蛋白激酶Cβ介导急性股动脉损伤后基质金属蛋白酶9的活化

目的 观察急性内皮剥脱损伤对血管壁基质金属蛋白酶(matrix metalloproteinase,MMP)9的活化及其可能的信号调控机制.方法 以C57BL/6小鼠为研究对象,实施股动脉内皮剥脱损伤后,分别制备股动脉总蛋白和膜蛋白提取液,用明胶酶谱法检测MMP 9活性,蛋白质印迹(Western Blot)检测MMP 9抗原表达和膜蛋白中蛋白激酶C(protein kinase C,PKC)多种异构体的表达水平;并以PKCβ抑制剂Ruboxistaurin预处理小鼠72 h后,观察急性血管损伤对MMP 9表达的影响.结果 内皮剥脱损伤4～24 h,小鼠股动脉MMP 9活性增加,最大诱导表达量在损伤后8 h,为对照组的(99.33±9.50)倍(F=51.49,P＜0.01);MMP 9抗原表达与其活性呈现平行变化.内皮剥脱损伤15～120 min,股动脉膜蛋白提取液中PKCβⅡ被快速诱导表达,30 min时达最大值,为对照组的(7.50±0.60)倍(F=207.06,P＜0.01).PKCβ抑制剂Ruboxistaurin可减少血管损伤诱导的MMP 9活化(F=53.96,P＜0.01).结论 在小鼠股动脉内皮剥脱模型中,MMP 9活化至少部分由PKCβ信号所介导,阻断PKCβ信号通路可望成为抑制血管损伤后MMP 9过度活化的干预靶点.

Abstract：

Objective To investigate the activation of matrix metalloproteinase (MMP)9 by acute arterial injury and the involved signaling mechanism in murine femoral artery. Methods In the C57BL/6 mice femoral artery denudation injury were performed. Total protein and membrane protein extracts were prepared from targeted arteries. The MMP 9 activity was measured by zymography assay, the expressions of MMP 9 antigen and protein kinase C (PKC) isoforms were measured by Western blot. Seventy-two hours after mice fed with PKCβ inhibitor (ruboxistaurin), the denudation injury triggered MMP 9 activation was reassessed. Results Within 4-24 hours after denudation injury, MMP 9 activity in femoral arteries was significantly increased, with a peak induction of (99.3±9.5) times the sham control (F=51.49,P＜0.01) at 8 h. MMP 9 antigen increased in parallel with MMP 9 activity. Within 15-120 minutes after denudation injury, there was a significant induction of PKCβⅡ in membrane fraction of femoral arteries, with a maximum induction of (7.50±0.60) times the sham control (F=207.06,P＜0.01)at 30 min. Injury-induced MMP 9 activation was significantly inhibited by ruboxistaurin. Conclusions MMP 9 activation is, at least in part, mediated by PKCβ in acute arterial denudation injury, it highlights the new target for therapeutic intervention to suppress the over-activation of MMP 9, which plays a critical role in restenosis.