恩格列净通过激活Nrf2信号减轻脂多糖致内皮细胞损伤

Empagliflozin alleviates lipopolysaccharide-induced endothelial cell injury by activating Nrf2 signaling

目的观察恩格列净(Empagliflozin,Em)对脂多糖(lipopolysaccharide,LPS)诱导的人脐静脉内皮细胞(humanumbilicalveinendothelialcells,HUVEC)损伤的作用及机制。方法将HUVEC随机分为4组:Control组、LPS(1μg/ml)组、LPS+Em(1μmol/L)组和LPS+Em+Nrf2 siRNA组,先使用Em和Nrf2 siRNA预处理12 h,然后再用LPS处理24 h。倒置显微镜下观察各组细胞形态变化,CCK-8法检测各组HUVEC活力,Western blot法检测核转录因子E2相关因子(nuclear factor erythroid2-related factor 2,Nrf2)、NADPH氧化酶2(NADPHOxidase2,NOX2)、NADPH氧化酶4(NADPH-Oxidase4,NOX4)、BCL2-相关X蛋白质(Bcl-2associatedX protein,Bax)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl2)的表达。DCFH-DA染色观察细胞活性氧(reactive oxygen species,ROS)含量的变化,TUNEL染色观察细胞凋亡程度,免疫荧光染色检测各组细胞Nrf2表达。结果与Control组相比,LPS组细胞活力降低(P<0.01),Bax表达增加而Bcl2表达减少(P<0.05),细胞凋亡率上升(P<0.05),NOX2和NOX4及ROS含量升高(P<0.05),Nrf2表达下降(P<0.05)。Em干预后可以提高细胞活力(P<0.01),降低Bax表达和细胞凋亡率(P<0.05),并增加Bcl2表达(P<0.05),降低NOX2和NOX4表达及ROS含量(P<0.05),同时上调Nrf2表达(P<0.05)。而给予Nrf2 siRNA处理后,可明显阻断Em对HUVEC的保护作用和对Nrf2信号的调控(P<0.05)。结论Em可以改善LPS引起的HUVEC损伤,其机制与激活Nrf2信号、降低氧化应激有关。

AIM To observe the protective effect and mechanism of empagliflozin(Em)against lipopolysaccharide(LPS)induced injury of human umbilical vein endothelial cells(HUVEC).METHODS HUVEC were randomly divided into 4 groups:control group,LPS(1μg/ml)group,LPS+Em(1μmol/L)group and LPS+Em+Nrf2 siRNA group.HUVEC were pretreated with Em and Nrf2 siRNA for 12h and then were treated with LPS for 24h.The changes of cell morphology were observed under inverted microscope and the viability of HUVEC was detected by CCK-8 method.Nuclear factor erythroid2-related factor 2(Nrf2),NADPH-Oxidase 2(NOX2),NADPH-Oxidase 4(NOX4),Bcl-2 associated X protein(BAX)and B-cell lymphoma-2(Bcl-2)were detected by Western blotting DCFH-DA staining was used to observe the changes in the content of ROS,TUNEL staining was used to observe the degree of apoptosis and immunofluorescence staining was used to detect the expression of Nrf2 in each group.RESULTS Compared with those in control group,the cell viability was decreased(P<0.01),the expression of Bax was increased(P<0.05),the expression of Bcl2 was decreased(P<0.05),the apoptosis rate was increased(P<0.05),the contents of NOX2,NOX4 and ROS were increased(P<0.05)and the expression of Nrf2 was decreased(P<0.05)in LPS group.Em treatment significantly increased the cell viability(P<0.01),decreased Bax expression and apoptosis rate(P<0.05),increased Bcl2 expression(P<0.05),decreased NOX2 and NOX4 expression and ROS content(P<0.05),and increased Nrf2 expression(P<0.05).However,Nrf2 siRNA treatment significantly blocked the protective effect of Em on HUVEC and the regulation of Nrf2 signaling(P<0.05).CONCLUSION Empagliflozin ameliorates lipopolysaccharide induced HUVEC injury and the mechanism is related to activating Nrf2 signaling and reducing oxidative stress.