目的 探讨氧化苦参碱（oxymatrine，OMT）对棕榈酸（palmitic acid，PA）诱导的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）胰岛素抵抗（insulin resistance，IR）作用及机制。方法 通过PA（0.25 mmol/L）诱导HUVECs建立IR模型后，进行OMT的量效关系和时效关系研究，筛选出OMT的最佳作用浓度和最佳作用时间。CCK-8法检测HUVECs活力；苏木素-伊红（HE）染色观察细胞形态；Western blotting检测细胞磷酸化磷脂酰肌醇3-激酶（phosphorylated phosphatidylinositol 3-kinase，p-PI3K）/蛋白激酶B（protein kinase B，Akt）/内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）通路、激活信号传导及转录激活因子（signal transducer and activator of transcription，STAT）蛋白抑制因子1（protein inhibitor of activated STAT 1，PIAS1）蛋白表达；试剂盒检测一氧化氮（nitric oxide，NO）水平。根据筛选出的OMT最佳浓度（4 μmol/L）和作用时间（48 h），敲低PIAS1进行机制探讨，Western blotting检测PIAS1、核因子-κB p65（nuclear factor-κB p65，NF-κB p65）蛋白表达；荧光探针2',7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorodihydrofluorescein diacetate，DCFH-DA）检测活性氧（reactive oxygen species，ROS）水平；ELISA法检测白细胞介素-6（interleukin-6，IL-6）水平；免疫荧光法观察NF-κB p65核转位情况。结果 随着OMT作用浓度和时间的增加，PI3K/Akt/eNOS通路和PIAS1蛋白表达量及NO水平均增加（P<0.05、0.01、0.001）。转染Ad-PIAS1-RNAi后，NF-κB p65蛋白表达量、ROS和IL-6水平均显著增加（P<0.01、0.001），NF-κB p65入核程度显著增强（P<0.05、0.01），而OMT可以部分逆转上述作用（P<0.05、0.01、0.001）。结论 OMT能改善PA体外诱导的血管内皮IR，其机制主要是通过上调PIAS1通路减轻高脂诱导的炎症反应和氧化应激。

Objective To explore the effect and mechanism of oxymatrine (OMT) on human umbilical vein endothelial cells (HUVECs) insulin resistance (IR) induced by palmitic acid (PA). Methods IR model was established by PA (0.25 mmol/L)-induced HUVECs. The dose-effect relationship and time-effect relationship of OMT were studied, and the optimal concentration and time of OMT were selected. The cell viability was examined by CCK-8. The cell morphology was observed by HE staining. The protein expressions of phosphorylated phosphatidylinositol 3-kinase (p-PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway and protein inhibitor of activated STAT 1 (PIAS1) were detected by Western blotting. Nitric oxide (NO) level was detected by kit. According to the optimal concentration (4 μmol/L) of OMT and action time (48 h), the mechanism of knocking down PIAS1 was discussed. PIAS1 and nuclear factor-κB p65 (NF-κB p65) protein expressions were determined by Western blotting. The generation of reactive oxygen species (ROS) was observed by fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Interleukin-6 (IL-6) level was detected by ELISA. NF-κB p65 nuclear translocation was determined by immunofluorescence. Results The protein expressions of PI3K/Akt/eNOS pathway and PIAS1 and NO level were significantly increased with the concentration and treatment time of OMT increased (P < 0.05, 0.01, 0.001). After infection with Ad-PIAS1-RNAi, NF-κB p65 protein expression and levels of ROS, IL6 were significantly increased (P < 0.01, 0.001), and the NF-κB p65 nuclear translocation was significantly enhanced (P < 0.05, 0.01). The above effects produced by Ad-PIAS1-RNAi were partially reversed by OMT intervention (P < 0.05, 0.01, 0.001). Conclusion OMT can improve IR by up-regulating PIAS1 pathway to alleviate the inflammation and oxidative stress induced by PA in vascular endothelium in vitro.

R285.5

国家自然科学基金资助项目（81960153）；南昌大学“大学生创新创业训练计划”项目（2022CX267）