Objective: To explore the association of cytoplasmic p53 with autophagy and apoptosis of primary aortic endothelial cells (MAECs) exposed to heat stress.
Methods: Cultured mouse MAECs were exposed to heat stress induced by incubation at 43 ℃ for 2 h, with the cells in routine culture condition (37 ℃, 5% CO2) as the control group. All the cells were further incubated for 1, 3, 6 or 9 h at 37 ℃ before treatment with the autophagy inhibitor 3-MA (5 mmol/L), the autophagy inducer rapamycin (20 μmol/L), or the p53 inhibitor PFT (10 μmol/L) for 1 h. After the treatments, the cell viability was measured with CCK8 method, cell apoptosis analyzed by flow cytometry, and the mitochondrial membrane potential detected with flow cytometry with JC-1 staining; the subcellular localization of p53 and the autophagy- associated protein LC3-Ⅱ was detected with immunofluorescence staining, and their protein expressions were analyzed using Western blotting.
Results: Compared with the control cells, MAECs exposed to heat stress showed significantly decreased viability (P < 0.05). At 6 h after the exposure, the cells exhibited significantly decreased mitochondrial membrane potential with increased apoptotic rate (P < 0.05). The cytoplasmic fraction of p53 expression decreased and its mitochondrial fraction increased gradually with time within 6 h after heat stress. Treatment with 3- MA further decreased the mitochondrial membrane potential and significantly increased the apoptotic rate of the exposed cells (P < 0.05), while rapamycin obviously reversed these heat stress-induced cell injuries (P < 0.05). PFT significantly enhanced the expression of LC3-Ⅱ and also inhibited heat stress-induced mitochondrial membrane potential reduction and cell apoptosis (P < 0.05).
Conclusions: Heat stress induces mitochondrial damage and apoptosis in MAECs possibly in relation with mitochondrial translocation of cytoplasmic p53 to result in autophagy inhibition.
目的: 观察热打击主动脉内皮细胞（MAEC）后核外p53与细胞自噬和细胞凋亡之间的关系，阐明热打击后细胞死亡模式及其分子机制。
方法: 体外原代分离培养小鼠主动脉内皮原代细胞（MAEC），建立小鼠主动脉内皮细胞热打击模型，对照组将细胞置于标准37 ℃、5% CO2细胞培养箱，热打击组将细胞置于43 ℃细胞培养箱中进行热打击2 h，热打击后继续在细胞培养箱进行复温（0、1、3、6、9 h），并分别使用自噬抑制剂3-MA（5 mmol/L），自噬诱导剂rapamycin（20 μmol/L），p53抑制剂PFT（10 μmol/L）预处理细胞1 h，处理后的细胞通过CCK8法检测细胞活力以及Annexin V-FITC/PI双染色方法检测细胞凋亡率，JC-1染色流式观察细胞线粒体膜电位改变，细胞免疫荧光染色及Western blotting观察p53亚细胞定位和LC3-Ⅱ蛋白表达。
结果: 与对照组（37 ℃）比较，热打击后（43 ℃）MAEC细胞活力显著下降（P < 0.05）；热打击后复温6 h线粒体膜电位明显下降，同时细胞凋亡率显著增高（P < 0.05）；热打击后随着复温时间（0 h、6 h）延长，胞浆p53表达逐渐减弱，而线粒体p53表达逐渐增强；自噬抑制剂3-MA可进一步诱导细胞线粒体膜电位下降，并显著增高细胞凋亡率，而自噬诱导剂rapamycin可逆转上述损伤作用（P < 0.05）。p53抑制剂PFT明显促进了自噬相关蛋白LC3-Ⅱ的表达，同时也明显抑制了热打击诱导的细胞线粒体膜电位降低和细胞凋亡（P < 0.05）。
结论: 热打击诱导MAEC细胞线粒体损伤及细胞凋亡，其机制与核外p53线粒体移位继而介导细胞自噬抑制有关。
Keywords: apoptosis; autophagy inhibition; cytoplasmic p53; heat stress; mitochondrial damage.