目的 探究大黃酸調(diào)控血管內(nèi)皮細(xì)胞糖脂代謝功能的機(jī)制。方法 用棕櫚酸干預(yù)人臍靜脈內(nèi)皮細(xì)胞（human umbilical vein endothelial cells，HUVECs），隨后用2.5、5.0、10.0 μmol/L大黃酸處理24 h。qRT-PCR檢測脂肪酸氧化（fatty acid oxidation，F(xiàn)AO）及糖酵解的關(guān)鍵酶[肉堿棕櫚酰轉(zhuǎn)移酶-1A（carnitine palmitoyltransferase-1A，CPT-1A）、葡萄糖轉(zhuǎn)運(yùn)蛋白1（glucose transporter protein 1，GLUT1）、6-磷酸果糖-2-激酶/果糖-2,6-雙磷酸酶3（6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3，PFKFB3）、己糖激酶2（hexokinase 2，HK2）]的表達(dá)情況；Seahorse XF96分析儀檢測FAO和糖酵解水平；ELISA法檢測一氧化氮（nitric oxide，NO）和血管內(nèi)皮生長因子（vascular endothelial growth factor，VEGF）水平；Western blotting及免疫熒光觀察一氧化氮合酶（endothelial nitric oxide synthase，eNOS）、VEGF、CPT-1A、PFKFB3表達(dá)情況；利用分子對接預(yù)測大黃酸的作用靶點(diǎn)。給予PPARα抑制劑norathyriol、FAO抑制劑etomoxir、糖酵解抑制劑3PO進(jìn)行干預(yù)，觀察大黃酸調(diào)節(jié)內(nèi)皮細(xì)胞糖脂代謝功能的變化。結(jié)果 棕櫚酸能顯著上調(diào)HUVECs糖脂代謝酶CPT-1A、GLUT1、PFKFB3、HK2的表達(dá)（P＜0.05、0.01）；與模型組比較，10.0 μmol/L大黃酸顯著上調(diào)CPT-1A的mRNA表達(dá)（P＜0.05），并顯著下調(diào)GLUT1、PFKFB3、HK2的mRNA表達(dá)（P＜0.01）。代謝表型實(shí)驗(yàn)發(fā)現(xiàn)，大黃酸可以促進(jìn)FAO并抑制糖酵解，加入PPARα抑制劑后上述現(xiàn)象發(fā)生逆轉(zhuǎn)。細(xì)胞功能評價(jià)結(jié)果顯示，大黃酸能促進(jìn)血管舒張因子NO并抑制病理性血管生成因子VEGF水平（P＜0.05、0.01）。分子對接發(fā)現(xiàn)大黃酸與PPARα的親和力較高。3PO聯(lián)用大黃酸可抑制糖酵解、促進(jìn)FAO，可以升高eNOS、NO水平，降低VEGF水平；大黃酸聯(lián)用etomoxir能抑制糖酵解及FAO，并恢復(fù)eNOS、NO水平，降低VEGF水平。結(jié)論 大黃酸通過激活PPARα促進(jìn)FAO并抑制糖酵解，進(jìn)而調(diào)節(jié)血管內(nèi)皮細(xì)胞NO、VEGF表達(dá)。

Objective To explore the mechanism of rhein in regulating function of glucose and lipid metabolism in vascular endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) was used for intervention with palmitic acid, followed by treatment with 2.5, 5.0, 10.0 μmol/L rhein for 24 h. qRT-PCR was used to detect the expressions of fatty acid oxidation (FAO) and key enzymes of glycolysis [carnitine palmitoyltransferase-1A (CPT-1A), glucose transporter protein 1 (GLUT1), 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and hexokinase 2 (HK2)]; Seahorse XF96 analyzer was used to detect FAO and glycolysis levels; ELISA was used to detect the levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF); Western blotting and immunofluorescence were used to observe the expressions of endothelial nitric oxide synthase (eNOS), VEGF, CPT1-A and PFKFB3; Molecular docking was used to predict the target of rhein. After intervened with PPARα inhibitor norathyriol, FAO inhibitor etomoxir and glycolysis inhibitor 3PO, the changes in regulation of endothelial cell glucose and lipid metabolism function by rhein was observed. Results Palmitic acid could significantly up-regulate the expressions of glucose and lipid metabolism enzymes (CPT-1A, GLUT1, PFKFB3, HK2) in HUVECs (P < 0.05, 0.01); Compared with model group, 10.0 μmol/L rhein significantly up-regulated the mRNA expression of CPT-1A (P < 0.05), and significantly down-regulated the mRNA expressions of GLUT1, PFKFB3 and HK2 (P < 0.01). Metabolic phenotype experiments found that rhein could promote FAO and inhibit glycolysis, and this phenomenon was reversed after the addition of PPARα inhibitors. The results of cellular functional evaluation showed that rhein could promote vasodilator factor NO and inhibit the level of pathological angiogenic factor VEGF (P < 0.05, 0.01). Molecular docking revealed a high affinity between rhein and PPARα. The combination of 3PO and rhein could inhibit glycolysis, promote FAO, increase eNOS and NO levels, and reduce VEGF level; The combination of rhein and etomoxir could inhibit glycolysis and FAO, restore eNOS and NO levels, and reduce VEGF level. Conclusion Rhein promotes FAO and inhibits glycolysis by activating PPARα, thereby regulating the expressions of NO and VEGF in vascular endothelial cells.

R285.5

國家自然科學(xué)基金面上項(xiàng)目（81873287）