热应激对大鼠胸主动脉内皮细胞生物钟基因 Bmal1和细胞周期蛋白表达水平的影响

Heat stress affects expression levels of circadian clock gene Bmal1 and cyclins in rat thoracic aortic endothelial cells

摘要

目的 研究热应激条件下大鼠胸主动脉结构的变化以及胸主动脉内皮细胞Bmal1和周期蛋白表达水平的变化。 方法 选取20只雄性SD大鼠，随机分为对照组和热应激组（各10只），用HE染色法观察胸主动脉的组织病理学变化，免疫组化法检测胸主动脉中Bmal1的表达。体外实验采用大鼠胸主动脉内皮细胞（RTAECs），分为对照组、阴性对照组、阴性热暴露组、热暴露组（HS）、Bmal1干扰（si-Bmal1）组和 si-Bmal1+HS 组。体内和体外实验均采用 Western blotting检测 Bmal1、细胞周期蛋白 CDK1、CDK4、CDK6 和 cyclin B1 以及凋亡相关蛋白 Bax和Bcl-2 的表达。TUNEL法检测胸主动脉中的细胞凋亡，流式细胞术检测RTAECs的细胞周期分布和凋亡水平变化。 结果 与对照组相比，热应激组大鼠血压升高，心率减慢，胸主动脉出现弹力纤维断裂，Bmal1、cyclin B1 和 CDK1 表达增加。体外结果显示，与对照组相比，热应激组 RTAECs 中 Bmal1、cyclin B1 和 CDK1 蛋白表达水平升高；si-Bmal1转染逆转了热应激引起的细胞周期蛋白表达改变并抑制热应激诱导的细胞凋亡，表现为 Bax 表达降低和 Bcl-2 表达升高（P<0.05）。 结论 热应激上调大鼠胸主动脉内皮细胞中生物钟基因Bmal1和细胞周期蛋白的表达，最终引发细胞凋亡。抑制Bmal1的表达可逆转热应激诱导的大鼠胸主动脉上皮细胞损伤，并在一定程度上缓解热应激引起的细胞周期变化。

Abstract

Objective To investigate the structural changes of rat thoracic aorta and changes in expression levels of Bmal1 and cyclins in thoracic aorta endothelial cells following heat stress. Methods Twenty male SD rats were randomized equally into control group and heat stress group. After exposure to 32 ℃ for 2 weeks in the latter group, the rats were examined for histopathological changes and Bmal1 expression in the thoracic aorta using HE staining and immunohistochemistry. In the cell experiments, cultured rat thoracic aortic endothelial cells (RTAECs) were incubated at 40 ℃ for 12 h with or without prior transfection with a Bmal1-specific small interfering RNA (si-Bmal1) or a negative sequence. In both rat thoracic aorta and RTAECs, the expressions of Bmal1, the cell cycle proteins CDK1, CDK4, CDK6, and cyclin B1, and apoptosis-related proteins Bax and Bcl-2 were detected using Western blotting. TUNEL staining was used to detect cell apoptosis in rat thoracic aorta, and the changes in cell cycle distribution and apoptosis in RTAECs were analyzed with flow cytometry. Results Compared with the control rats, the rats exposed to heat stress showed significantly increased blood pressures and lowered heart rate with elastic fiber disruption and increased expressions of Bmal1, cyclin B1 and CDK1 in the thoracic aorta (P<0.05). In cultured RTAECs, heat stress caused significant increase of Bmal1, cyclin B1 and CDK1 protein expression levels, which were obviously lowered in cells with prior si-Bmal1 transfection. Bmal1 knockdown also inhibited heat stress-induced increase of apoptosis in RTAECs as evidenced by decreased expression of Bax and increased expression of Bcl-2. Conclusion Heat stress upregulates Bmal1 expression and causes alterations in expressions of cyclins to trigger apoptosis of rat thoracic aorta endothelial cells, which can be partly alleviated by suppressing Bmal1 expression.

关键词

热应激 / 生物钟基因 / Bmal1 / 胸主动脉内皮细胞 / 周期蛋白 / 细胞凋亡

Key words

heat stress / circadian clock genes / Bmal1 / thoracic aortic endothelial cells / cyclins / apoptosis

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