水蛭素对高磷诱导的大鼠血管平滑肌细胞钙化的影响

Effect of Hirudin on Calcification of Rat Vascular Smooth Muscle Cells Induced by High Phosphorus

关键词:  水蛭素  血管平滑肌细胞  血管钙化  高磷

Key words:  hirudin  vascular smooth muscle cells  vascular calcification  high phosphorus

摘要:

目的 探讨水蛭素对高磷诱导的血管平滑肌细胞（vascular smooth muscle cells，VSMCs）钙化的影响及机制。方法 采用β-甘油磷酸诱导体外培养的大鼠VSMCs，建立钙化模型，加入不同浓度水蛭素进行干预，实验设置5个组：对照组、钙化组、低浓度水蛭素组、中浓度水蛭素组、高浓度水蛭素组。通过茜素红S染色及细胞内钙含量检测各组细胞钙化情况；用qRT-PCR及Western blotting检测各组细胞α-SMA、RUNX2、BMP2的mRNA和蛋白表达量及细胞内碱性磷酸酶（alkaline phosphatase，ALP）活性以测定各组细胞转分化情况。结果 与对照组比较，高磷诱导的钙化组中茜素红S染色可见明显橘红色钙化结节，钙含量检测显示细胞内钙含量明显增加（P<0.01），细胞内ALP活性升高（P<0.01），α-SMA的mRNA及蛋白表达量降低（P<0.01），RUNX2、BMP2的mRNA及蛋白表达量升高（P<0.01）。与钙化组比较，水蛭素组茜素红S染色结果显示的橘红色钙化结节呈浓度依赖性减少，细胞内钙含量及ALP活性呈浓度依赖性降低（P<0.05或P<0.01），α-SMA的mRNA及蛋白表达量呈浓度依赖性升高（P<0.05或P<0.01），RUNX2、BMP2的mRNA及蛋白表达量呈浓度依赖性降低（P<0.05或P<0.01）。结论 水蛭素通过抑制高磷诱导大鼠VSMCs转分化来减轻VSMCs的钙化，其机制可能与上调α-SMA的表达，下调ALP活性、RUNX2及BMP2的表达有关。

Abstract:

OBJECTIVE To explore the effect and mechanism of hirudin on calcification of vascular smooth muscle cells (VSMCs) induced by high phosphorus. METHODS Rat VSMCs cultured in vitro were induced by β-glycerophosphoric acid to establish calcification model, and different concentrations of hirudin were added to intervene. The experiment set five groups: control group, calcification group, low concentration hirudin group, medium concentration hirudin group and high concentration hirudin group. The cellular calcification was detected by alizarin red S staining and intracellular calcium content. The activity of alkaline phosphatase(ALP) and the mRNA and protein expression of α-SMA, RUNX2, BMP2 which were assayed by qRT-PCR and Western blotting to detect the transdifferentiation of cells in each group. RESULTS Compared with the control group, alizarin red S staining showed obvious orange calcium nodules in high phosphorus-induced calcification group, the detection of calcium content showed that the calcium content in cells increased significantly(P<0.01), intracellular ALP activity increased(P<0.01), mRNA and protein expression of α-SMA decreased(P<0.01), mRNA and protein expression of RUNX2 and BMP2 increased(P<0.01). Compared with calcification group, the number of orange-red calcium nodules stained by alizarin red S in hirudin group decreased in a concentration-dependent manner, the intracellular calcium content and ALP activity decreased in a concentration-dependent manner(P<0.05 or P<0.01), the mRNA and protein expression of α-SMA increased in a concentration-dependent manner(P<0.05 or P<0.01), the mRNA and protein expression of RUNX2 and BMP2 decreased significantly in a concentration-dependent manner(P<0.05 or P<0.01). CONCLUSION Hirudin attenuates the calcification of VSMCs by inhibiting the transdifferentiation of VSMCs induced by high phosphorus. The mechanism may be related to the up-regulation of α-SMA expression and down-regulation of ALP activity, RUNX2 and BMP2 expression.