Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.
Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.
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柠檬酸二乙酯和柠檬酸钠降低纳米羟基磷灰石晶体对小鼠血管平滑肌细胞的细胞毒性作用
DOI：
10.2147/ijn.s145386
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发表时间：
2017
影响因子：
8
通讯作者：
Gui BS
中科院分区：
文献类型：
--
作者：
Zhang CY;Sun XY;Ouyang JM;Gui BS
目的探讨纳米羟基磷灰石（nano-HAp）对小鼠主动脉平滑肌细胞（MOVASs）的损伤机制及柠檬酸二乙酯（Et 2Cit）和柠檬酸钠（Na 3Cit）的抗损伤作用，为开发同时具有抗凝和抑制血管钙化作用的药物提供实验依据。方法采用细胞增殖检测试剂盒检测细胞活力的变化，采用乳酸脱氢酶（LDH）试剂盒检测LDH的释放量。DCFH-DA染色和JC-1染色检测细胞内活性氧（ROS）和线粒体损伤。Annexin V染色检测细胞凋亡和坏死。分别使用Fluo-4/AM和吖啶橙子测定细胞内钙浓度和溶酶体完整性。结果Nano-HAp可降低细胞活力，破坏细胞膜，导致大量LDH释放。Nano-HAp进入细胞后破坏线粒体，产生大量活性氧，诱导细胞凋亡。此外，nano-HAp增加细胞内Ca 2+浓度，导致溶酶体破裂和细胞坏死。加入抗凝剂Et 2Cit或Na 3Cit后，细胞活力和线粒体膜电位增加，而LDH释放量、ROS和凋亡率降低。Et 2 Cit和Na 3 Cit也可与Ca+螯合，抑制nano-HAp引起的细胞内Ca 2+升高，防止溶酶体破裂，减少细胞坏死。高浓度的Et 2Cit和Na 3Cit表现出强的抑制作用。在相同浓度下，Na_3Cit的抑制作用强于Et_2Cit。结论Et 2Cit和Na 3Cit均能显著降低nano-HAp对MOVAS的细胞毒性，抑制nano-HAp晶体诱导的MOVAS细胞凋亡和坏死。柠檬酸盐的螯合功能导致抗凝和与HAp结合。Et 2Cit和Na 3Cit可能作为抗凝剂在减少nano-HAp引起的血管壁损伤中发挥作用。
Objective This study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et2Cit) and sodium citrate (Na3Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification. Methods The change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively. Results Nano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca2+ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et2Cit or Na3Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et2 Cit and Na3Cit could also chelate with Ca+ to inhibit the intracellular Ca2+ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et2Cit and Na3Cit exhibited strong inhibitory effects. The inhibitory capacity of Na3Cit was stronger than that of Et2Cit at similar concentrations. Conclusion Both Et2Cit and Na3Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et2Cit and Na3Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.