[Role of cytoplasmic p53-mediated suppression of autophagy in heat stress-induced injury of cultured mouse aortic endothelial cells]

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。

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.

To explore the association of cytoplasmic p53 with autophagy and apoptosis of primary aortic endothelial cells (MAECs) exposed to heat stress.

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。