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EP300介导苹果酸酶1巴豆酰化修饰促进肾小管上皮细胞铁死亡

EP300-mediated crotonylation of malic enzyme 1 promotes ferroptosis in renal tubular epithelial cells

    摘要
  • 摘要:
    目的 探讨E1A结合蛋白p300（E1A binding protein p300, EP300）介导的苹果酸酶1（malic enzyme 1, ME1）巴豆酰化（crotonylation, Kcr）修饰在肾小管上皮细胞铁死亡中的作用。
    方法 将C57BL/6小鼠分为假手术组（Sham 组）、DMSO溶剂对照＋缺血再灌注（ischemia-reperfusion, IR）损伤模型组（Vehi＋IR组）、EP300抑制剂C646＋IR损伤模型组（C646＋IR组），每组12只。评估各组肾组织的病理变化，检测肾功能、铁死亡、纤维化等指标。另取小鼠建立IR损伤模型，分析Kcr修饰水平、EP300表达随IR损伤时间的变化。构建体外HK-2细胞实验模型，分为对照组、缺氧/再复氧（hypoxia/reoxygenation, H/R）组、C646＋H/R组。HK-2细胞转染ME1-K337R质粒，突变337赖氨酸（K）为精氨酸（R）模拟去Kcr修饰。使用小干扰RNA靶向EP300转染细胞，获得EP300敲低细胞（siEP300）。检测铁死亡相关蛋白和纤维化表型表达。
    结果 IR损伤后14 d，小鼠蛋白质Kcr水平和EP300表达升高。与Sham组比较，Vehi＋IR组肾脏组织结构明显破坏，肾间质大量红色胶原沉积，谷胱甘肽、还原型烟酰胺腺嘌呤二核苷酸磷酸、谷胱甘肽过氧化物酶4表达水平降低（P＜0.01），丙二醛和血肌酐升高（P＜0.01），ME1酶活性降低（P＜0.001），纤连蛋白1、平滑肌肌动蛋白α表达升高（P＜0.001）。与Vehi＋IR组比较，C646＋IR组肾脏病理损伤减轻，肾功能改善，铁死亡和纤维化程度均减轻（P＜0.05）。与对照组相比，H/R处理后HK-2细胞铁死亡和纤维化加重（P＜0.05）；与H/R组比较，C646＋H/R组铁死亡和纤维化均减轻（P＜0.05）。转染ME1-K337R质粒和EP300敲低细胞的ME1 Kcr降低，铁死亡和纤维化减轻。
    结论 EP300通过介导ME1 Kcr修饰促进肾小管上皮细胞铁死亡，抑制EP300有助于减轻IR损伤后慢性化转归。

     

    Abstract:
    Objective To explore the role of E1A binding protein p300 (EP300)-mediated crotonylation (Kcr) modification of malic enzyme 1 (ME1) in ferroptosis of renal tubular epithelial cells.
    Methods C57BL/6 mice were divided into Sham operation group (Sham group), DMSO vehicle + ischemia-reperfusion (IR) injury group (Vehi + IR group), and EP300 inhibitor C646 + IR injury group (C646 + IR group), with 12 mice in each group. Pathological changes in renal tissues were evaluated in each group, and indicators of renal function, ferroptosis, and fibrosis were assessed. Additional mice were used to establish the IR injury model to analyze changes in Kcr modification levels and EP300 expression over the course of IR injury. The in vitro HK-2 cell experimental model was established and divided into control group, hypoxia/reoxygenation (H/R) group, and C646 + H/R group. Corresponding HK-2 cells were transfected with the ME1-K337R plasmid, in which lysine 337 (K) was mutated to arginine (R) to mimic de-Kcr modification. HK-2 cells were transfected with small interfering RNA targeting EP300 to obtain EP300 knockdown cells (siEP300). The expression of ferroptosis-related proteins and fibrosis phenotypes was detected.
    Results At 14 d after IR injury, the protein Kcr level and EP300 expression were increased in mice. Compared with the Sham group, the Vehi+IR group showed markedly disrupted renal tissue structure, extensive red collagen deposition in the renal interstitium, decreased expression levels of glutathione, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione peroxidase 4 (GPX4, P＜0.01), increased malondialdehyde and serum creatinine levels (P＜0.01), decreased ME1 enzymatic activity (P＜0.001), and increased expression of fibronectin 1 and smooth muscle actin-α (P＜0.001). Compared with the Vehi+IR group, the C646+IR group showed alleviated renal pathological injury, improved renal function, and reduced ferroptosis and fibrosis (P＜0.05). Compared with the control group, HK-2 cells subjected to H/R treatment exhibited aggravated ferroptosis and altered cell polarity (P＜0.05); compared with the H/R group, the C646+H/R group showed reduced ferroptosis and fibrosis (P＜0.05). Transfection with the ME1-K337R plasmid and siEP300 showed reduced ME1 Kcr, ferroptosis, and fibrosis.
    Conclusions EP300 promotes ferroptosis in renal tubular epithelial cells by mediating ME1 crotonylation, and inhibition of EP300 helps alleviate the chronic progression following IR injury.

     

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