枸橼酸二乙酯、枸橼酸钠和膦甲酸钠对高钙诱导小鼠血管平滑肌细胞钙化的抑制

在高钙及不同浓度枸橼酸二乙酯(Et2Cit)、枸橼酸钠(Na3Cit)和膦甲酸钠(PFA)存在下培养小鼠血管平滑肌细胞(MOVAS)14 d,分别通过茜素红染色、免疫荧光和annexin V染色检测细胞的分化、凋亡和细胞钙沉积量,研究了Et2Cit、Na3Cit和PFA对高钙诱导MOVAS细胞钙化的抑制效果及可能的作用机制。结果表明,Et2Cit、Na3Cit和PFA均能减少高钙诱导的MOVAS钙化,减少细胞外钙化斑块和钙沉积量。这些抑制剂均能抑制平滑肌细胞向成骨样细胞表型转化,导致向成骨细胞转化的标记物碱性磷酸酶(ALP)活性降低。抑制效果均存在浓度依赖性。当抑制剂浓度相同时,其抑制效果从大到小为:PFA>Na3Cit>Et2Cit。低浓度的Et2Cit和Na3Cit可通过减少细胞凋亡来抑制钙化,但高浓度的Et2Cit、Na3Cit和PFA自身具有毒性,这增加了细胞的凋亡。作为血液抗凝剂的Et2Cit和Na3Cit可以有效地抑制MOVAS的钙化。

Inhibition of Diethyl Citrate, Sodium Citrate, and Phosphonoformic Acid on Mouse Aortic Smooth Muscle Cells Calcification Induced by High Calcium

Mouse aortic smooth muscle cells (MOVAS) were cultured for 14 d under the conditions of high calcium and different concentrations of diethyl citrate (Et₂Cit), sodium citrate (Na₃Cit), and phosphonoformic acid (PFA). Alizarin red staining, immunofluorescence and annexin V staining were used to detect cell transdifferentiation, apoptosis, and cell calcium deposition. The inhibitory effects of Et₂Cit, Na₃Cit, and PFA on calcium-induced calcification of MOVAS cells and their possible mechanisms were studied. The results showed that Et₂Cit, Na₃Cit, and PFA can all reduce MOVAS calcification induced by high calcium and reduce extracellular calcified plaque and calcium deposition. These inhibitors can inhibit the transdifferentiation of MOVAS to osteoblast-like cells, resulting in a decrease in the activity of alkaline phosphatase (ALP), a marker transformed to osteogenic differentiation. Their inhibitory effects exhibited concentration dependence. The inhibitory effects of the inhibitors at the same concentration showed the following trend:PFA > Na₃Cit > Et₂Cit. Low concentrations of Na₃Cit and Et₂Cit inhibit calcification by reducing cell apoptosis, but high concentrations of Na₃Cit, Et₂Cit, and PFA increased cell apoptosis due to their own toxicity. Et₂Cit and Na₃Cit as blood anticoagulants can effectively inhibit MOVAS calcification induced by high calcium.