Department of Cardiovascular Surgery,the First Affiliated Hospital of Nanjing Medical University,Nanjing,China.210029
目的：探究DEHP暴露对PI3K/AKT信号通路和HCAECs的影响，为寻求DEHP暴露致HCAECs损伤的治疗提供理论基础。方法：根据相关文献研究，用不同浓度DEHP使HCAECs染毒，设置对照组（DMSO），实验组（DEHP 128、256、384、512μM）。用CCK-8法测定细胞增殖能力；体外成管实验评估细胞成管能力；流式细胞术评估细胞凋亡水平；Western-Blot检测细胞中P-PI3K、PI3K、P-AKT、AKT和凋亡相关蛋白BAX、Bcl-2、cleaved caspase-3和caspase-3水平；结果：CCK-8结果显示，DEHP以浓度和时间依赖的方式抑制HCAECs的增殖能力（p<0.05）。体外成管实验结果显示，实验组细胞形态多呈现为孤立的圆形、卵圆形，当DEHP浓度≥256μM时，其形成交点数目较对照组减少（p<0.05）。流式细胞实验结果显示，实验组凋亡比例较对照组上升（p<0.05）。Western-blot结果显示实验组Bcl-2表达水平较对照组降低，同时BAX和cleaved caspase-3/caspase-3的水平升高（p<0.05）；当DEHP≥256μM时，P-PI3K/PI3K、P-AKT/AKT比值较对照组降低（p<0.05）。结论：DEHP可通过抑制PI3K/AKT/信号通路诱导HCAECs凋亡、抑制其增殖、血管形成能力。
Objective: To investigate the effects of DEHP exposure on PI3K/AKT signaling pathway and HCAECs, and to provide a theoretical basis for the treatment of HCAECs damaged by DEHP exposure. Methods: According to relevant literature research, HCAECs were treated with different concentrations of DEHP and divided into control group (DMSO) and experimental group (DEHP 128, 256, 384, 512 μM). The ability of cell proliferation was measured by CCK- 8 assay, the ability of cell tube formation was evaluated by in vitro tube formation assay, the level of apoptosis was evaluated by flow cytometry, and the levels of P-PI3K, PI3K, P-AKT, AKT and apoptosis-related proteins BAX, Bcl-2, cleaved caspase-3 and caspase-3 were detected by Western-Blot. Results: CCK-8 assay showed that DEHP inhibited the proliferation ability of HCAECs in a concentration- and time-dependent manner (p<0.05). The results of in vitro tube formation assay showed that the morphology of cells in the experimental group was mostly isolated round and oval. The number of intersections formed was reduced compared with the control group (p<0.05) when the DEHP concentration was ≥256 μM. The results of flow cytometry showed that the proportion of apoptosis in the experimental group was higher than that in the control group (p<0.05). Western-blot results showed that the expression level of Bcl-2 in the experimental group was lower than that in the control group, while the levels of BAX and cleaved caspase-3/caspase-3 increased (p<0.05). And the ratio of P-PI3K/PI3K and P-AKT/AKT were lower than those in the control group (p<0.05) when DEHP≥256μM. Conclusion: DEHP can induce apoptosis and inhibit the proliferation and angiogenesis of HCAECs by inhibiting PI3K/AKT/ signal pathway.