调。AngⅡ诱导白色脂肪细胞的脂肪因子表达谱改变。Western blot结果显示,与对照组相比,AngⅡ组的促炎脂肪因子瘦素(LEP)、抵抗素(RETN)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的蛋白表达均上调,抗炎脂肪因子脂联素(ADPN)和网膜素1(ITLN1)的蛋白表达下调。此外,AngⅡ使p53和SAT1蛋白表达增加。抑制p53表达可改善AngⅡ处理下白色脂肪细胞的铁死亡水平和脂肪因子表达水平。Western blot结果显示,与AngⅡ组相比,AngⅡ+PFT-α组的p53和SAT1蛋白表达下调,SLC7A11和GPX4蛋白表达上调,ACSL4蛋白表达下调；AngⅡ+PFT-α组的ADPN蛋白表达上调,TNF-α、LEP和RETN的蛋白表达均下调。 [结论]AngⅡ通过激活p53/SAT1信号通路诱导白色脂肪细胞铁死亡。

Abstract:

Aim To investigate the effect and mechanism of angiotensin Ⅱ (AngⅡ) on ferroptosis in white adipocytes. Methods The 3T3-L1 preadipocytes were differentiated into white adipocytes by inducer stimulation. The experiment was divided into control group, AngⅡ group, AngⅡ+ Fer-1(ferroptosis inhibitor) group and AngⅡ+ PFT-α (p53 inhibitor) group. AngⅡwas used to treat cells. RT-qPCR and Western blot were used to detect the expression levels of ferroptosis factors and adipokines. JC-1 kit was used to detect mitochondrial membrane potential (MMP) level.Iron ion kit was used to detect intracellular iron content. Glutathione (GSH) kit was used to detect GSH content. Fer-1 and AngⅡ were added to treat cells to detect the the changes of ferroptosis level. The expression of p53 and spermidine/spermine N1-acetyltransferase 1 (SAT1) protein was detected. Subsequently, PFT-α and AngⅡ were added to co-treat cells to detect the changes of p53 and SAT1 protein expression, and to observe the effect of inhibiting p53 expression on the expression levels of ferroptosis factors and adipokines. Results 3T3-L1 cells were successfully differentiated into white adipocytes by stimulator-induced differentiation. AngⅡ induced ferroptosis in white adipocytes. RT-qPCR results showed that compared with control group, the mRNA expression of anti-ferroptosis factor glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and iron regulatory protein 1 (IRP-1) was down-regulated in AngⅡ group, and the mRNA expression of pro-ferroptosis factor acyl-CoA synthetase of long-chain family member 4 (ACSL4) was up-regulated. Western blot results showed that compared with control group, the protein expression of SLC7A11 and GPX4 was down-regulated in AngⅡ group, and the protein expression of ACSL4 was up-regulated. AngⅡ treatment increased the content of intracellular iron ions and decreased the levels of GSH and MMP. Compared with AngⅡ group, the mRNA expression of IRP-1 and SLC7A11 was up-regulated in AngⅡ+Fer-1 group. AngⅡ induced changes in the expression profile of adipokines in white adipocytes. Western blot results showed that compared with control group, the protein expression of pro-inflammatory adipokine leptin (LEP), resistin (RETN), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was up-regulated in AngⅡ group, and the protein expression of anti-inflammatory adipokine adiponectin (ADPN) and omentin 1 (ITLN1) was down-regulated. In addition, AngⅡ increased the protein expression of p53 and SAT1. Inhibition of p53 expression can improve the level of ferroptosis and adipokine expression in white adipocytes treated with AngⅡ. Western blot results showed that compared with AngⅡ group, the protein expression of p53 and SAT1 was down-regulated in AngⅡ + PFT-α group, the protein expression of SLC7A11 and GPX4 was up-regulated, and the protein expression of ACSL4 was down-regulated. The protein expression of ADPN was up-regulated in AngⅡ+ PFT-α group, and the protein expression of TNF-α, LEP and RETN was down-regulated. Conclusion AngⅡ induces ferroptosis in white adipocytes through activating the p53/SAT1 signaling pathway.