Comparison of the Inhibitory Mechanisms of Diethyl Citrate, Sodium Citrate, and Phosphonoformic Acid on Calcification Induced by High Inorganic Phosphate Contents in Mouse Aortic Smooth Muscle Cells
Comparison of the Inhibitory Mechanisms of Diethyl Citrate, Sodium Citrate, and Phosphonoformic Acid on Calcification Induced by High Inorganic Phosphate Contents in Mouse Aortic Smooth Muscle Cells
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DOI：
10.1097/fjc.0000000000000537
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发表时间：
2017-09
影响因子：
3
通讯作者：
Chaoyang Duan;Chong-Yu Zhang;Xin-Yuan Sun;B. Gui;Da Guo;J. Ouyang
中科院分区：
文献类型：
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作者：
Chaoyang Duan;Chong-Yu Zhang;Xin-Yuan Sun;B. Gui;Da Guo;J. Ouyang
目的：探讨柠檬酸二乙酯（Et 2Cit）、柠檬酸钠（Na 3Cit）和膦酰基甲酸（PFA）对高浓度无机磷（Pi）诱导的小鼠主动脉平滑肌细胞（MOVAS）钙化的抑制作用及差异，寻找具有抗凝和抑制血管钙化（VC）作用的药物。方法：在高Pi条件下，随着Et 2Cit、Na 3Cit和PFA浓度的增加，评估存活和固定MOVAS 14天。通过茜素红染色和钙沉积量检测MOVAS上的钙化;通过膜联蛋白V染色检测细胞凋亡;通过测量平滑肌谱系基因（-SMA）表达和碱性磷酸酶活性检测细胞转分化。结果：Et 2Cit、Na 3Cit和PFA与MOVAS共孵育可显著降低PI诱导的MOVAS细胞凋亡率，低浓度抑制剂可降低凋亡率。3种抑制剂均能抑制高磷诱导的碱性磷酸酶活性，增加β-平滑肌肌动蛋白基因的表达。因此，MOVAS向成骨细胞样细胞的转分化被阻断。抑制效果均存在浓度依赖性。各抑制剂在相同浓度下的抑制效果表现为PFA > Na 3Cit> Et 2Cit。结论：Et 2Cit、Na 3Cit和PFA均能阻止MOVAS的钙化，抑制血管平滑肌细胞的骨软骨细胞转化。因此，Et 2Cit和Na 3Cit作为抗凝剂在临床应用中可能缓解VC。
Objective:This study aimed to investigate the differences and inhibitory effects of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid (PFA) on calcification induced by high inorganic phosphate (Pi) contents in mouse aortic smooth muscle cells (MOVAS) and to develop drugs that can induce anticoagulation and inhibit vascular calcification (VC). Methods:Alive and fixed MOVAS were assessed for 14 days in the presence of high Pi with increasing Et2Cit, Na3Cit, and PFA concentrations. Calcification on MOVAS was measured through Alizarin red staining and the deposited calcium amount; apoptosis was detected by annexin V staining; and cell transdifferentiation was examined by measuring smooth muscle lineage gene (-SMA) expression and alkaline phosphatase activity. Results:Coincubation of MOVAS with Et2Cit, Na3Cit, and PFA significantly decreased Pi-induced VC in live MOVAS, and the apoptotic rate was reduced by low inhibitor concentrations. The 3 inhibitors could prevent the alkaline phosphatase activity induced by high Pi contents and increased the expression of –smooth muscle actin genes. Thus, the transdifferentiation of MOVAS into osteoblast-like cells was blocked. Their inhibitory effects exhibited concentration dependence. The inhibitory effect of each inhibitor at the same concentration showed the following trend: PFA > Na3Cit > Et2Cit. Conclusions:Et2Cit, Na3Cit, and PFA prevented the calcification of MOVAS and inhibited the osteochondrocytic conversion of vascular smooth muscle cells. Thus, Et2Cit and Na3Cit as anticoagulants may alleviate VC in clinical applications.