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

Pathologically Elevated-Cyclic Stretch Suppressed Vascular Smooth Muscle Cell Mitochondrial Biogenesis by Down-Regulating PGC1α Expression

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 down-regulated 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.