目的 观察羟基红花黄色素A（HSYA）对缺氧复氧诱导的人脐静脉内皮细胞EA.hy926凋亡的影响。方法 噻唑蓝（MTT）比色法检测不同缺氧（8、12 h）、复氧（4、8、12 h）时间对细胞活力的影响以及不同浓度HSYA（0.1、1、10、100 μmol/L）对缺氧12 h、复氧8 h后细胞活力的影响。Western blotting法检测HSYA对缺氧12 h，复氧8 h后EA.hy926细胞中B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的天冬氨酸蛋白水解酶-3（cleaved Caspase-3）、cleaved Caspase-9蛋白表达的影响。实时荧光定量PCR（qRT-PCR）检测HSYA对缺氧12 h复氧8 h后EA.hy926细胞中Bax、Bcl-2 mRNA表达的影响。Hoechest染色、流式细胞术检测HSYA对缺氧12 h复氧8 h后EA.hy926细胞凋亡的影响。结果 与对照组比较，缺氧8、12 h，复氧4、8、12 h后EA.hy926细胞活力均显著下降，其中缺氧12 h、复氧8 h后细胞活力下降最为显著（P<0.001），且Bax、cleaved Caspase-9、cleaved Caspase-3蛋白表达水平显著上升、Bcl-2蛋白表达水平显著下降。与模型组比较，HSYA 10 μmol/L能够明显提高缺氧复氧后细胞活力（P<0.01），并显著上调Bcl-2蛋白表达水平，下调Bax、cleaved Caspase-9、cleaved Caspase-3蛋白表达水平。结论 HSYA能有效抑制缺氧复氧导致的EA.hy926凋亡，其机制可能与下调Bax、cleaved Caspase-9、cleaved Caspase-3蛋白表达、上调Bcl-2蛋白表达有关。

Objective To observe the effect of hydroxysafflower yellow A (HSYA) on the apoptosis of human umbilical vein endothelial cells EA.hy926 induced by hypoxia-reoxygenation. Methods MTT colorimetry method was used to detect the effects of different hypoxia time (8, 12 h) and different reoxygenation time (4, 8, 12 h) on the cell viability. And after the cell had been in the status of hypoxia for 12 h and reoxygenation for 8 h, this method was adapted once again to evaluate the effects of different concentrations of HSYA (0.1, 1, 10, and 100 μmol/L) on cell viability in different time stages. After the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, Western blotting was used to test its effects on the expressions of the following proteins in different time stages, which contained Bcl-2, Bax, cleaved Caspase-3, and activated cleaved Caspase-9. This method was also used to detect whether it had an improvement effect on the above proteins after the pr-treat the cells with HSYA. Real-time PCR was used to evaluate the mRNA expressions of Bax, Bcl-2 after the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, and this method was also used to test the effect of HSYA on the expressions of Bax, Bcl-2 after the same time stages. Hoechest staining and flow cytometry were used to detect the apoptosis situation of the cell after it was in the status of hypoxia for 12 h and reoxygenation for 8 h. And this method was also adapted to detect the effect of HSYA on apoptosis of the cell after the same time stage. Results Compared with the control group, the EA.hy926 cell viability decreased significantly after hypoxia for 8, 12 h, reoxygenation for 4, 8, and 12 h (P < 0.01). The cell viability decreased the most significantly after hypoxia for 12 h and reoxygenation for 8 h (P < 0.01), and during this period, the expression of Bax, cleaved Caspase-9, cleaved Caspase-3 protein increased significantly, and Bcl-2 protein was decreased significantly. Compared with the H/R group, HSYA (10 μmol/L) significantly increased the cell viability (P < 0.01) after hypoxia-reoxygenation, and significantly up-regulated the protein expression of Bcl-2, and down-regulated the protein expressions of Bax, cleaved Caspase-9, and cleaved Caspase-3. Conclusion Hydroxysafflor yellow A can effectively inhibit the apoptosis of EA.hy926 induced by hypoxia and reoxygenation. The mechanism may be related to the down-regulation of Bax, cleaved Caspase-9, cleaved Caspase-3 protein as well as the up-regulation of Bcl-2 protein.

国家自然科学基金资助项目（81574044）；江苏省高等学校自然科学研究面上项目（18KJB330003）；江苏省研究生科研与实践创新计划（KYCX19_1292）