Zhu Ping, Chen Ji-Mei, Guo Hui-Ming, Fan Xiao-Ping, Zhang Xiao-Shen, Fan Rui-Xin, Zheng Shao-Yi, Wu Ruo-Bin, Xiao Xue-Jun, Huang Huan-Lei, Zhu Xiao-Lan, Liu Huai-Pu, Long Guang, Chen Yan-Fang, Zhuang Jian
Cardiovascular Surgery Department, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academic of Medical Sciences, Guangzhou, People's Republic of China.
Ann Vasc Surg. 2012 Feb;26(2):268-75. doi: 10.1016/j.avsg.2011.10.006.
To investigate the effects of matrine on the vascular smooth muscle cell (VSMC) migration modulated by disturbed flow and their underlying molecular mechanisms in vitro.
Isolated rat aortic VSMCs were grown to confluence on 20- × 80-mm fibronectin-coated glass cover slides, and then, denuded zones were made at the position calculated to be the oscillating flow-reattachment zone and also in the downstream laminar flow region. VSMCs were treated with different doses of matrine (0, 10, 20, 30, and 40 mg/L), or PD98059 (30 μM), ML-7 (10 μM) combined with matrine (40 mg/L) for 30 minutes before and during the experiments. Then, the wounded monolayers were kept under static conditions or were subjected to laminar or disturbed flow for 21 hours or 10 hours. The VSMC migration was assessed by microscopic images. The extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) proteins were determined by Western blot.
Disturbed flow significantly increased phosphorylation of ERK1/2. Selective inhibition of ERK1/2 phosphorylation by inhibitor PD98059 and matrine significantly suppressed VSMC migration under disturbed flow. Disturbed flow significantly enhanced phosphorylation of MLCK, whereas both matrine and PD98059 inhibited the phosphorylation of MLCK under disturbed flow. The complete inhibition of MLCK phosphorylation using the selective MLCK inhibitor ML-7 significantly inhibited VSMC migration under disturbed flow.
Matrine inhibits VSMC migration under disturbed flow, in part, by downregulation of ERK1/2-MLCK signaling pathway.
在体外研究苦参碱对紊乱血流调节的血管平滑肌细胞（VSMC）迁移的影响及其潜在分子机制。
将分离的大鼠主动脉VSMC接种于涂有纤连蛋白的20×80mm玻璃盖玻片上，待细胞汇合后，在计算出的振荡血流再附着区位置以及下游层流区域制造无细胞区。在实验前及实验过程中，用不同剂量的苦参碱（0、10、20、30和40mg/L）或PD98059（30μM）、ML-7（10μM）与苦参碱（40mg/L）联合处理VSMC 30分钟。然后，将损伤的单层细胞置于静态条件下，或进行层流或紊乱血流处理21小时或10小时。通过显微镜图像评估VSMC迁移。采用蛋白质印迹法测定细胞外信号调节激酶1/2（ERK1/2）和肌球蛋白轻链激酶（MLCK）蛋白。
紊乱血流显著增加ERK1/2的磷酸化。抑制剂PD98059和苦参碱选择性抑制ERK1/2磷酸化，显著抑制紊乱血流条件下的VSMC迁移。紊乱血流显著增强MLCK的磷酸化，而苦参碱和PD98059均抑制紊乱血流条件下MLCK的磷酸化。使用选择性MLCK抑制剂ML-7完全抑制MLCK磷酸化，显著抑制紊乱血流条件下的VSMC迁移。
苦参碱部分通过下调ERK1/2-MLCK信号通路抑制紊乱血流条件下的VSMC迁移。
