BMP信号通路在华法林诱导的大鼠血管平滑肌细胞钙化中的作用

The Role of BMP Signaling Pathway in Warfarin-induced Calcification of Rat Vascular Smooth Muscle Cells

目的探讨骨形态发生蛋白(BMP)信号通路在华法林诱导的大鼠血管平滑肌细胞(VSMC)钙化中的作用。方法体外分离培养大鼠胸主动脉VSMC,将其随机分为正常对照组、高磷组、华法林(10μmol/L)干预组及华法林(10μmol/L)+维生素K(10μmol/L)干预组。对细胞进行钙含量和碱性磷酸酶(ALP)活性检测,茜素红染色检测钙结节,Western blot检测细胞中Runx2蛋白的表达变化,RT-PCR检测细胞中骨形态发生蛋白2(BMP-2)、Smad1及Runx2的表达变化。结果茜素红染色结果显示,与正常对照组和高磷组相比,华法林干预组钙化结节明显增多;钙含量测定结果与茜素红染色结果基本一致。与正常对照组和高磷组相比,华法林干预组ALP活性明显增加(P<0.05),Runx2 mRNA和蛋白表达水平及BMP-2、Smad1 mRNA表达明显增高(P<0.05);与华法林干预组相比,华法林+维生素K干预组细胞钙含量、ALP活性及BMP-2、Smad1、Runx2的表达均明显降低(P<0.05)。结论BMP信号通路参与了华法林促进的大鼠VSMC钙化,其可能的作用机制是介导了VSMC的表型转化。

Aim To explore whether warfarin promotes calcification of rat vascular smooth muscle cells（ VSMC）through the bone morphogenetic protein（ BMP） signaling pathway. Methods Vascular smooth muscle cells were obtained from rat aortic,and identified by immunocytochemistry,then randomly divided into control group,high phosphorus group,warfarin（ 10 μmol / L） group,warfarin（ 10 μmol / L） ＋vitamin K（ 10 μmol / L） group. Calcification staining,calcium content and alkaline phosphatase（ ALP） activity were measured,the expression of Runx2 protein was detected by Western blot and the expression of BMP-2,Smad1,Runx2 mRNA was detected by RT-PCR. Results The results of alizarin red stain were shown that the number of calcified nodules in the warfarin group was significantly higher than that in the control and high phosphorus group（ P〈 0. 05）. The results of calcium content were in line with that of alizarin red stain. The ALP activity in the warfarin group was significantly higher than that in the control and high phosphorus group（ P〈 0.05）. The results of RT-PCR and Western blot were shown that the expression level of Runx2,BMP-2 and Smad1 in the warfarin group was significantly higher than that in the control and high phosphorus group（ P〈 0.05）. However,the calcification content,ALP activity and the expression level of BMP-2,Smad1 and Runx2 in the warfarin＋vitamin K group were significantly lower than those in the warfarin group（ P〈 0.05）. Conclusion The BMP pathway was involved in warfarin-induced vascular calcification of rat vascular smooth muscle cells,which may be achieved by promoting the transdifferentiation of vascular smooth muscle cells.