黄芪多糖通过活化 AMPK-eNOS 信号通路减轻游离脂肪酸对人血管内皮细胞的损伤

摘要

目的：研究黄芪多糖对游离脂肪酸所诱导的血管内皮细胞损伤的作用及机制。方法：培养人脐静脉内皮细胞（HUVECs），实验分为正常对照组、黄芪多糖组[黄芪多糖（200 mg／L）处理24 h]、游离脂肪酸组[游离脂肪酸（0.25 mmol／L）共同处理24 h]、游离脂肪酸加黄芪多糖组[游离脂肪酸（0.25 mmol／L）和黄芪多糖（200 mg／L）共同处理24 h]和compound C组[游离脂肪酸（0.25 mmol／L）、黄芪多糖（200 mg／L）及AMPK阻断剂compound C （10μmol／L）处理24 h]。采用MTT法检测细胞活性，硝酸还原酶法测定培养液中一氧化氮（ NO）含量，Western blot法测细胞总腺苷酸活化蛋白激酶（ AMPK ）、磷酸化腺苷酸活化蛋白激酶（ p-AMPK ）、内皮型一氧化氮合酶（ eNOS）及磷酸化内皮型一氧化氮合酶（ p-eNOS）的蛋白水平。结果：黄芪多糖组各项指标较正常对照组无显著差异。 MTT结果显示游离脂肪酸组的细胞活性较正常对照组明显下降，游离脂肪酸加黄芪多糖组的细胞活性较游离脂肪酸组明显好转，compound C组的细胞活性与游离脂肪酸组无显著差异。游离脂肪酸组的NO含量及p-AMPK、p-eNOS水平较对照组明显减少，黄芪多糖能显著抑制游离脂肪酸所致的NO含量及p-AMPK、p-eNOS水平的减少， compound C则可阻断黄芪多糖的作用。各组AMPK及eNOS表达均无明显差异。结论：黄芪多糖可以减轻游离脂肪酸诱导的血管内皮细胞损伤，其机制与AMPK-eNOS信号通路有关。%[ ABSTRACT] AIM:To study the protective effect of Astragalus polysaccharides ( APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs).METHODS: Cultured HUVECs were divided into control group, APS group [ APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and com-pound C group [ free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h] .The cell viability was detected by MTT assay.Nitric oxide ( NO) content in the medium was determined by nitrate reductase assay.The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phos-phorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase ( p-eNOS) were measured by Western blot.RESULTS: No significant difference of all indexes between APS group and control group was observed.The cell viability in free fatty acid group de-creased significantly compared with control group.The cell viability in free fatty acid plus APS group was significantly im-proved as compared with free fatty acid group.The cell viability in compound C group was almost the same as that in free fatty acid group.The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group.No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed.No significant difference of the AMPK and eNOS protein levels in all groups was found.CONCLUSION:APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.