氧化低密度脂蛋白经AMPK/mTOR信号通路诱导HUVECs自噬

氧化低密度脂蛋白（oxygenized low density lipoprotein, ox-LDL）诱导人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）损伤有助于动脉粥样硬化（atherosclerosis, AS）的发展。但ox-LDL对HUVECs自噬的影响及机制尚不清楚。为探究其机制，采用体外培养HUVECs，建立ox-LDL损伤模型。透射电子显微镜观察HUVECs中自噬体的变化;Western印迹法检测p-AMPK、AMPK、p-mTOR、mTOR及Beclin1、LC3-II、P62的表达。结果显示，与对照组比较，透射电子显微镜下观察到ox-LDL组的自噬体明显增多。Western印迹结果显示，与对照组比较，ox-LDL组Beclin1(0.81±0.04 vs. 1.83±0.11，P＜0.01)、LC3-II(0.80±0.06 vs. 1.61±0.06, P＜0.01)和P62(0.65±0.10 vs. 1.64±0.17, P＜0.01)表达显著增高。ox-LDL和BafilomycinA1共同干预组Beclin-1(3.15±0.15 vs. 3.17±0.13, P>0.05)、LC3-II(2.95±0.12 vs. 2.96±0.12, P >0.05)和P62(3.26±0.15 vs. 3.19±0.15, P>0.05)表达与BafilomycinA1组无显著差异，ox-LDL未使自噬起始增加，可能是降解受损导致自噬体的积累。与对照组比较，ox-LDL增加p-AMPK (0.47±0.03 vs. 0.96±0.03, P＜0.01)表达，并降低p-mTOR(0.86±0.04 vs. 0.25±0.05, P＜0.01)表达。单独阻断mTOR时， Beclin-1(0.81±0.05 vs. 2.19±0.17, P＜0.01)、LC3-II(0.76±0.13 vs 2.00±0.05, P＜0.01)和P62(0.74±0.12 vs. 1.94±0.11, P＜0.01)表达显著增加。亮氨酸（Leucine）可增加p-mTOR(0.87±0.11 vs. 1.67±0.07, P＜0.01)表达，并降低Beclin-1(0.81±0.05 vs. 0.37±0.03, P＜0.01)、LC3-II（0.76±0.13 vs. 0.41±0.02, P＜0.01）和P62（0.76±0.10 vs. 0.44±0.04, P＜0.01）表达，但ox-LDL可使Leucine预处理后的p-mTOR（1.67±0.11 vs. 0.82±0.02, P＜0.01）表达显著降低，并且Beclin-1（0.37±0.03 vs. 0.78±0.04, P＜0.01）、LC3-II（0.41±0.02 vs. 0.78±0.02, P＜0.01）和P62（0.44±0.04 vs. 0.74±0.04, P＜0.01）表达显著增加。说明mTOR参与ox-LDL诱导的自噬。与ox-LDL组相比，ox-LDL和Si-AMPK共同处理组p-mTOR(0.25±0.05 vs. 0.46±0.03, P＜0.01)表达增加以及Beclin-1(1.97±0.04 vs. 1.26±0.12, P＜0.01)、LC3-II(1.42±0.10 vs. 0.95±0.05, P＜0.01)和P62(1.58±0.09 vs. 0.98±0.11, P＜0.01)表达降低。以上结果表明，ox-LDL通过AMPK/mTOR途径诱导HUVECs发生自噬，并且导致自噬体的积累。

Oxidized Low Density Lipoprotein Induces HUVEC Autophagy by the AMPK/mTOR Signaling Pathway

Oxidized low density lipoprotein (ox-LDL) induces injury of human umbilical vein endothelial cells (HUVECs) and contributes to the development of atherosclerosis (AS). However, the effect and mechanism of ox-LDL on autophagy in HUVECs remain unclear. In order to explore its mechanism，HUVECs were cultured in vitro and an ox-LDL injury model was established. The changes of autophagy in HUVECs were observed by transmission electron microscopy. The expressions of p-AMPK, AMPK，p-mTOR, mTOR，Beclin1, LC3-II and P62 were detected by Western blotting. The results showed that the autophagic levels in the ox-LDL group increased significantly compared with the control group under transmission electron microscopy. Western blotting results showed that the expressions of Beclin1 (0.81±0.04 vs.1.83±0.11, P<0.01)，LC3-II (0.80±0.06 vs.1.61±0.06, P<0.01) and P62 (0.65±0.10 vs．1.64±0.17, P<0.01) in the ox-LDL group were significantly higher than those in the control group. There was no significant difference in Beclin-1 (3.15± 0.15 vs. 3.17± 0.13, P > 0.05), LC3-II (2.95 ±0.12 vs. 2.96± 012, P>0.05) and P62 (3.26± 0.15 vs. 3.19± 0.15, P>0.05) expression levels between the ox-LDL and Bafilomycin A1 co-treatment group and the Bafilomycin A1 group. Ox-LDL did not increase the initiation of autophagy, which may be due to the accumulation of autophagy due to degradation damage. In addition, ox-LDL increased the expression of p-AMPK (0.47±0.03 vs. 0.96±0.03, P<0.01) and decreased the expression of p-mTOR (0.86±0.04 vs. 0.25±0.05, P<0.01). When mTOR was blocked alone, Beclin-1(0.81±0.05 vs. 2.19±0.17, P<0.01)，LC3-II (0.76±0.13 vs． 2.00±0.05, P<0.01) and P62 (0.74±0.12 vs. 1.94±0.11, P<0.01) expression increased significantly. Leucine increased the expression of p-mTOR (0.87±0.11 vs. 1.67±0.07, P<0.01) and decreased the expression of Beclin-1 (0.81±0.05 vs. 0.37±0.03, P<0.01), LC3-II (0.76±0.13 vs．0.41±0.02, P<0.01), P62 (0.76±0.10 vs．0.44±0.04, P<0.01). However, ox-LDL significantly reduced the expression of p-mTOR (1.67±0.11 vs. 0.82±0.02, P<0.01) after Leucine pretreatment，and Beclin-1 (0.37±0.03 vs.0.78±0.04, P<0.01), LC3-II (0.41 ± 0.02 vs. 0.78 ± 0.02, P<0.01), P62 (0.44 ± 0.04 vs. 0.74 ± 0.04, P<0.01) expression increased significantly. Compared with the ox-LDL group, the expression of p-mTOR (0.25±0.05 vs. 0.46±0.03, P<0.01) increased and Beclin-1 (1.97±0.04 vs. 1.26±0.12, P<0.01), LC3-II (1.42±0.10 vs. 0.95±0.05, P<0.01), P62 (1.58±0.09 vs. 0.98±0.11, P<0.01) decreased in ox-LDL and si-AMPK co-treatment groups. Compared with the ox-LDL group, ox-LDL and si-AMPK co-treatment group enhanced the expression of p-mTOR (0.25±0.05 vs. 0.46±0.03, P<0.01) and decreased the expression of Beclin-1 (1.97±0.04 vs. 1.26±0.12, P<0.01), LC3-II (1.42±0.10 vs. 0.95±0.05, P<0.01) and P62 (1.58±0.09 vs．0.98±0.11, P<0.01). In conclusion，the results indicate that ox-LDL induces autophagy in HUVECs through the AMPK/mTOR pathway, which leads to the accumulation of autophagy and promotes the development of atherosclerosis.