Tacrolimus alleviates Ox-LDL damage through inducing vascular endothelial autophagy.

Abstract

To study the protective effect of tacrolimus on vascular endothelium injured by oxidized low-density lipoprotein (ox-LDL) and its mechanism. Human umbilical vein endothelial cells were used as objects of study, and divided into control group, tacrolimus group and autophagy inhibition group. Control group received no ox-LDL, while tacrolimus group and autophagy inhibition group were treated with ox-LDL (100 μg/mL) for 3 h. Tacrolimus group was pre-treated with tacrolimus (100 nM) for 0.5 h, and the autophagy inhibition group was pre-treated with 3-methyladenine (3-MA) (10 mM) and tacrolimus (100 nM) for 0.5 h. The cell viability was detected via cell counting kit 8 (CCK8) assay, the cell apoptosis ratio was detected via flow cytometry and Hoechst staining, and the releases of superoxide dismutase (SOD), reactive oxygen species (ROS) and other cytokines were detected using the kit. Moreover, the autophagy level was detected via LC3 fluorescence staining, and the autophagy- and apoptosis-related molecules were detected via polymerase chain reaction (PCR) and Western blotting. In the absence of ox-LDL, neither tacrolimus nor 3-MA had an effect on the cell viability. After the addition of ox-LDL, the cell viability was significantly decreased, whereas tacrolimus could alleviate such damage to cells. Flow cytometry and Hoechst staining proved that tacrolimus could reduce the proportion of apoptotic cells induced by ox-LDL, while PCR and Western blotting confirmed the decreased expression of apoptosis-related proteins in tacrolimus group. 3-MA could up-regulate the ratio of apoptosis and the expressions of apoptosis-related proteins. The detection of SOD and ROS showed that ox-LDL could induce the cell oxidative stress injury, whereas tacrolimus could inhibit such an effect. The addition of 3-MA inhibited the effect of tacrolimus. Besides, LC3 fluorescence staining, PCR and Western blotting revealed that ox-LDL could induce the autophagy, while tacrolimus could enhance the autophagy. After the addition of 3-MA, the intracellular autophagy level was significantly inhibited. Tacrolimus protects vascular endothelial cells from ox-LDL damage through inducing the autophagy.