目的 研究委陵菜酸和蔷薇酸对缺氧诱导的内皮细胞损伤的保护作用，并探究其抗凋亡的机制。方法 将人脐静脉内皮细胞（EA.hy926）置于37 ℃、95% N₂、5% CO₂的培养箱培养2 h，建立缺氧损伤模型。给予委陵菜酸和蔷薇酸干预后，采用MTT法检测细胞增殖活力；检测细胞上清液中乳酸脱氢酶（lactate dehydrogenase，LDH）活性；检测细胞内丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）水平及超氧化物歧化酶（superoxide dismutase, SOD）、过氧化氢酶（catalase，CAT）活力；采用台盼蓝染色观察细胞存活率；采用Hoechst/PI双染观察细胞形态学变化；采用流式细胞术检测细胞凋亡率；采用Western blotting检测凋亡相关蛋白[B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、细胞色素C（cytochrome-C，Cyt-C）、半胱氨酸天冬氨酸蛋白酶-9（cystein-asparate protease-9，Caspase-9）、cleaved Caspase-3、pro-Caspase-3）]及蛋白激酶B（protein kinase B，Akt）通路相关蛋白（Akt、p-Akt）和丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路相关蛋白[胞外信号调节激酶1/2（extracellular regulated protein kinase 1/2，ERK1/2）、p-ERK1/2、p38、p-p38、Janus激酶（Janus kinase，JNK）、p-JNK]的表达。结果 与模型组比较，委陵菜酸和蔷薇酸显著提高缺氧诱导的细胞活力（P＜0.05、0.01），降低LDH释放量及MDA水平（P＜0.05、0.01），升高SOD、CAT活性及GSH水平（P＜0.05、0.01），减少台盼蓝染色蓝染细胞数（P＜0.05），减少细胞膜破裂及Hoechst/PI双染红染的细胞数，降低细胞凋亡率（P＜0.05）。Western blotting结果显示，委陵菜酸和蔷薇酸显著上调Bc1-2、pro-Caspase-3表达（P＜0.01），下调Bax、Cyt-C、cleaved-Caspase-3、Caspase-9、p-p38、p-JNK表达（P＜0.01）；委陵菜酸显著上调p-ERK1/2表达（P＜0.01），蔷薇酸对p-ERK1/2表达无明显影响。结论 委陵菜酸和蔷薇酸可显著改善缺氧诱导的血管内皮细胞缺氧损伤，抑制细胞凋亡。其抗凋亡机制均涉及线粒体凋亡信号途径，委陵菜酸的抗凋亡机制可能与调控ERK1/2、p38、JNK信号通路有关，蔷薇酸的抗凋亡机制可能与调控p38、JNK信号通路有关。

Objective To study the protective effects of tormentic acid (TA) and euscaphic acid (EA) on hypoxia-induced endothelial cell damage, and explore the mechanism of anti-apoptosis. Methods Human umbilical vein endothelial cells (EA.hy926) were cultured in 37 ℃, 95% N₂ and 5% CO₂ incubator for 2 h to establish an hypoxia injury model. After intervention with TA and EA, the cell proliferation activity was detected by MTT assay; The activity of lactate dehydrogenase (LDH) in the cell supernatant was detected; The levels of malondialdehyde (MDA), glutathione (GSH) and the activities of superoxide dismutase (SOD), catalase (CAT) in cells were detected; Trypan blue staining was used to observe cell survival rate; Cellular morphological changes were observed by Hoechst/PI double staining; Flow cytometry was used to detect cell apoptosis rate; Western blotting was used to detect the expressions of apoptosis related proteins [B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome C (Cyt-C), cysteine aspartate protease-9 (Caspase-9), cleaved Caspase-3, pro-Caspase-3)], as well as protein kinase B (Akt) pathway related proteins (Akt, p-Akt) and mitogen-activated protein kinase (MAPK) signaling pathway related proteins [extracellular regulated protein kinase 1/2 (ERK1/2), p-ERK1/2, p38, p-p38, Janus kinase (JNK) and p-JNK]. Results Compared with model group, TA and EA significantly increased hypoxia induced cell viability (P < 0.05, 0.01), reduced LDH release and MDA level (P < 0.05, 0.01), increased activities of SOD, CAT and GSH level (P < 0.05, 0.01), reduced the number of cells stained with trypan blue (P < 0.05), decreased cell membrane rupture and the number of cells with Hoechst/PI double staining, and reduced cell apoptosis rate (P < 0.05). The Western blotting results showed that TA and EA significantly up-regulated the expressions of Bc1-2 and pro-Caspase-3 (P < 0.01), down-regulated the expressions of Bax, Cyt-C, cleaved-Caspase-3, Caspase-9, p-p38 and p-JNK (P < 0.01); TA significantly up-regulated p-ERK1/2 expression (P < 0.01), while EA had no significant effect on p-ERK1/2 expression. Conclusion TA and EA could significantly improve hypoxia injury of vascular endothelial cells and inhibit cell apoptosis. The anti-apoptotic mechanism is involved the mitochondrial apoptosis signaling pathway. The anti-apoptotic mechanism of TA may be related to the regulation of ERK1/2, p38 and JNK signaling pathways, while the anti-apoptotic mechanism of EA may be related to the regulation of p38 and JNK signaling pathways.

R285.5

国家自然科学基金资助项目（81073152）；天津市自然科学基金资助项目（20JCQNJC01260）