目的 研究委陵菜酸和薔薇酸對(duì)缺氧誘導(dǎo)的內(nèi)皮細(xì)胞損傷的保護(hù)作用，并探究其抗凋亡的機(jī)制。方法 將人臍靜脈內(nèi)皮細(xì)胞（EA.hy926）置于37 ℃、95% N₂、5% CO₂的培養(yǎng)箱培養(yǎng)2 h，建立缺氧損傷模型。給予委陵菜酸和薔薇酸干預(yù)后，采用MTT法檢測(cè)細(xì)胞增殖活力；檢測(cè)細(xì)胞上清液中乳酸脫氫酶（lactate dehydrogenase，LDH）活性；檢測(cè)細(xì)胞內(nèi)丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）水平及超氧化物歧化酶（superoxide dismutase, SOD）、過氧化氫酶（catalase，CAT）活力；采用臺(tái)盼藍(lán)染色觀察細(xì)胞存活率；采用Hoechst/PI雙染觀察細(xì)胞形態(tài)學(xué)變化；采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率；采用Western blotting檢測(cè)凋亡相關(guān)蛋白[B淋巴細(xì)胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相關(guān)X蛋白（Bcl-2 associated X protein，Bax）、細(xì)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）通路相關(guān)蛋白（Akt、p-Akt）和絲裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信號(hào)通路相關(guān)蛋白[胞外信號(hào)調(diào)節(jié)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]的表達(dá)。結(jié)果 與模型組比較，委陵菜酸和薔薇酸顯著提高缺氧誘導(dǎo)的細(xì)胞活力（P＜0.05、0.01），降低LDH釋放量及MDA水平（P＜0.05、0.01），升高SOD、CAT活性及GSH水平（P＜0.05、0.01），減少臺(tái)盼藍(lán)染色藍(lán)染細(xì)胞數(shù)（P＜0.05），減少細(xì)胞膜破裂及Hoechst/PI雙染紅染的細(xì)胞數(shù)，降低細(xì)胞凋亡率（P＜0.05）。Western blotting結(jié)果顯示，委陵菜酸和薔薇酸顯著上調(diào)Bc1-2、pro-Caspase-3表達(dá)（P＜0.01），下調(diào)Bax、Cyt-C、cleaved-Caspase-3、Caspase-9、p-p38、p-JNK表達(dá)（P＜0.01）；委陵菜酸顯著上調(diào)p-ERK1/2表達(dá)（P＜0.01），薔薇酸對(duì)p-ERK1/2表達(dá)無(wú)明顯影響。結(jié)論 委陵菜酸和薔薇酸可顯著改善缺氧誘導(dǎo)的血管內(nèi)皮細(xì)胞缺氧損傷，抑制細(xì)胞凋亡。其抗凋亡機(jī)制均涉及線粒體凋亡信號(hào)途徑，委陵菜酸的抗凋亡機(jī)制可能與調(diào)控ERK1/2、p38、JNK信號(hào)通路有關(guān)，薔薇酸的抗凋亡機(jī)制可能與調(diào)控p38、JNK信號(hào)通路有關(guān)。

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

國(guó)家自然科學(xué)基金資助項(xiàng)目（81073152）；天津市自然科學(xué)基金資助項(xiàng)目（20JCQNJC01260）