氧化低密度脂蛋白经AMPK/mTOR信号通路诱导HUVECs自噬
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摘要
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、m TOR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,oxLDL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±012,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m TOR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pm TOR(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m TOR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/m TOR途径诱导HUVECs发生自噬,并且导致自噬体的积累。
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,pm TOR,m TOR,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 m TOR 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-m TOR( 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/m TOR pathway,which leads to the accumulation of autophagy and promotes the development of atherosclerosis.
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,pm TOR,m TOR,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 m TOR 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-m TOR( 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/m TOR pathway,which leads to the accumulation of autophagy and promotes the development of atherosclerosis.
引文
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[16]Zhao X,Li Y,Lin X,et al.Ozone induces autophagy in rat chondrocytes stimulated with IL-1beta through the AMPK/m TORsignaling pathway[J].J Pain Res,2018,11:3003-3017
[17]Zhao W,Zhang L,Chen R,et al.SIRT3 protects against acute kidney injury via AMPK/m TOR-regulated autophagy[J].Front Physiol,2018,9:1526
[18]Kim YC,Guan KL.m TOR:a pharmacologic target for autophagy regulation[J].J Clin Invest,2015,125(1):25-32
[19]Wang X,Li L,Niu X,et al.m TOR enhances foam cell formation by suppressing the autophagy pathway[J].DNA Cell Biol,2014,33(4):198-204
[20]Kurumbail RG,Calabrese MF.Structure and regulation of AMPK[J].Exp Suppl,2016,107:3-22
[21]Hardie DG,Ross FA,Hawley SA.AMPK:a nutrient and energy sensor that maintains energy homeostasis[J].Nat Rev Mol Cell Biol,2012,13(4):251-262
[22]Viollet B,Horman S,Leclerc J,et al.AMPK inhibition in health and disease[J].Crit Rev Biochem Mol Biol,2010,45(4):276-295
[23]Wang Y,Lin Z,Huang H,et al.AMPK is required for PM2.5-induced autophagy in human lung epithelial A549 cells[J].Int JClin Exp Med,2015,8(1):58-72
[24]Zhang J,Zhao P,Quan N,et al.The endotoxemia cardiac dysfunction is attenuated by AMPK/m TOR signaling pathway regulating autophagy[J].Biochem Biophys Res Commun,2017,492(3):520-527
[25]Fan X,Wang J,Hou J,et al.Berberine alleviates ox-LDLinduced inflammatory factors by up-regulation of autophagy via AMPK/m TOR signaling pathway[J].J Transl Med,2015,13:92
[2]Gimbrone MA Jr,García-Carde1a G.Endothelial cell dysfunction and the pathobiology of atherosclerosis[J].Circ Res,2016,118(4):620-636
[3]Gao S,Zhao D,Wang M,et al.Association between circulating oxidized LDL and atherosclerotic cardiovascular disease:A Metaanalysis of observational studies[J].Can J Cardiol,2017,33(12):1624-1632
[4]Liao X,Sluimer JC,Wang Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553
[5]Perrotta I,Aquila S.The role of oxidative stress and autophagy in atherosclerosis[J].Oxid Med Cell Longev,2015,2015:130315
[6]Li GH,Lin XL,Zhang H,et al.Ox-Lp(a)transiently induces HUVEC autophagy via an ROS-dependent PAPR-1-LKB1-AMPK-mTOR pathway[J].Atherosclerosis,2015,243(1):223-235
[7]Singh R,Kaushik S,Wang Y,et al.Autophagy regulates lipid metabolism[J].Nature,2009,458(7242):1131-1135
[8]De Meyer GR,Grootaert MO,Michiels CF,et al.Autophagy in vascular disease[J].Circ Res,2015,116(3):468-479
[9]Nussenzweig SC,Verma S,Finkel T.The role of autophagy in vascular biology[J].Circ Res,2015,116(3):480-488
[10]Hou X,Hu Z,Xu H,et al.Advanced glycation endproducts trigger autophagy in cadiomyocyte via RAGE/PI3K/AKT/m TORpathway[J].Cardiovasc Diabetol,2014,13:78
[11]Ravanan P,Srikumar IF,Talwar P.Autophagy:The spotlight for cellular stress responses[J].Life Sci,2017,188:53-67
[12]Levine B,Klionsky DJ.Development by self-digestion:molecular mechanisms and biological functions of autophagy[J].Dev Cell,2004,6(4):463-477
[13]Ma Y,Huang Z,Zhou Z,et al.A novel antioxidant Mito-Tempol inhibits ox-LDL-induced foam cell formation through restoration of autophagy flux[J].Free Radic Biol Med,2018,129:463-472
[14]Gomez-Sanchez R,Yakhine-Diop SM,Rodriguez-Arribas M,et al.mRNA and protein dataset of autophagy markers(LC3 and p62)in several cell lines[J].Data Brief,2016,7:641-647
[15]Zhao J,Goldberg AL.Coordinate regulation of autophagy and the ubiquitin proteasome system by MTOR[J].Autophagy,2016,12(10):1967-1970
[16]Zhao X,Li Y,Lin X,et al.Ozone induces autophagy in rat chondrocytes stimulated with IL-1beta through the AMPK/m TORsignaling pathway[J].J Pain Res,2018,11:3003-3017
[17]Zhao W,Zhang L,Chen R,et al.SIRT3 protects against acute kidney injury via AMPK/m TOR-regulated autophagy[J].Front Physiol,2018,9:1526
[18]Kim YC,Guan KL.m TOR:a pharmacologic target for autophagy regulation[J].J Clin Invest,2015,125(1):25-32
[19]Wang X,Li L,Niu X,et al.m TOR enhances foam cell formation by suppressing the autophagy pathway[J].DNA Cell Biol,2014,33(4):198-204
[20]Kurumbail RG,Calabrese MF.Structure and regulation of AMPK[J].Exp Suppl,2016,107:3-22
[21]Hardie DG,Ross FA,Hawley SA.AMPK:a nutrient and energy sensor that maintains energy homeostasis[J].Nat Rev Mol Cell Biol,2012,13(4):251-262
[22]Viollet B,Horman S,Leclerc J,et al.AMPK inhibition in health and disease[J].Crit Rev Biochem Mol Biol,2010,45(4):276-295
[23]Wang Y,Lin Z,Huang H,et al.AMPK is required for PM2.5-induced autophagy in human lung epithelial A549 cells[J].Int JClin Exp Med,2015,8(1):58-72
[24]Zhang J,Zhao P,Quan N,et al.The endotoxemia cardiac dysfunction is attenuated by AMPK/m TOR signaling pathway regulating autophagy[J].Biochem Biophys Res Commun,2017,492(3):520-527
[25]Fan X,Wang J,Hou J,et al.Berberine alleviates ox-LDLinduced inflammatory factors by up-regulation of autophagy via AMPK/m TOR signaling pathway[J].J Transl Med,2015,13:92