Wang Y, Yu Y, Zhang H X, Wu X L
Department of Blood Purification, Fuzhou General Hospital of PLA, Fuzhou 350025, China.
Zhonghua Yi Xue Za Zhi. 2018 May 15;98(18):1446-1451. doi: 10.3760/cma.j.issn.0376-2491.2018.18.016.
To observe the expression of protein kinase B (Akt) / mammalian target of rapamycin (mTOR) induced by high phosphorus in rat vascular smooth muscle cells (VSMC) calcification model, and its modulation on the expression of core binding factor alpha 1 (Cbfα1). Rat VSMC cells were cultured and then divided into two groups: normal phosphorus group (Pi 1.3 mmol/L) and high phosphorus group (Pi 2.6 mmol/L). At day 7, calcium deposition was detected by Alizarin stain. The mRNA levels of Cbfα1 and osteopontin (OPN) were determined by real-time PCR. The protein expressions of p-Akt (ser473), p-mTOR (S2448), Cbfα1 and OPN were quantified by Western blot. Then, VSMC cultured with high phosphorus were treated with Akt inhibitor (Wortmannin) and mTOR inhibitor (Rapamycin) with different concentrations. After 24 h, the mRNA levels of Cbfα1 and OPN were determined and after 48 h, the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN were quantified. Also, at day 7, calcium deposition was also visualized by Alizarin stain. After 7 days, compared with normal phosphorus group, calcium deposition was more obvious in high phosphorus group. The mRNA expressions of Cbfα1 and OPN increased significantly and the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN up-regulated significantly in high phosphorus group (all <0.05). After treated with Wortmannin or Rapamycin for 24 h, compared with high phosphorus group, the mRNA expressions of Cbfα1 and OPN decreased significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all <0.05) and high phosphorus + Rapamycin (1, 10 and 100 ng/ml) groups (all <0.05). After treated with Wortmannin or Rapamycin for 48 h, compared with high phosphorus group, the protein expressions of p-Akt, Cbfα1 and OPN down-regulated significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all <0.05). It showed a dose-dependent down-regulation of p-mTOR, Cbfα1 and OPN in high phosphorus + Rapamycin (1, 10, 100 ng/ml) groups (all <0.05). After 7 days, compared with high phosphorus group, calcium deposition decresased significantly in high phosphorus + Wortmannin and high phosphorus + Rapamycin groups. High phosphorus can induce VSMC calcification. Akt and mTOR are involved in VSMC calcification induced by high phosphorus through the activation of Cbfα1.
观察高磷诱导的大鼠血管平滑肌细胞（VSMC）钙化模型中蛋白激酶B（Akt）/雷帕霉素靶蛋白（mTOR）的表达，及其对核心结合因子α1（Cbfα1）表达的调控。培养大鼠VSMC细胞，然后分为两组：正常磷组（Pi 1.3 mmol/L）和高磷组（Pi 2.6 mmol/L）。第7天，通过茜素染色检测钙沉积。通过实时PCR测定Cbfα1和骨桥蛋白（OPN）的mRNA水平。通过蛋白质印迹法定量p-Akt（ser473）、p-mTOR（S2448）、Cbfα1和OPN的蛋白表达。然后，用不同浓度的Akt抑制剂（渥曼青霉素）和mTOR抑制剂（雷帕霉素）处理高磷培养的VSMC。24小时后，测定Cbfα1和OPN的mRNA水平，48小时后，定量p-Akt、p-mTOR、Cbfα1和OPN的蛋白表达。同样，在第7天，也通过茜素染色观察钙沉积。7天后，与正常磷组相比，高磷组钙沉积更明显。高磷组Cbfα1和OPN的mRNA表达显著增加，p-Akt、p-mTOR、Cbfα1和OPN的蛋白表达显著上调（均<0.05）。用渥曼青霉素或雷帕霉素处理24小时后，与高磷组相比，高磷+渥曼青霉素（30、50和100 nmol/L）组和高磷+雷帕霉素（1、10和100 ng/ml）组中Cbfα1和OPN的mRNA表达显著降低（均<0.05）。用渥曼青霉素或雷帕霉素处理48小时后，与高磷组相比，高磷+渥曼青霉素（30、50和100 nmol/L）组中p-Akt、Cbfα1和OPN的蛋白表达显著下调（均<0.05）。高磷+雷帕霉素（1、10、100 ng/ml）组中p-mTOR、Cbfα1和OPN呈剂量依赖性下调（均<0.05）。7天后，与高磷组相比，高磷+渥曼青霉素组和高磷+雷帕霉素组钙沉积显著减少。高磷可诱导VSMC钙化。Akt和mTOR通过激活Cbfα1参与高磷诱导的VSMC钙化。
