ROCK1及其相关信号分子参与张应变调控的血管平滑肌细胞增殖

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain

目的探讨Rho相关卷曲蛋白激酶1(Rho-associated coiled-coil containing protein kinase 1,ROCK1)及其相关信号分子在感受张应变机械刺激、调控血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖功能中的作用。方法应用张应变加载系统对体外培养VSMCs施加牵张幅度10%、频率1.25 Hz生理性周向张应变;Brdu检测VSMCs增殖水平;Western blotting检测力学加载后VSMCs的ROCK1表达水平以及蛋白激酶C(protein kinase C,PKC)α/βII、蛋白激酶D(protein kinase D,PKD)、胞外信号调节激酶(extracellular regulated protein kinase,ERK)磷酸化水平;采用RNA干扰技术(RNA interference,RNAi)检测ROCK1对VSMC增殖和PKCα/βII、PKD、ERK磷酸化的调控作用。结果 10%生理性张应变加载12、24 h显著抑制VSMCs的ROCK1表达,并显著抑制PKD和ERK的磷酸化;10%生理性张应变加载12 h显著抑制PKCα/βⅡ的磷酸化,但加载24 h PKCα/βⅡ的磷酸化与静止对照组相比无显著差异。RNAi抑制VSMCs的ROCK1表达后,VSMCs增殖水平显著降低,同时PKCα/βⅡ和PKD磷酸化水平显著降低,但ERK磷酸化无明显变化。结论 10%生理性张应变可能通过抑制ROCK1表达调控PKCα/βⅡ和PKD的磷酸化水平,从而影响VSMCs增殖,维持血管稳定性。探讨张应变力学刺激调控血管细胞功能的细胞内信号转导网络,对心血管生理和疾病病理机制研究具有一定意义。

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 （ROCKI） and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the prolif- eration of vascular smooth muscle cells （VSMCs）. Methods Physiological cyclic strain with magnitude of 10% and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system. The proliferation level of VSMCs was analyzed by BrdU ELISA; the expression level of ROCK1, phosphorylations of protein kinase C （PKC） α/βII, protein kinase D （PKD） and extracellular regulated protein kinase （ERK） in VSMCs modulated by cyclic strain were detected with Western blotting ; the expression of ROCK1 was specifically repressed by using RNA interference （RNAi）. Results Compared with the static control, 10% cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK. The phosphorylation of PKCα/βII decreased significantly under 10% cyclic strain for 12 h, but returned to normal level after loading for 24 h. Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation, suppressed phosphorylations of PKCα/βII and PKD, but no obvious changes were found in phosphorylation of ERK. Conclusions Physiological cyclic strain with magnitude of 10% may repress the phosphorylation of PKCα/βII and PKD via inhibiting the ex- pression of ROCK1, and subsequently affects VSMC proliferation and maintains vascular hemostasis. The inves- tigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.