Department of Cardiology, Qilu Hospital of Shandong University, Qingdao, China.
Eur Rev Med Pharmacol Sci. 2018 May;22(10):3199-3206. doi: 10.26355/eurrev_201805_15081.
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.
研究他克莫司（tacrolimus）对氧化型低密度脂蛋白（ox-LDL）损伤血管内皮细胞的保护作用及其机制。
以人脐静脉内皮细胞为研究对象，分为对照组、他克莫司组和自噬抑制组。对照组不接受 ox-LDL，而他克莫司组和自噬抑制组用 100μg/mL ox-LDL 处理 3 小时。他克莫司组先用他克莫司（100nM）预处理 0.5 小时，自噬抑制组先用 3-甲基腺嘌呤（3-MA）（10mM）和他克莫司（100nM）预处理 0.5 小时。通过细胞计数试剂盒 8（CCK8）法检测细胞活力，通过流式细胞术和 Hoechst 染色检测细胞凋亡率，通过试剂盒检测超氧化物歧化酶（SOD）、活性氧（ROS）等细胞因子的释放。此外，通过 LC3 荧光染色检测自噬水平，通过聚合酶链反应（PCR）和 Western 印迹检测自噬和凋亡相关分子。
在没有 ox-LDL 的情况下，他克莫司和 3-MA 对细胞活力均无影响。加入 ox-LDL 后，细胞活力明显下降，而他克莫司可减轻 ox-LDL 对细胞的损伤。流式细胞术和 Hoechst 染色证明，他克莫司可降低 ox-LDL 诱导的细胞凋亡比例，而 PCR 和 Western 印迹证实他克莫司组中凋亡相关蛋白的表达减少。3-MA 可上调细胞凋亡比例和凋亡相关蛋白的表达。SOD 和 ROS 的检测表明，ox-LDL 可诱导细胞氧化应激损伤，而他克莫司可抑制这种作用。加入 3-MA 可抑制他克莫司的作用。此外，LC3 荧光染色、PCR 和 Western 印迹显示，ox-LDL 可诱导自噬，而他克莫司可增强自噬。加入 3-MA 后，细胞内自噬水平明显受到抑制。
他克莫司通过诱导自噬来保护血管内皮细胞免受 ox-LDL 损伤。
