目的 探讨高血压背景下病理性高张应变对血管平滑肌细胞( vascular smooth muscle cells,VSMCs)线粒体生物发生的影响,以及 PGC1α 蛋白在这一过程中的作用。 方法 采用 Flexcell-5000T 体外细胞张应变加载系统对VSMCs 施加频率为 1. 25 Hz、幅度分别为 5% 和 15% 的周期性张应变,模拟正常生理情况和高血压病理情况下的力学环境;通过蛋白免疫印迹(Western blotting)和荧光定量 PCR(qPCR)方法检测正常生理和高血压病理力学条件下VSMCs 的 PGC1α 蛋白表达,以及柠檬酸合酶和线粒体 DNA(mitochondrial DNA,mtDNA) 拷贝数变化情况;应用PGC1α 特异性激活剂 ZLN005 和有效干扰片段 siRNA 检测 PGC1α 表达上调或下调对柠檬酸合酶和 mtDNA 拷贝数的影响。 结果 与 5% 生理性周期性张应变相比,15% 病理性高张应变显著抑制 VSMCs 的 PGC1α 和柠檬酸合酶的表达,mtDNA 拷贝数显著降低。 与对照组相比,对 VSMCs 转染 PGC1α 干扰片段 siRNA 或孵育 PGC1α 特异性激活剂 ZLN005,分别下调和上调 PGC1α 蛋白表达,VSMCs 的柠檬酸合酶表达和 mtDNA 拷贝数也相应地降低和增加。在加载生理性周期性张应变条件下,对 VSMCs 转染 PGC1α 干扰片段 siRNA 显著下调其蛋白表达;对 VSMCs 施加PGC1α 特异性激活剂 ZLN005 显著上调 PGC1α 蛋白表达,柠檬酸合酶表达和 mtDNA 拷贝数也被增加。 结论 高血压背景下病理性周期性高张应变通过抑制 VSMCs 的 PGC1α 蛋白显著下调 VSMCs 的柠檬酸合酶表达和 mtDNA拷贝数,进而导致 VSMCs 线粒体功能障碍。 PGC1α 可能是缓解高血压病理进程的潜在治疗靶向分子。
Objective To investigate the effect of pathologically elevated-cyclic stretch induced by hypertension on mitochondrial biogenesis of vascular smooth muscle cells (VSMCs), and the role of PGC1α in this process. Methods The Flexcell-5000T stretch loading system in vitro was applied to VSMCs with a frequency of 1. 25 Hz and an amplitude of 5% or 15% to simulate the mechanical environment under normal physiological or hypertensive pathological conditions respectively. Western blotting and qPCR were used to detect the expression of PGC1α, citrate synthase and mitochondrial DNA (mtDNA) copy number in VSMCs under normal physiological or hypertensive pathological conditions. VSMCs were treated with PGC1α specific activator ZLN005 to promote PGC1α expression or specific interfering fragment siRNA to inhibit PGC1α expression in order to detect the effect on citrate synthase and mtDNA copy number. Results Compared with 5% physiological cyclic stretch, 15% pathologically elevated-cyclic stretch significantly suppressed the expression of PGC1α, citrate synthase and mtDNA copy number in VSMCs. Compared with control group, the protein expression of PGC1α was significantly decreased and increased respectively. When VSMCs transfected with PGC1α siRNA or incubated PGC1α activator ZLN005, the expression of citrate synthase and mtDNA copy number were also significantly downregulated and up-regulated in VSMCs accordingly. Under physiological cyclic stretch conditions, the protein level of PGC1α was significantly down-regulated by PGC1α siRNA, which also significantly down-regulated citrate synthase expression and mtDNA copy number. The protein expression of PGC1α was significantly up-regulated by ZLN005, which also enhanced the expression of citrate synthase and mtDNA copy number. Conclusions The pathological cyclic stretch induced by hypertension significantly down-regulated the expression of citrate synthase and mtDNA copy number via suppressing the expression of PGC1α, resulting in mitochondrial dysfunction of VSMCs. PGC1α may be a potential therapeutic target molecule to alleviate the progression of hypertension.
张守敏,李之音,田文浩,陶雨婷,齐颖新,韩 悦.周期性高张应变通过下调 PGC1α 表达抑制血管平滑肌细胞线粒体生物发生[J].医用生物力学,2023,38(1):156-163复制